Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant -Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an -Alkylguanine Adductome.

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, -methylguanine -methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA -alkylguanines (-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple -alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different -alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known -MethylG (-MeG) levels.

For Better and For Worse: Frequent Gamblers Use Dual Counterfactuals to Justify Continued Gambling.

How might frequent gamblers convince themselves to keep playing despite persistent losses or after a win that should be savored? The purpose of this research is to examine the unexplored question of how frequent gamblers' use counterfactual thinking to motivate their desire to continue gambling. Using a sample of n = 69 high and n = 69 low frequency gamblers in a field setting, we found that infrequent gamblers tended to consider how the perceived outcome of losing "could have been better" (i.e.

Functionally additive fixed positive and negative charges in the CFTR channel pore control anion binding and conductance.

Ion channels use charged amino-acid residues to attract oppositely charged permeant ions into the channel pore. In the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel, a number of arginine and lysine residues have been shown to be important for Cl permeation. Among these, two in close proximity in the pore-Lys and Arg-are indispensable for anion binding and high Cl conductance, suggesting that high positive charge density is required for pore function.

Two positively charged amino acid side-chains in the inner vestibule of the CFTR channel pore play analogous roles in controlling anion binding and anion conductance.

Positively charged amino acid side-chains play important roles in anion binding and permeation through the CFTR chloride channel. One pore-lining lysine residue in particular (K95) has been shown to be indispensable for anion binding, conductance, and selectivity. Here, we use functional investigation of CFTR to show that a nearby arginine (R134) plays a functionally analogous role.

Electrostatic Tuning of Anion Attraction from the Cytoplasm to the Pore of the CFTR Chloride Channel.

Anions enter from the cytoplasm into the channel pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel not via a central pathway but via a single lateral portal or fenestration. High Cl conductance is dependent on electrostatic attraction of cytoplasmic Cl ions by four positively charged amino acid side-chains located within this portal. Here we use a mutagenic approach to investigate the functional effects of transplanting or supplementing these positive charges at nearby portal-lining sites.

Contribution of the eighth transmembrane segment to the function of the CFTR chloride channel pore.

Our molecular understanding of the cystic fibrosis transmembrane conductance regulator (CFTR)-the chloride channel that is mutated in cystic fibrosis-has been greatly enhanced by a number of recent atomic-level structures of the protein in different conformations. One surprising aspect of these structures was the finding that the eighth of CFTR's 12 membrane-spanning segments (TM8) appeared close to the channel pore. Although functional evidence supports a role for other TMs in forming the pore, such a role for TM8 has not previously been reported.

Pomegranate Juice Supplementation Alters Utero-Placental Vascular Function and Fetal Growth in the eNOS Mouse Model of Fetal Growth Restriction.

The eNOS mouse provides a well-characterized model of fetal growth restriction (FGR) with altered uterine and umbilical artery function and reduced utero- and feto-placental blood flow. Pomegranate juice (PJ), which is rich in antioxidants and bioactive polyphenols, has been posited as a beneficial dietary supplement to promote cardiovascular health. We hypothesized that maternal supplementation with PJ will improve uterine and umbilical artery function and thereby enhance fetal growth in the eNOS mouse model of FGR.

Functional organization of cytoplasmic portals controlling access to the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel pore.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl channel that apparently has evolved from an ancestral active transporter. Key to the CFTR's switch from pump to channel function may have been the appearance of one or more "lateral portals." Such portals connect the cytoplasm to the transmembrane channel pore, allowing a continuous pathway for the electrodiffusional movement of Cl ions.

Conformational change of the extracellular parts of the CFTR protein during channel gating.

Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing.

Vitamin D attenuates sphingosine-1-phosphate (S1P)-mediated inhibition of extravillous trophoblast migration.

Introduction: Failure of trophoblast invasion and remodelling of maternal blood vessels leads to the pregnancy complication pre-eclampsia (PE). In other systems, the sphingolipid, sphingosine-1-phosphate (S1P), controls cell migration therefore this study determined its effect on extravillous trophoblast (EVT) function.

Methods: A transwell migration system was used to assess the behaviour of three trophoblast cell lines, Swan-71, SGHPL-4, and JEG3, and primary human trophoblasts in the presence or absence of S1P, S1P pathway inhibitors and 1,25(OH)D.

Exposure to omentum adipose tissue conditioned medium from obese pregnant women promotes myometrial artery dysfunction.

Aim: Underlying mechanisms of poor pregnancy outcome in obese (OB) mothers (body mass index [BMI] ≥ 30 kg/m ) are unknown. Our studies demonstrate that OB pregnant women have altered myometrial artery (MA) function related to the thromboxane and nitric oxide pathways. In obesity, increased central fat mass is associated with an altered endocrine milieu.

Contribution of a leucine residue in the first transmembrane segment to the selectivity filter region in the CFTR chloride channel.

The anion selectivity and conductance of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are determined predominantly by interactions between permeant anions and the narrow region of the channel pore. This narrow region has therefore been described as functioning as the "selectivity filter" of the channel. Multiple pore-lining transmembrane segments (TMs) have previously been shown to contribute to the selectivity filter region.

The atrial natriuretic peptide (ANP) knockout mouse does not exhibit the phenotypic features of pre-eclampsia or demonstrate fetal growth restriction.

The ANP knockout mouse is reported to exhibit pregnancy-associated hypertension, proteinuria and impaired placental trophoblast invasion and spiral artery remodeling, key features of pre-eclampsia (PE). We hypothesized that these mice may provide a relevant model of human PE with associated fetal growth restriction (FGR). Here, we investigated pregnancies of ANP wild type (ANP(+/+)), heterozygous (ANP(+/-)) and knockout (ANP(-/-)) mice.

Breaks in Play: Do They Achieve Intended Aims?

Breaks in play represent a responsible gambling strategy designed to disrupt states of dissociation and enhance the likelihood of drawing attention to a player's session behaviour and expenditure with respect to time and money. The aim of the break in play is to motivate the player to modify or cease gambling so the activity remains within affordable levels. The aim of this study was to investigate whether imposed breaks in play in the absence of accompanying warning messages were effective in reducing cravings.

Mast cells protect against Pseudomonas aeruginosa-induced lung injury.

Pseudomonas aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortality in immune-compromised individuals. Maintaining the integrity of the respiratory epithelium is critical for an effective host response to P. aeruginosa.

Maternal obesity impairs specific regulatory pathways in human myometrial arteries.

Obese women (body mass index ≥30 kg/m(2)) are at greater risk than normal weight women of pregnancy complications associated with maternal and infant morbidity, particularly the development of cardiovascular disease and metabolic disorders in later life; why this occurs is unknown. Nonpregnant, obese individuals exhibit systemic vascular endothelial dysfunction. We tested the hypothesis that obese pregnant women have altered myometrial arterial function compared to pregnant women of normal (18-24 kg/m(2)) and overweight (25-29 kg/m(2)) body mass index.

Sildenafil citrate increases fetal weight in a mouse model of fetal growth restriction with a normal vascular phenotype.

Fetal growth restriction (FGR) is defined as the inability of a fetus to achieve its genetic growth potential and is associated with a significantly increased risk of morbidity and mortality. Clinically, FGR is diagnosed as a fetus falling below the 5(th) centile of customised growth charts. Sildenafil citrate (SC, Viagra™), a potent and selective phosphodiesterase-5 inhibitor, corrects ex vivo placental vascular dysfunction in FGR, demonstrating potential as a therapy for this condition.

Organic cation transporters in human nasal primary culture: expression and functional activity.

Background: The majority of drugs cross epithelial cells by either passive diffusion or via carrier-mediated drug transporters. The aim of this study was to investigate the transport characteristics, protein expression and localization of organic cation transporters in human nasal epithelium.

Methods & Results: The expression, localization and transport characteristics of the transporters were investigated using permeation, PCR and immunohistochemistry.

Maternal obesity and its effect on placental cell turnover.

Background: Maternal obesity is a frequent obstetric risk factor, linked with short- and long-term consequences for mother and child, including foetal overgrowth, growth restriction and stillbirth. The mechanisms underlying these pathologies remain unknown but likely involve the placenta.

Aims: To study placental cell turnover in relation to maternal body mass index (BMI).

Optimization of human nasal epithelium primary culture conditions for optimal proton oligopeptide and organic cation transporters expression in vitro.

Aim: To investigate the effect of key tissue culture conditions on cell growth, gene expression and functional uptake of peptide and organic cation transporter substrates in the human nasal epithelium (HNE).

Methods: HNE were cultured on different growth surfaces (polystyrene plastic, collagen film, and hydrated collagen gel) and were maintained with three popular nasal tissue culture media supplements [DMEM/F12 supplemented with Ultroser(®) G (2%), FBS (10%) and NuSerum(®) (10%)], respectively. The expression of gene transcripts for organic cation and peptide transporters were screened using qPCR and substrate uptake studies.

Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle.

Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function.

Reduced expression of psoriasin in human airway cystic fibrosis epithelia.

Psoriasin is a low molecular weight Ca(2+)-binding protein with known antimicrobial activity. Since human airway epithelial cells produce a number of powerful antimicrobial agents as part of their host defence, we investigated whether psoriasin was expressed in human bronchial epithelial cell lines. Expression was investigated in 16HBE14o- cells, derived from a normal individual, and compared to CFBE41o- cells, derived from a cystic fibrosis patient.

Pseudohalide anions reveal a novel extracellular site for potentiators to increase CFTR function.

Background And Purpose: There is great interest in the development of potentiator drugs to increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis. We tested the ability of several anions to potentiate CFTR activity by a novel mechanism.

Experimental Approach: Patch clamp recordings were used to investigate the ability of extracellular pseudohalide anions (Co(CN)(6) (3-) , Co(NO(2) )(6) (3-) , Fe(CN)(6) (3-) , IrCl(6) (3-) , Fe(CN)(6) (4-) ) to increase the macroscopic conductance of mutant CFTR in intact cells via interactions with cytoplasmic blocking anions.

The prostaglandin E₂ type 4 receptor participates in the response to acute oxidant stress in airway epithelial cells.

Oxidative stress is implicated in the pathogenesis of many inflammatory pulmonary diseases, including cystic fibrosis (CF). Delineating how oxidative stress stimulates CF transmembrane conductance regulator (CFTR) in airway epithelial cells is useful, both to increase the understanding of airways host defense and suggest therapeutic approaches to reduce the oxidant stress burden in the CF lung. Using the airway epithelial cell line Calu-3, we investigated the hypothesis that hydrogen peroxide (H₂O₂), which stimulates anion efflux through CFTR, does so via the production of prostaglandin E₂ (PGE₂).

The moderating role of self-monitoring on the interpersonal aspects of attitude ambivalence.

Extant research has found a relation between holding conflicting attitudes with a familiar person (interpersonal discrepancy) and subjective attitude ambivalence. In 2 studies, we investigated the role of interpersonal discrepancy in the experience of attitude ambivalence as a function of self-monitoring and level of liking of the other person. Building on balance theory, we proposed and found that high (vs.