A validated assay by liquid chromatography-tandem mass spectrometry for the simultaneous quantification of elvitegravir and rilpivirine in HIV positive patients.

Because of the large variability in the pharmacokinetics of anti-HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC-MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20-mM ammonium acetate/MeOH 50:50. After reverse-phase chromatography, quantification of RPV and EVG, using matrix-matched calibration samples, is performed by electrospray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic-labeled compounds RPV-(13) C6 and EVG-D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long-term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter-day CV%: 3-6.3%) and accurate (3.8-7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, -20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in-source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti-HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service. Copyright © 2013 John Wiley & Sons, Ltd.

Low adiponectin is associated with increased ambulatory pulse pressure and activation of the renin-angiotensin system in subjects of African descent

Purpose: Adiponectin, arterial stiffness, as well components of the renin-angiotensin system are associated with cardiovascular risk. This study was aimed to investigate whether plasma adiponectin was directly linked with pulse pressure (PP), as a marker for arterial stiffness, and the renin-angiotensin system (RAS). Methods and materials: A family-based study in subjects of African descent enriched with hypertensive patients was carried out in the Seychelles. Fasting plasma adiponectin was determined by ELISA, plasma renin activity according to the antibody-trapping principle and plasma aldosterone by radioimmunoassay. Daytime ambulatory blood pressure (BP) was measured using Diasys Integra devices. PP was calculated as the difference between systolic and diastolic BP. The association of adiponectin with PP, plasma renin activity and plasma aldosterone were analyzed using generalized estimating equations with a gaussian family link and an exchangeable correlation structure to account for familial aggregation. Results: Data from 335 subjects from 73 families (152 men, 183 women) were available. Men and women had mean (SD) age of 45.4 ± 11.1 and 47.3 ± 12.4 years, BMI of 26.3 ± 4.4 and 27.8 ± 5.1 kg/m2, daytime systolic/diastolic BP of 132.6 ± 15.4 / 86.1 ± 10.9 and 130 ± 17.6 / 83.4 ± 11.1 mmHg, and daytime PP of 46.5 ± 9.9 and 46.7 ± 10.7 mmHg, respectively. Plasma adiponectin was 4.4± 3.04 ng/ml in men and 7.39 ± 5.44 ng/ml in women (P <0.001). After adjustment for age, sex and BMI, log-transformed adiponectin was negatively associated with daytime PP (-0.009 ± 0.003, P = 0.004), plasma renin activity (-0.248 ± 0.080, P = 0.002) and plasma aldosterone (-0.004 ± 0.002, P = 0.014). Conclusion: Low adiponectin is associated with increased ambulatory PP and RAS activation in subjects of African descent. Our data are consistent with the observation that angiotensin II receptor blockers increase adiponectin in humans.

Association of eGFR-Related Loci Identified by GWAS with Incident CKD and ESRD.

Family studies suggest a genetic component to the etiology of chronic kidney disease (CKD) and end stage renal disease (ESRD). Previously, we identified 16 loci for eGFR in genome-wide association studies, but the associations of these single nucleotide polymorphisms (SNPs) for incident CKD or ESRD are unknown. We thus investigated the association of these loci with incident CKD in 26,308 individuals of European ancestry free of CKD at baseline drawn from eight population-based cohorts followed for a median of 7.2 years (including 2,122 incident CKD cases defined as eGFR <60ml/min/1.73m(2) at follow-up) and with ESRD in four case-control studies in subjects of European ancestry (3,775 cases, 4,577 controls). SNPs at 11 of the 16 loci (UMOD, PRKAG2, ANXA9, DAB2, SHROOM3, DACH1, STC1, SLC34A1, ALMS1/NAT8, UBE2Q2, and GCKR) were associated with incident CKD; p-values ranged from p = 4.1e-9 in UMOD to p = 0.03 in GCKR. After adjusting for baseline eGFR, six of these loci remained significantly associated with incident CKD (UMOD, PRKAG2, ANXA9, DAB2, DACH1, and STC1). SNPs in UMOD (OR = 0.92, p = 0.04) and GCKR (OR = 0.93, p = 0.03) were nominally associated with ESRD. In summary, the majority of eGFR-related loci are either associated or show a strong trend towards association with incident CKD, but have modest associations with ESRD in individuals of European descent. Additional work is required to characterize the association of genetic determinants of CKD and ESRD at different stages of disease progression.

Impairment of the Bacterial Biofilm Stability by Triclosan

The accumulation of the widely-used antibacterial and antifungal compound triclosan (TCS) in freshwaters raises concerns about the impact of this harmful chemical on the biofilms that are the dominant life style of microorganisms in aquatic systems. However, investigations to-date rarely go beyond effects at the cellular, physiological or morphological level. The present paper focuses on bacterial biofilms addressing the possible chemical impairment of their functionality, while also examining their substratum stabilization potential as one example of an important ecosystem service. The development of a bacterial assemblage of natural composition – isolated from sediments of the Eden Estuary (Scotland, UK) – on non-cohesive glass beads (,63 mm) and exposed to a range of triclosan concentrations (control, 2 – 100 mg L21) was monitored over time by Magnetic Particle Induction (MagPI). In parallel, bacterial cell numbers, division rate, community composition (DGGE) and EPS (extracellular polymeric substances: carbohydrates and proteins) secretion were determined. While the triclosan exposure did not prevent bacterial settlement, biofilm development was increasingly inhibited by increasing TCS levels. The surface binding capacity (MagPI) of the assemblages was positively correlated to the microbial secreted EPS matrix. The EPS concentrations and composition (quantity and quality) were closely linked to bacterial growth, which was affected by enhanced TCS exposure. Furthermore, TCS induced significant changes in bacterial community composition as well as a significant decrease in bacterial diversity. The impairment of the stabilization potential of bacterial biofilm under even low, environmentally relevant TCS levels is of concern since the resistance of sediments to erosive forces has large implications for the dynamics of sediments and associated pollutant dispersal. In addition, the surface adhesive capacity of the biofilm acts as a sensitive measure of ecosystem effects

Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry I. Screening analysis.

The general strategy to perform anti-doping analyses of urine samples starts with the screening for a wide range of compounds. This step should be fast, generic and able to detect any sample that may contain a prohibited substance while avoiding false negatives and reducing false positive results. The experiments presented in this work were based on ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry. Thanks to the high sensitivity of the method, urine samples could be diluted 2-fold prior to injection. One hundred and three forbidden substances from various classes (such as stimulants, diuretics, narcotics, anti-estrogens) were analysed on a C(18) reversed-phase column in two gradients of 9min (including two 3min equilibration periods) for positive and negative electrospray ionisation and detected in the MS full scan mode. The automatic identification of analytes was based on retention time and mass accuracy, with an automated tool for peak picking. The method was validated according to the International Standard for Laboratories described in the World Anti-Doping Code and was selective enough to comply with the World Anti-Doping Agency recommendations. In addition, the matrix effect on MS response was measured on all investigated analytes spiked in urine samples. The limits of detection ranged from 1 to 500ng/mL, allowing the identification of all tested compounds in urine. When a sample was reported positive during the screening, a fast additional pre-confirmatory step was performed to reduce the number of confirmatory analyses.

Identity and combinations of arbuscular mycorrhizal fungal isolates influence plant resistance and insect preference

1. Accumulating evidence indicates that plant resistance against above-ground herbivores can be affected by the presence of arbuscular mycorrhizal fungi (AMF) in association with the host plant. Little is known, however, about how AMF composition can influence herbivore choice to feed on a particular plant. 2. Unravelling the preference-performance hypothesis in a multitrophic context is needed to expand our knowledge of complex multitrophic interactions in natural systems. If given mycorrhizal fungal genotypes increase attractiveness for a herbivore (reduced plant resistance), then the benefits of increased unpalatability provided by the mycorrhizal fungi (increased plant resistance) might be outweighed by the increased herbivore recruitment. 3. This was addressed by designing three experiments to test the effects of different AMF genotypes, inoculated either alone or in combination, to measure intraspecific AMF effects on plant resistance and insect herbivore preference. Using strawberry (Fragaria vesca L.) plants that were colonised by eight different combinations of Rhizophagus irregularis isolates, we measured effects on plant growth, insect growth and survival, as well as feeding preferences of a generalist herbivore caterpillar (Spodoptera littoralis Boisduval). 4. Overall, it was found that: (i) AMF influenced plant resistance in an AMF genotype-specific manner; (ii) some AMF inoculations decreased insect performance; (iii) insects preferentially chose to feed more on leaves originating from non-mycorrhizal plants; but also that (iv) in a whole plant bioassay, insects preferentially chose the biggest plant, regardless of their mycorrhizal status. 5. Therefore, AMF-mediated trade-offs between growth and resistance against herbivores have been shown. Such trade-offs, particularly driven by plant attractiveness to herbivores, buffer the positive effects of the mycorrhizal symbiosis on enhanced plant growth.

Survival in soil and detection of co-transformed Trichoderma harzianum by nested PCR

The objective of this work was to evaluate the survival of two Trichoderma harzianum co-transformants, TE 10 and TE 41, carrying genes for green fluorescent protein (egfp) and for resistance to benomyl, during four weeks in a contained soil microcosm. Selective culture media were used to detect viable fungal material, whose identity was confirmed by the observation of the fluorescent phenotype by direct epifluorence microscopy. PCR using two nested primer pairs specific to the egfp gene was also used to detect the transformed fungi. Although it was not possible to reliably detect the egfp gene directly from soil extracts, an enrichment step involving selective culture of soil samples in liquid medium prior to DNA extraction enabled the consistent detection of the T. harzianum co-transformants by nested PCR for the duration of the incubation period.

Sublimation of new matrix candidates for high spatial resolution imaging mass spectrometry of lipids: enhanced information in both positive and negative polarities after 1,5-diaminonapthalene deposition.

Matrix sublimation has demonstrated to be a powerful approach for high-resolution matrix-assisted laser desorption ionization (MALDI) imaging of lipids, providing very homogeneous solvent-free deposition. This work presents a comprehensive study aiming to evaluate current and novel matrix candidates for high spatial resolution MALDI imaging mass spectrometry of lipids from tissue section after deposition by sublimation. For this purpose, 12 matrices including 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,6-dihydroxyacetphenone (DHA), 2',4',6'-trihydroxyacetophenone (THAP), 3-hydroxypicolinic acid (3-HPA), 1,8-bis(dimethylamino)naphthalene (DMAN), 1,8,9-anthracentriol (DIT), 1,5-diaminonapthalene (DAN), p-nitroaniline (NIT), 9-aminoacridine (9-AA), and 2-mercaptobenzothiazole (MBT) were investigated for lipid detection efficiency in both positive and negative ionization modes, matrix interferences, and stability under vacuum. For the most relevant matrices, ion maps of the different lipid species were obtained from tissue sections at high spatial resolution and the detected peaks were characterized by matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. First proposed for imaging mass spectrometry (IMS) after sublimation, DAN has demonstrated to be of high efficiency providing rich lipid signatures in both positive and negative polarities with high vacuum stability and sub-20 μm resolution capacity. Ion images from adult mouse brain were generated with a 10 μm scanning resolution. Furthermore, ion images from adult mouse brain and whole-body fish tissue sections were also acquired in both polarity modes from the same tissue section at 100 μm spatial resolution. Sublimation of DAN represents an interesting approach to improve information with respect to currently employed matrices providing a deeper analysis of the lipidome by IMS.

Roles of astrocytic sodium dynamics in the neurometabolic coupling : a study by cellular imaging

RESUME LARGE PUBLIC Le système nerveux central est principalement composé de deux types de cellules :les neurones et les cellules gliales. Ces dernières, bien que l'emportant en nombre sur les neurones, ont longtemps été considérées comme des cellules sans intérêts par les neuroscientifiques. Hors, les connaissances modernes à leurs sujets indiquent qu'elles participent à la plupart des tâches physiologiques du cerveau. Plus particulièrement, elles prennent part aux processus énergétiques cérébraux. Ceux-ci, en plus d'être vitaux, sont particulièrement intrigants puisque le cerveau représente seulement 2 % de la masse corporelle mais consomme environ 25 % du glucose (substrat énergétique) corporel. Les astrocytes, un type de cellules gliales, jouent un rôle primordial dans cette formidable utilisation de glucose par le cerveau. En effet, l'activité neuronale (transmission de l'influx nerveux) est accompagnée d'une augmentation de la capture de glucose, issu de la circulation sanguine, par les astrocytes. Ce phénomène est appelé le «couplage neurométabolique » entre neurones et astrocytes. L'ion sodium fait partie des mécanismes cellulaires entrant en fonction lors de ces processus. Ainsi, dans le cadre de cette thèse, les aspects dynamiques de la régulation du sodium astrocytaire et leurs implications dans le couplage neurométabolique ont été étudiés par des techniques d'imagerie cellulaires. Ces études ont démontré que les mitochondries, machineries cellulaires convertissant l'énergie contenue dans le glucose, participent à la régulation du sodium astrocytaire. De plus, ce travail de thèse a permis de découvrir que les astrocytes sont capables de se transmettre, sous forme de vagues de sodium se propageant de cellules en cellules, un message donnant l'ordre d'accroître leur consommation d'énergie. Cette voie de signalisation leur permettrait de fournir de l'énergie aux neurones suite à leur activation. RESUME Le glutamate libéré dans la fente synaptique pendant l'activité neuronale, est éliminé par les astrocytes environnants. Le glutamate est co-transporté avec des ions sodiques, induisant une augmentation intracellulaire de sodium (Na+i) dans les astrocytes. Cette élévation de Na+i déclenche une cascade de mécanismes moléculaires qui aboutissent à la production de substrats énergétiques pouvant être utilisés par les neurones. Durant cette thèse, la mesure simultanée du sodium mitochondrial (Na+mit) et cytosolique par des techniques d'imagerie utilisant des sondes fluorescentes spécifiques, a indiqué que les variations de Na+i induites par le transport du glutamate sont transmises aux mitochondries. De plus, les voies d'entrée et de sortie du sodium mitochondrial ont été identifiées. L'échangeur de Na+ et de Ca2+ mitochondrial semble jouer un rôle primordial dans l'influx de Na+mit, alors que l'efflux de Na+mit est pris en charge par l'échangeur de Na+ et de H+ mitochondrial. L'étude du Na+mit a nécessité l'utilisation d'un système de photoactivation. Les sources de lumière ultraviolette (UV) classiques utilisées à cet effet (lasers, lampes à flash) ayant plusieurs désavantages, une alternative efficace et peu coûteuse a été développée. Il s'agit d'un système compact utilisant une diode électroluminescente (LED) à haute puissance et de longueur d'onde de 365nm. En plus de leurs rôles dans le couplage neurométabolique, les astrocytes participent à la signalisation multicellulaire en transmettant des vagues intercellulaires de calcium. Ce travail de thèse démontre également que des vagues intercellulaires de sodium peuvent être évoquées en parallèle à ces vagues calciques. Le glutamate, suite à sa libération par un mécanisme dépendent du calcium, est réabsorbé par les transporteurs au glutamate. Ce mécanisme a pour conséquence la génération de vagues sodiques se propageant de cellules en cellules. De plus, ces vagues sodiques sont corrélées spatialement avec une consommation accrue de glucose par les astrocytes. En conclusion, ce travail de thèse a permis de montrer que le signal sodique astrocytaire, déclenché en réponse au glutamate, se propage à la fois de façon intracellulaire aux mitochondries et de façon intercellulaire. Ces résultats suggèrent que les astrocytes fonctionnent comme un réseau de cellules nécessaire au couplage énergétique concerté entre neurones et astrocytes et que le sodium est un élément clé dans les mécanismes de signalisations cellulaires sous-jacents. SUMMARY Glutamate, released in the synaptic cleft during neuronal activity, is removed by surrounding astrocytes. Glutamate is taken-up with Na+ ions by specific transporters, inducing an intracellular Na+ (Na+i) elevation in astrocytes which triggers a cascade of molecular mechanisms that provides metabolic substrates to neurons. Thus, astrocytic Na+i homeostasis represents a key component of the so-called neurometabolic coupling. In this context, the first part of this thesis work was aimed at investigating whether cytosolic Na+ changes are transmitted to mitochondria, which could therefore influence their function and contribute to the overall intracellular Na+ regulation. Simultaneous monitoring of both mitochondrial Na+ (Na+mit) and cytosolic Na+ changes with fluorescent dyes revealed that glutamate-evoked cytosolic Na+ elevations are indeed transmitted to mitochondria. The mitochondrial Na+/Ca2+ exchangers have a prominent role in the regulation of Na+mit influx pathway, and Na+mit extrusion appears to be mediated by Na+/H+ exchangers. To demonstrate the implication of Na+/Ca2+ exchangers, this study has required the technical development of an UV-flash photolysis system. Because light sources for flash photolysis have to be powerful and in the near UV range, the use of UV lasers or flash lamps is usually required. As an alternative to these UV sources that have several drawbaks, we developped a compact, efficient and lowcost flash photolysis system which employs a high power 365nm light emitting diode. In addition to their role in neurometabolic coupling, astrocytes participate in multicellular signaling by transmitting intercellular Ca2+ waves. The third part of this thesis show that intercellular Na+ waves can be evoked in parallel to Ca2+ waves. Glutamate released by a Ca2+ wave-dependent mechanism is taken up by glutamate transporters, resulting in a regenerative propagation of cytosolic Na+ increases. Na+ waves in turn lead to a spatially correlated increase in glucose uptake. In conclusion, the present thesis demonstrates that glutamate-induced Na+ changes occurring in the cytosol of astrocytes propagate to both the mitochondrial matrix and the astrocytic network. These results furthermore support the view that astrocytic Na+ is a signal coupled to the brain energy metabolism.

Carnatic music: Svara, Gamaka, Motif and Raga identity

Over the last century in Karnatik music, the method ofunderstanding raga has been to break it down into its variouscomponents, svara, scale, gamaka, and phrases. In this paper, an attempt is made to define the abstract concept of raga in its entirety within the aesthetics of Karnatik music considering the various components and their symbiotic relationship. This paper also attempts to prove that theidentity of a raga exists as a whole. Section 2 explains theconcept of a fundamental musical note or svara. Section 3 illustrates the concept of gamaka or inflections. Section 4 delves into the concept of raga in detail and then flows into Section 5 which enunciates the identity of a raga in terms of svara, gamaka, and phraseology. The paper concludes in Section 6, and Section 7 gives the references.

Genetic distance estimated by RAPD markers and its relationship with hybrid performance in maize

The objective of this work was to evaluate the genetic diversity of 16 maize inbred lines, and to determine the correlation between genetic distance and hybrid performance, using random amplified polymorphic DNA (RAPD) molecular markers. Twenty-two different random primers were used, which resulted in the amplification of 265 fragments, 237 (84.44%) of them being polymorphic. A genetic similarity matrix was created from the RAPD data, using Jaccard coefficient, and a dendrogram was constructed. Hybrid analyses were carried out using random block design and Griffing method VI for diallel crossings. The genetic associations showed five distinct heterotic groups. Correlations between genetic divergences detected by RAPD, as well as the means observed in the diallel crossings were positive and significant for plant height, ear height, prolificacy, and grain weight. The correlation of genetic divergences, detected by RAPD, and the specific combining ability between heterotic group associations, showed significance in all characteristics under study, except prolificacy. A direct relationship between genetic divergence and productivity was found in 79.2% of the 120 hybrids confirming the hypothesis that genetic divergence is directly related to the performance of hybrids and is efficient in predicting it.

Lentiviral vector-mediated gene transfer in adult mouse photoreceptors is impaired by the presence of a physical barrier

RESUME L'utilisation de la thérapie génique dans l'approche d'un traitement des maladies oculaires dégénératives, plus particulièrement de la rétinite pigmentaire, semble être très prometteuse (Acland et al. 2001). Parmi les vecteurs développés, les vecteurs lentiviraux (dérivé du virus humain HIV-1), permettent la transduction des photorécepteurs après injection sous-rétinienne chez la souris durant les premiers jours de vie. Cependant l'efficacité du transfert de gène est nettement plus limitée dans ce type cellulaire après injection chez l'adulte (Kostic et al. 2003). L'objet de notre étude est de déterminer si la présence d'une barrière physique produite au cours du développement, située entre les photorécepteurs et l'épithélium pigmentaire ainsi qu'entre les photorécepteurs eux-mêmes, est responsable de: la diminution de l'entrée en masse du virus dans les photorécepteurs, minimisant ainsi son efficacité chez la souris adulte. De précédentes recherches, chez le lapin, ont décrit la capacité d'enzymes spécifiques comme la Chondroïtinase ABC et la Neuraminidase X de modifier la structure de la matrice entourant les photorécepteurs (Inter Photoreceptor Matrix, IPM) par digestion de certains de ses constituants suite à leur injection dans l'espace sous-rétinien (Yao et al. 1990). Considérant l'IPM comme une barrière physique, capable de réduire l'efficacité de transduction des photorécepteurs chez la souris adulte, nous avons associé différentes enzymes simultanément à l'injection sous-rétinienne de vecteurs lentiviraux afin d'améliorer la transduction virale en fragilisant I'IPM, la rendant ainsi plus perméable à la diffusion du virus. L'injection sous-rétinienne de Neuraminidase X et de Chondroïtinase ABC chez la souris induit des modifications structurales de l'IPM qui se manifestent respectivement par la révélation ou la disparition de sites de liaison de la peanut agglutinin sur les photorécepteurs. L'injection simultanée de Neuraminidase X avec le vecteur viral contenant le transgène thérapeutique augmente significativement le nombre de photorécepteurs transduits (environ cinq fois). Nous avons en fait démontré que le traitement enzymatique augmente principalement la diffusion du lentivirus dans l'espace situé entre l'épithélium pigmentaire et les photorécepteurs. Le traitement à la Chondroïtinase ABC n'entraîne quant à elle qu'une légère amélioration non significative de la transduction. Cette étude montre qu'une meilleure connaissance de l'IPM ainsi que des substances capables de la modifier (enzymes, drogues etc.) pourrait aider à élaborer de nouvelles stratégies afin d'améliorer la distribution de vecteurs viraux dans la rétine adulte.

Exhaled breath condensate as a matrix for combustion-based nanoparticle exposure and health effect evaluation

Health assessment and medical surveillance of workers exposed to combustion nanoparticles are challenging. The aim was to evaluate the feasibility of using exhaled breath condensate (EBC) from healthy volunteers for (1) assessing the lung deposited dose of combustion nanoparticles and (2) determining the resulting oxidative stress by measuring hydrogen peroxide (H2O2) and malondialdehyde (MDA). Methods: Fifteen healthy nonsmoker volunteers were exposed to three different levels of sidestream cigarette smoke under controlled conditions. EBC was repeatedly collected before, during, and 1 and 2 hr after exposure. Exposure variables were measured by direct reading instruments and by active sampling. The different EBC samples were analyzed for particle number concentration (light-scattering-based method) and for selected compounds considered oxidative stress markers. Results: Subjects were exposed to an average airborne concentration up to 4.3×10(5) particles/cm(3) (average geometric size ∼60-80 nm). Up to 10×10(8) particles/mL could be measured in the collected EBC with a broad size distribution (50(th) percentile ∼160 nm), but these biological concentrations were not related to the exposure level of cigarette smoke particles. Although H2O2 and MDA concentrations in EBC increased during exposure, only H2O2 showed a transient normalization 1 hr after exposure and increased afterward. In contrast, MDA levels stayed elevated during the 2 hr post exposure. Conclusions: The use of diffusion light scattering for particle counting proved to be sufficiently sensitive to detect objects in EBC, but lacked the specificity for carbonaceous tobacco smoke particles. Our results suggest two phases of oxidation markers in EBC: first, the initial deposition of particles and gases in the lung lining liquid, and later the start of oxidative stress with associated cell membrane damage. Future studies should extend the follow-up time and should remove gases or particles from the air to allow differentiation between the different sources of H2O2 and MDA.

Regulation of the transforming growth factor beta-responsive transcription factor CTF-1 by calcineurin and calcium/calmodulin-dependent protein kinase IV.

Transforming growth factor beta (TGF-beta) is a pluripotent peptide hormone that regulates various cellular activities, including growth, differentiation, and extracellular matrix protein gene expression. We previously showed that TGF-beta induces the transcriptional activation domain (TAD) of CTF-1, the prototypic member of the CTF/NF-I family of transcription factors. This induction correlates with the proposed role of CTF/NF-I binding sites in collagen gene induction by TGF-beta. However, the mechanisms of TGF-beta signal transduction remain poorly understood. Here, we analyzed the role of free calcium signaling in the induction of CTF-1 transcriptional activity by TGF-beta. We found that TGF-beta stimulates calcium influx and mediates an increase of the cytoplasmic calcium concentration in NIH3T3 cells. TGF-beta induction of CTF-1 is inhibited in cells pretreated with thapsigargin, which depletes the endoplasmic reticulum calcium stores, thus further arguing for the potential relevance of calcium mobilization in TGF-beta action. Consistent with this possibility, expression of a constitutively active form of the calcium/calmodulin-dependent phosphatase calcineurin or of the calcium/calmodulin-dependent kinase IV (DeltaCaMKIV) specifically induces the CTF-1 TAD and the endogenous mouse CTF/NF-I proteins. Both calcineurin- and DeltaCaMKIV-mediated induction require the previously identified TGF-beta-responsive domain of CTF-1. The immunosuppressants cyclosporin A and FK506 abolish calcineurin-mediated induction of CTF-1 activity. However, TGF-beta still induces the CTF-1 TAD in cells treated with these compounds or in cells overexpressing both calcineurin and DeltaCaMKIV, suggesting that other calcium-sensitive enzymes might mediate TGF-beta action. These results identify CTF/NF-I as a novel calcium signaling pathway-responsive transcription factor and further suggest multiple molecular mechanisms for the induction of CTF/NF-I transcriptional activity by growth factors.