Repression of NHE1 Expression by PPAR gamma Activation Is a Potential New Approach for Specific Inhibition of the Growth of Tumor Cells In vitro and In vivo

Ligand-induced activation of peroxisome proliferator-activated receptor gamma (PPAIR gamma) inhibits proliferation in cancer cells in vitro and in vivo; however, the downstream targets remain undefined. We report the identification of a peroxisome proliferator response element in the promoter region of the Na+/ H transporter gene NHE1, the overexpression of which has been associated with carcinogenesis. Exposure of breast cancer cells expressing high levels of PPAR gamma to its natural and synthetic agonists resulted in downregulation of NHE1 transcription as well as protein expression. Furthermore, the inhibitory effect of activated PPAR gamma on tumor colony-forming ability was abrogated on overexpression of NHE1, whereas small interfering RNA-mediated gene silencing of NHE1 significantly increased the sensitivity of cancer cells to growth-inhibitory stimuli. Finally, histopathologic analysis of breast cancer biopsies obtained from patients with type II diabetes treated with the synthetic agonist rosiglitazone showed significant repression of NHE1 in the tumor tissue. These data provide evidence for tumor-selective downregulation of NHE1 by activated PPAR gamma in vitro and in pathologic specimens from breast cancer patients and could have potential implications for the judicious use of low doses of PPAR gamma ligands in combination chemotherapy regimens for an effective therapeutic response. [Cancer Res 2009;69(22):8636-44]

Effect of hydrophobic nanopatches within an ionic surface on the structure of liquids

The structures of liquid water and isopropanol have been studied as a function of the size of a hydrophobic patch present in a model hydrophilic surface via molecular dynamics simulations. A significant anisotropy extending into the first few solvent layers is found over the patch which suggests implications for many real-world systems in which nanoscale heterogeneity is found.

The Gastrointestinal Peptide Obestatin Induces Vascular Relaxation Via Specific Activation of Endothelium-Dependent Nitric Oxide Signalling.

Background and purpose: Obestatin is a recently-discovered gastrointestinal peptide with established metabolic actions, which is linked to diabetes and may exert cardiovascular benefits. Here we aimed to investigate the specific effects of obestatin on vascular relaxation. Experimental approach: Cumulative relaxation responses to obestatin peptides were assessed in isolated rat aorta and mesenteric artery (n=8) in the presence/absence of selective inhibitors. Complementary studies were performed in cultured bovine aortic endothelial cells (BAEC). Key results: Obestatin peptides elicited concentration-dependent relaxation in both aorta and mesenteric artery. Responses to full-length obestatin(1-23) were greater than those to obestatin(1-10) and obestatin(11-23). Obestatin(1-23)-induced relaxation was attenuated by endothelial denudation, L-NAME (NO synthase inhibitor), high extracellular K(+) , GDP-ß-S (G protein inhibitor), MDL-12,330A (adenylate cyclase inhibitor), wortmannin (PI3K inhibitor), KN-93 (CaMKII inhibitor), ODQ (guanylate cyclase inhibitor) and iberiotoxin (BK(Ca) blocker), suggesting that it is mediated by an endothelium-dependent NO signalling cascade involving an adenylate cyclase-linked G protein-coupled receptor, PI3K/Akt, Ca(2+) -dependent eNOS activation, soluble guanylate cyclase and modulation of vascular smooth muscle K(+) . Supporting data from BAEC indicated that nitrite production, intracellular Ca(2+) and Akt phosphorylation were increased after exposure to obestatin(1-23). Relaxations to obestatin(1-23) were unaltered by inhibitors of candidate endothelium-derived hyperpolarising factors (EDHFs) and combined SK(Ca) /IK(Ca) blockade, suggesting that EDHF-mediated pathways were not involved. Conclusions and Implications: Obestatin produces significant vascular relaxation via specific activation of endothelium-dependent NO signalling. These actions may be important in normal regulation of vascular function and are clearly relevant to diabetes, a condition characterised by endothelial dysfunction and cardiovascular complications.

IMMUNOREACTIVITY TO 2 SPECIFIC REGIONS OF CHROMOGRANIN-A IN THE NERVOUS-SYSTEM OF ASCARIS-SUUM - AN IMMUNOCYTOCHEMICAL STUDY

Antisera to a highly conserved region of chromogranin A (sequence KELTAE) and to a hexapeptide (sequence KGQELE) adjacent to the putative C-terminus of pancreastatin, a peptide whose sequence is found within the chromogranin A molecule, have been used to examine the localisation of immunoreactivity (IR) to these peptides in Ascaris suum. IR to both peptides was found in the nerve rings and nerve cords. In addition, KGQELE-IR was also observed in the pharyngeal neurones and in a network of fibres on the surface of the female gonoduct. The staining was specific in that it could be abolished by preincubation of the antisera with the appropriate antigen. The two antisera appeared to be staining different subsets of neurones, suggesting that (at least) two peptides were being recognised. The widespread distribution of IR to both peptides throughout the nervous system of the parasite suggests that the peptides carrying the epitopes recognised by the antisera are of fundamental importance to the functioning of the parasite's nervous system.

Conformation-specific crosslinking of mitochondrial complex I

Complex I is the only component of the eukaryotic respiratory chain of which no high-resolution structure is yet available. A notable feature of mitochondrial complex I is the so-called active/de-active conformational transition of the idle enzyme from the active (A) to the de-active, (D) form. Using an amine- and sulfhydryl-reactive crosslinker of 6.8 Å length (SPDP) we found that in the D-form of complex I the ND3 subunit crosslinked to the 39 kDa (NDUFA9) subunit. These proteins could not be crosslinked in the A-form. Most likely, both subunits are closely located in the critical junction region connecting the peripheral hydrophilic domain to the membrane arm of the enzyme where the entrance path for substrate ubiquinone is and where energy transduction takes place.

Organization specific predictors of job satisfaction: findings from a Canadian multi-site quality of work life cross-sectional survey

Background: Organizational features can affect how staff view their quality of work life. Determining staff perceptions about quality of work life is an important consideration for employers interested in improving employee job satisfaction. The purpose of this study was to identify organization specific predictors of job satisfaction within a health care system that consisted of six independent health care organizations.

Methods: 5,486 full, part and causal time (non-physician) staff on active payroll within six organizations (2 community hospitals, 1 community hospital/long-term care facility, 1 long-term care facility, 1 tertiary care/community health centre, and 1 visiting nursing agency) located in five communities in Central West Ontario, Canada were asked to complete a 65-item quality of work life survey. The self-administered questionnaires collected staff perceptions of: co-worker and supervisor support; teamwork and communication; job demands and decision authority; organization characteristics; patient/resident care; compensation and benefits; staff training and development; and impressions of the organization. Socio-demographic data were also collected.

Results: Depending on the organization, between 15 and 30 (of the 40 potential predictor) variables were found to be statistically associated with job satisfaction (univariate analyses). Logistic regression analyses identified the best predictors of job satisfaction and these are presented for each of the six organizations and for all organizations combined.

Conclusions: The findings indicate that job satisfaction is a multidimensional construct and although there appear to be some commonalities across organizations, some predictors of job satisfaction appear to be organization and context specific.

In utero exposure to cigarette chemicals induces sex-specific disruption of one-carbon metabolism and DNA methylation in the human fetal liver

Background: Maternal smoking is one of the most important modifiable risk factors for low birthweight, which is strongly associated with increased cardiometabolic disease risk in adulthood. Maternal smoking reduces the levels of the methyl donor vitamin B12 and is associated with altered DNA methylation at birth. Altered DNA methylation may be an important mechanism underlying increased disease susceptibility; however, the extent to which this can be induced in the developing fetus is unknown.

Methods: In this retrospective study, we measured concentrations of cobalt, vitamin B12, and mRNA transcripts encoding key enzymes in the 1-carbon cycle in 55 fetal human livers obtained from 11 to 21 weeks of gestation elective terminations and matched for gestation and maternal smoking. DNA methylation was measured at critical regions known to be susceptible to the in utero environment. Homocysteine concentrations were analyzed in plasma from 60 fetuses.

Results: In addition to identifying baseline sex differences, we found that maternal smoking was associated with sex-specific alterations of fetal liver vitamin B12, plasma homocysteine and expression of enzymes in the 1-carbon cycle in fetal liver. In the majority of the measured parameters which showed a sex difference, maternal smoking reduced the magnitude of that difference. Maternal smoking also altered DNA methylation at the imprinted gene IGF2 and the glucocorticoid receptor (GR/NR3C1).

Conclusions: Our unique data strengthen studies linking in utero exposures to altered DNA methylation by showing, for the first time, that such changes are present in fetal life and in a key metabolic target tissue, human fetal liver. Furthermore, these data propose a novel mechanism by which such changes are induced, namely through alterations in methyl donor availability and changes in 1-carbon metabolism.

Utilisation of a novel ProteaseTag™ activity immunoassay for the specific measurement of neutrophil elastase in patients with Cystic Fibrosis

Neutrophil elastase (NE), a biomarker of infection and inflammation, correlates with the severity of several respiratory diseases including cystic fibrosis (CF) however, its detection and quantification in biological samples is confounded by a lack of robust methodologies. Standard assays using chromogenic or fluorogenic substrates are not specific when added to complex samples containing multiple proteolytic and hydrolytic enzymes, resulting in an over-estimation of the target protease. ELISA systems measure total protein levels which can be a mixture of latent, active and protease-inhibitor complexes. We have therefore developed a novel immunoassay (NE-Tag ELISA), incorporating an activity dependent ProteaseTag™ and a specific antibody step, which is selective and specific for the capture of active NE. The objective of this study was to clinically validate NE-Tag ELISA for the detection of active NE in sputum from CF patients. Sputum (n=45) was recovered from CF patients hospitalised for acute exacerbation. Sol was recovered and analysed for NE activity using the NE-Tag ELISA and two fluorogenic substrate-based assays [1. Suc-AAPV-AMC (Sigma) and 2. InnozymeTM Immunocapture assay (Calbiochem)]. NE activity between assays and with a range of clinical parameters was correlated.A highly significant correlation was shown between assays. NE activity (NE-Tag) further correlated appropriately with clinical parameters: inversely with FEV1 (p = 0.036) and positively with CRP (p = 0.035), neutrophils and total white cell counts (p < 0.001). The InnozymeTM assay showed similar correlations with the clinical parameters (with the exception of CRP). No correlations with any of the clinical parameters were observed when NE was measured using the standard fluorogenic substrate.

Genome-wide gene pathway analysis of psychotic illness symptom dimensions based on a new schizophrenia-specific model of the OPCRIT

Empirically derived phenotypic measurements have the potential to enhance gene-finding efforts in schizophrenia. Previous research based on factor analyses of symptoms has typically included schizoaffective cases. Deriving factor loadings from analysis of only narrowly defined schizophrenia cases could yield more sensitive factor scores for gene pathway and gene ontology analyses. Using an Irish family sample, this study 1) factor analyzed clinician-rated Operational Criteria Checklist items in cases with schizophrenia only, 2) scored the full sample based on these factor loadings, and 3) implemented genome-wide association, gene-based, and gene-pathway analysis of these SCZ-based symptom factors (final N= 507). Three factors emerged from the analysis of the schizophrenia cases: a manic, a depressive, and a positive symptom factor. In gene-based analyses of these factors, multiple genes had q<. 0.01. Of particular interest are findings for PTPRG and WBP1L, both of which were previously implicated by the Psychiatric Genomics Consortium study of SCZ; results from this study suggest that variants in these genes might also act as modifiers of SCZ symptoms. Gene pathway analyses of the first factor indicated over-representation of glutamatergic transmission, GABA-A receptor, and cyclic GMP pathways. Results suggest that these pathways may have differential influence on affective symptom presentation in schizophrenia.

Specific role of neuronal nitric-oxide synthase when tethered to the plasma membrane calcium pump in regulating the beta-adrenergic signal in the myocardium

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates beta-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser(16) phospholamban (PLB) phosphorylation as well as Ser(22) and Ser(23) cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the beta-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.

Utilisation of a novel ProteaseTag™ activity immunoassay for the specific measurement of neutrophil elastase in BAL from children with cystic fibrosis

Introduction: Neutrophil elastase (NE) is a serine protease implicated in the pathogenesis of several respiratory diseases including cystic fibrosis (CF). The presence of free NE in BAL is a predictor of subsequent bronchiectasis in children with CF (Sly et al, 2013, NEJM 368: 1963-1970). Furthermore, children with higher levels of sputum NE activity (NEa) tend to experience a more rapid decline in FEV1 over time even after adjusting for age, gender and baseline FEV1 (Sagel et al, 2012, AJRCCM 186: 857-865). Its detection and quantification in biological samples is however confounded by a lack of robust methodologies. Standard assays using chromogenic or fluorogenic substrates are not specific when added to complex samples containing multiple proteolytic and hydrolytic enzymes. ELISA systems measure total protein levels which can be a mixture of latent, active and protease-inhibitor complexes. We have therefore developed a novel assay (ProteaseTag™ Active NE Immunoassay), which couples an activity dependent NE-Tag with a specific antibody step, resulting in an assay which is both selective and specific for NEa. Aims: To clinically validate ProteaseTag™ Active NE for the detection of free NEa in BAL from children with CF. Methods: A total of 95 paediatric BAL samples [CF (n=76; 44M, 32F) non-CF (n=19; 12M, 7F)] collected through the Study of Host Immunity and Early Lung Disease in CF (SHIELD CF) were analysed for NEa using ProteaseTag™ Active NE (ProAxsis Ltd) and a fluorogenic substrate-based assay utilising Suc-AAPV-AMC (Sigma). IL-8 was measured by ELISA (R&D Systems). Results were analysed to show comparisons in free NEa between CF and non-CF samples alongside correlations with a range of clinical parameters. Results: NEa measured by ProteaseTag™ Active NE correlated significantly with age (r=0.3, p=0.01) and highly significantly with both IL-8 (r=0.4, p=<0.0001) and the absolute neutrophil count (ANC) (r=0.4, p=<0.0001). These correlations were not observed when NEa was measured by the substrate assay even though a significant correlation was found between the two assays (r=0.8, p<0.0001). A trend towards significance was found between NEa in the CF and non-CF groups when measured by ProteaseTag™ Active NE (p=0.07). Highly significant differences were found with the other inflammatory parameters between the 2 groups (IL-8: p=0.0002 and ANC: p=0.006). Conclusion: NEa as a primary efficacy endpoint in clinical trials or as a marker of inflammation within the clinic has been hampered by the lack of a robust and simple to use assay. ProteaseTag™ Active NE has been shown to be a specific and superior tool in the measurement of NEa in paediatric CF BAL samples (supporting data from previous studies using adult CF expectorated samples). The technology is currently being transferred to a lateral flow device for use at Point of Care. Acknowledgements: This work was supported by the National Children’s Research Centre, Dublin (SHIELD CF) and grants from the Medical Research Council and Cystic Fibrosis Foundation Therapeutics.

Molecularly imprinted polymer based on MWCNTs-QDs as fluorescent biomimetic sensor for specific recognition of target protein

A novel molecularly imprinted optosensing material based on multi-walled carbon nanotube-quantum dots (MWCNT-QDs) has been designed and synthesized for its high selectivity, sensitivity and specificity in the recognition of a target protein bovine serum albumin (BSA). Molecularly imprinted polymer coated MWCNT-QDs using BSA as the template (BMIP-coated MWCNT-QDs) exhibits a fast mass-transfer speed with a response time of 25 min. It is found that the BSA as a target protein can significantly quench the luminescence of BMIP-coated MWCNT-QDs in a concentration-dependent manner that is best described by a Stem-Volmer equation. The K-SV for BSA is much higher than bovine hemoglobin and lysozyme, implying a highly selective recognition of the BMIP-coated MWCNT-QDs to BSA. Under optimal conditions, the relative fluorescence intensity of BMIP-coated MWCNT-QDs decreases linearly with the increasing target protein BSA in the concentration range of 5.0 x 10(-7)-35.0 x 10(-7) M with a detection limit of 80 nM.

Sex-specific disruption of murine midbrain astrocytic and dopaminergic developmental trajectories following antenatal GC treatment

The mammalian midbrain dopaminergic systems arising in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) are critical for coping behaviours and are implicated in neuropsychiatric disorders where early life challenges comprise significant risk factors. Here, we aimed to advance our hypothesis that glucocorticoids (GCs), recognised key players in neurobiological programming, target development within these systems, with a novel focus on the astrocytic population. Mice received antenatal GC treatment (AGT) by including the synthetic GC, dexamethasone, in the mothers' drinking water on gestational days 16-19; controls received normal drinking water. Analyses of regional shapes and volumes of the adult SNc and VTA demonstrated that AGT induced long-term, dose-dependent, structural changes that were accompanied by profound effects on astrocytes (doubling/tripling of numbers and/or density). Additionally, AGT induced long-term changes in the population size and distribution of SNc/VTA dopaminergic neurons, confirming and extending our previous observations made in rats. Furthermore, glial/neuronal structural remodelling was sexually dimorphic and depended on the AGT dose and sub-region of the SNc/VTA. Investigations within the neonatal brain revealed that these long-term organisational effects of AGT depend, at least in part, on targeting perinatal processes that determine astrocyte density and programmed cell death in dopaminergic neurons. Collectively, our characterisation of enduring, AGT-induced, sex-specific cytoarchitectural disturbances suggests novel mechanistic links for the strong association between early environmental challenge (inappropriate exposure to excess GCs) and vulnerability to developing aberrant behaviours in later life, with translational implications for dopamine-associated disorders (such as schizophrenia, ADHD, autism, depression), which typically show a sex bias