Liraglutide, leptin and their combined effects on feeding: additive intake reduction through common intracellular signalling mechanisms.

AIM: To investigate the behavioural and intracellular mechanisms by which the glucagon like peptide-1 (GLP-1) receptor agonist, liraglutide, and leptin in combination enhance the food intake inhibitory and weight loss effects of either treatment alone. METHODS: We examined the effects of liraglutide (a long-acting GLP-1 analogue) and leptin co-treatment, delivered in low or moderate doses subcutaneously (s.c.) or to the third ventricle, respectively, on cumulative intake, meal patterns and hypothalamic expression of intracellular signalling proteins [phosphorylated signal transducer and activator of transcription-3 (pSTAT3) and protein tyrosine phosphatase-1B (PTP1B)] in lean rats. RESULTS: A low-dose combination of liraglutide (25 µg/kg) and leptin (0.75 µg) additively reduced cumulative food intake and body weight, a result mediated predominantly through a significant reduction in meal frequency that was not present with either drug alone. Liraglutide treatment alone also reduced meal size; an effect not enhanced with leptin co-administration. Moderate doses of liraglutide (75 µg/kg) and leptin (4 µg), examined separately, each reduced meal frequency, cumulative food intake and body weight; only liraglutide reduced meal size. In combination these doses did not further enhance the anorexigenic effects of either treatment alone. Ex vivo immunoblot analysis showed elevated pSTAT3 in the hypothalamic tissue after liraglutide-leptin co-treatment, an effect which was greater than that of leptin treatment alone. In addition, s.c. liraglutide reduced the expression of PTP1B (a negative regulator of leptin receptor signalling), revealing a potential mechanism for the enhanced pSTAT3 response after liraglutide-leptin co-administration. CONCLUSIONS: Collectively, these results show novel behavioural and molecular mechanisms underlying the additive reduction in food intake and body weight after liraglutide-leptin combination treatment.

MAG-EPA resolves lung inflammation in an allergic model of asthma.

BACKGROUND: Asthma is a chronic disease characterized by airways hyperresponsiveness, inflammation and airways remodelling involving reversible bronchial obstruction. Omega-3 fatty acids and their derivatives are known to reduce inflammation in several tissues including lung. OBJECTIVES: The effects of eicosapentaenoic acid monoacylglyceride (MAG-EPA), a newly synthesized EPA derivative, were determined on the resolution of lung inflammation and airway hyperresponsiveness in an in vivo model of allergic asthma. METHODS: Ovalbumin (OVA)-sensitized guinea-pigs were treated or not with MAG-EPA administered per os. Isometric tension measurements, histological analyses, homogenate preparation for Western blot experiments or total RNA extraction for RT-PCR were performed to assess the effect of MAG-EPA treatments. RESULTS: Mechanical tension measurements revealed that oral MAG-EPA treatments reduced methacholine (MCh)-induced bronchial hyperresponsiveness in OVA-sensitized guinea-pigs. Moreover, MAG-EPA treatments also decreased Ca(2+) hypersensitivity of bronchial smooth muscle. Histological analyses and leucocyte counts in bronchoalveolar lavages revealed that oral MAG-EPA treatments led to less inflammatory cell recruitment in the lung of OVA-sensitized guinea-pigs when compared with lungs from control animals. Results also revealed a reduction in mucin production and MUC5AC expression level in OVA-sensitized animals treated with MAG-EPA. Following MAG-EPA treatments, the transcript levels of pro-inflammatory markers such as IL-5, eotaxin, IL-13 and IL-4 were markedly reduced. Moreover, per os MAG-EPA administrations reduced COX2 over-expression in OVA-sensitized animals. CONCLUSION AND CLINICAL RELEVANCE: We demonstrate that MAG-EPA reduces airway hyperresponsiveness and lung inflammation in OVA-sensitized animals, a finding consistent with a decrease in IL-4, IL-5, IL-13, COX-2 and MUC5AC expression levels in the lung. The present data suggest that MAG-EPA represents a new potential therapeutic strategy for resolving inflammation in allergic asthma.

Anticytoskeletal autoantibody to microfilament anchorage sites recognizes novel focal contact proteins.

Actin microfilaments are anchored to the plasma membrane at focal contacts. Using an indirect immunofluorescence method, we detected an autoantibody reactive with focal contacts in PtK2, HEp-2, and BHK-21 cells in serum from two patients with early systemic sclerosis. With double immunofluorescence, using the actin-binding drug phalloidin, we localized the plaques decorated by these sera specifically at the termini of microfilament bundles. The reactive antigens were identified by immunoblotting as proteins of 80,000- and 75,300-mol wt in PtK2, and of 53,500-mol wt in HEp-2 and BHK-21 cells. The 53,500-mol wt protein was also identified in rat skeletal, myocardial, and smooth muscle tissues. The detergent solubility of these proteins suggested that they may be linked to the plasma membrane. The autoantigens were immunologically distinct from vinculin and alpha-actinin, two major proteins also known to be concentrated at the ends of microfilament bundles. Our observations suggest that this novel anticytoskeletal autoantibody may identify a novel family of vertebrate cell proteins involved in the linkage of microfilaments to the plasma membrane at focal contacts.

Effect of acute changes in glomerular filtration rate on Na+/H+ exchange in rat renal cortex.

Studies were undertaken in Munich-Wistar rats to assess the influence of changes in filtered bicarbonate (FLHCO3), induced by changes in GFR, on Na+/H+ exchange activity in renal brush border membrane vesicles (BBMV). Whole-kidney and micropuncture measurements of GFR, FLHCO3, and whole-kidney and proximal tubule HCO3 reabsorption (APRHCO3) were coupled with BBMV measurements of H+ gradient-driven 22Na+ uptake in each animal studied. 22Na+ uptake was measured at three Na+ concentration gradients to allow calculation of Vmax and Km for Na+/H+ exchange. GFR was varied by studying animals under conditions of hydropenia, plasma repletion, and acute plasma expansion. The increase in GFR, FLHCO3, and APRHCO3 induced by plasma administration correlated directly with an increase in the Vmax for Na+/H+ exchange in BBMV. The Km for sodium was unaffected. In the plasma-expanded rats, the Vmax for Na+/H+ exchange was 22% greater than in the hydropenic rats (P less than 0.025) whereas APRHCO3 was 86% greater (P less than 0.001). These results indicate that increases in FLHCO3, induced by acute increases in GFR, stimulate Na+/H+ exchange activity in proximal tubular epithelium. This stimulation is a mechanism which can, in part, account for the delivery dependence of proximal bicarbonate reabsorption.

Monoclonal antibodies as probes for the transmembrane structure of neutral endopeptidase 24.11 ('enkephalinase').

The neutral endopeptidase (EC 3.4.24.11) ('enkephalinase') is a membrane-bound metalloendopeptidase that is present in large amounts in the microvilli of the kidney proximal tubules. By immunizing mice with purified rabbit kidney brush-border membranes, we have obtained four different monoclonal antibodies that recognize this enzyme in dot-blot and Western-blot assays and can be used for immunoprecipitation of neutral endopeptidase from crude kidney solubilizates. One of these monoclonal antibodies (2B12) allows the labeling of proximal tubule cells with colloidal gold particles. This monoclonal antibody also binds to native brush-border membrane vesicles (which are mostly in the right-side-out configuration) and recognizes an epitope which is destroyed after reduction and alkylation of the protein. By contrast, all three other monoclonal antibodies (21G10, 23B11 and 22E2) compete for another epitope of neutral endopeptidase that is not exposed at the extracytoplasmic surface either in intact cells or in sealed brush-border vesicles. Permeabilization of the vesicles with digitonin, however, restores the full binding activity. Binding of these antibodies is not altered by prior reduction and alkylation of the protein. Taken together, these results strongly suggest that the 2B12 monoclonal antibody binds a conformational epitope located on the ectodomain of the enzyme, whereas the three others (21G10, 23B11 and 22E2) bind to a common or to overlapping epitopes located on the cytosolic domain. These results also demonstrate unambiguously the transmembrane nature of neutral endopeptidase.

Sampling phasic dopamine signaling with fast-scan cyclic voltammetry in awake, behaving rats.

Fast-scan cyclic voltammetry (FSCV) is an electrochemical technique that permits the in vivo measurement of extracellular fluctuations in multiple chemical species. The technique is frequently utilized to sample sub-second (phasic) concentration changes of the neurotransmitter dopamine in awake and behaving rats. Phasic dopamine signaling is implicated in reinforcement, goal-directed behavior, and locomotion, and FSCV has been used to investigate how rapid changes in striatal dopamine concentration contribute to these and other behaviors. This unit describes the instrumentation and construction, implantation, and use of components required to sample and analyze dopamine concentration changes in awake rats with FSCV.

In vivo modulation of murine collagen induced arthritis.

The effects of in vivo modulation of murine collagen induced arthritis with monoclonal anti-CD4 antibodies, monoclonal anti-Ia antibodies, and gamma interferon are reviewed. We detail the mechanism of action of monoclonal anti-CD4 antibody on humoral and cell mediated immune responses and discuss the implications for designing therapeutic strategies. To further explore the induction of collagen induced arthritis, a syngeneic cell transfer system using collagen primed T lymphocytes is described. This cell transfer system provides an opportunity to study the role of CD4 positive T lymphocytes in arthritis induction during a short, defined time period.

The BioTools Suite. A comprehensive suite of platform-independent bioinformatics tools.

The BioTools Suite is a set of three comprehensive, platform-independent software packages (PepTool, GeneTool, and ChromaTool) developed for sequence assembly and analysis. In addition to supporting a large number of standard bioinformatics functions, these programs also incorporate a number of useful innovations including uniform graphical-user interface (GUI) design, direct internet connectivity, a novel approach to feature annotation, and a variety of enhanced algorithms for large scale proteome and genome analysis. This article describes the key features, recent changes, and general operation of all three programs.

Maturation of visual receptive field properties in the rat superior colliculus.

Visually responsive neurons were recorded in the superficial layers of rat superior colliculus from postnatal day 12 to 28. Receptive field properties such as size, type (ON, OFF, ON-OFF and motion sensitive) and direction selectivity were analyzed to disclose changes during maturation. Although some aspects of sensory properties are modified during development (latency, receptive field sizes, and proportions of receptive field types), a high level of sophistication is also present in young animals even before eyelid opening. For instance, direction selective and direction biased cells, which require complex synaptic relations, are already observed when the first light evoked responses emerge in the superior colliculus (P13), strongly suggesting that this property develops without visual experience. Furthermore, direction selectivity is present in the colliculus prior to the appearance of visually evoked activity in the cortex. This indicates that direction selectivity can not be attributable to incoming cortical afferents. This study provides the first direct evidence that, unlike the cat, the rat's cortico-tectal pathway is only weakly involved in the establishment of direction selectivity in collicular neurons.

ON and OFF field potentials in the rat superior colliculus during development.

The present investigation is aimed at characterizing the development of ON and OFF visually evoked responses in the rat superior colliculus from postnatal day 13 (P13) to postnatal day 25. Depth profiles of field potentials reveal that ON and OFF long latency biphasic field potentials are already present when collicular cells are first responsive to light (P13). There is an inversion in the polarity of these responses as the electrode penetrates the collicular layers, suggesting a synaptic organization similar to the one found in adult animals. At P15, OFF field potentials begin to exhibit oscillatory activity. Local cobalt injections within the superior colliculus abolishes these OFF oscillations, suggesting a postsynaptic origin. Fast Fourier transform (FFT) analysis of the OFF field potentials demonstrates that oscillatory activity increases in frequency during development. This increase is thought to reflect the myelination and stabilization of synaptic connections that occur during this period. To our knowledge, this is the first report of OFF oscillatory responses in the superior colliculus.

Spontaneous uterine infections are associated with elevated prostaglandin F(2)alpha metabolite concentrations in postpartum dairy cows.

Postpartum Holstein (n=21) and Jersey (n=4) cows were used to determine if uterine infections are associated with elevated plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F(2)alpha (PGFM). Based upon clinical examinations and bacterial content of intrauterine fluid samples, cows detected with uterine infections between 21 and 28 d post partum were used (infected; n=14). These cows were matched with herdmates that were free of infection (control; n=11). Beginning on the day the cows were assigned to the experiment (Day 1), blood samples were collected on alternate days for the next 14 to 15 d. Plasma samples were stored at -20 degrees C until assayed. From Day 1 until the end of the experiment, uterine fluid samples were collected transcervically twice weekly for aerobic bacterial culture. Endometrial biopsies were collected between Days 6 and 8 and Days 13 and 15. Control cows did not show signs of uterine infection throughout the trial, and bacterial cultures indicated that there were no significant bacterial populations in the uteri of the control cows. The uteri of infected cows harbored numerous microbes. Actinomyces pyogenes was most prominent. Various species of Streptococcus and Pasteurella were also prevalent in the infected cows. Escherichia coli was present in the uterus of both infected and control cows. Biopsies showed that infected cows had more (P<0.05) neutrophils, plasma cells and lymphocytes in the endometrium than did the control cows. As determined by plasma progesterone concentrations, 83% of the control and 50% of the infected cows had functional luteal tissue during the 2-wk sampling period. Plasma PGFM profiles were linear (P<0.03) and did not differ between treatment groups (P>0.01). However, average plasma PGFM concentrations were greater (P<0.0001) in infected than in control cows. These data indicate that plasma PGFM concentrations are greater in postpartum cows with spontaneous uterine infections then in herdmates free of infection.

[Disease prevention and health promotion for children and youth: current status and contemporary issues in CLSC-based nursing practice]. / Prévention et promotion de la santé pour les enfants et les jeunes: description et enjeux de la pratique infirmière en CLSC.

This article aims to provide an overview of the disease prevention and health promotion work carried out by CLSC nurses working with infants, children and youth. A second objective is to present our synthesis of four recent studies on the state of prevention-promotion in these organizations. This is followed by our analysis of several contemporary issues related to nursing practice in this intervention sector. While disease prevention and health promotion is a major component of the practice of CLSC nurses who work with infants, children and youth, it is an intervention sector that has been weakened by numerous changes affecting these organizations in the wake of the reform. The discussion thus proposes several courses of research and action in the hopes of strengthening this major component of the CLSC mission.

'Access arrangements' for biobanks: a fine line between facilitating and hindering collaboration.

This decade is witnessing the proliferation of large-scale population-based biobanks. Many biobanks have reached the stage of offering access to their collection of data and samples to the scientific community. This, however, requires that access arrangements be established to govern the relationship between biobanks and users. Access arrangements capture the convergence of all normative elements in the life cycle of a biobank: policies, laws, common practices, commitments made by the biobank to participants, the expectations of funders, and the needs of the scientific community. Furthermore, access arrangements shape new legal agreements between 'biobankers' and researchers to ensure appropriate, regulated and efficient use of biobank materials. This paper begins by examining the particularities of access arrangements, identifying the key elements of these new regulatory instruments. Second, the paper looks at various strategies used by biobanks to regulate access and surveys the underlying motivations of these strategies and the impact they can have on potential international collaboration. Third, an example of the challenges encountered in creating access policy is illustrated using the case of CARTaGENE, a biobank based in Montreal, Canada. Last, the paper presents how Public Population Project in Genomics (P(3)G) facilitates the work of biobankers and improves collaboration throughout the international human genomics research community.

Framing genomics, public health research and policy: points to consider.

Genetic information can be used to target interventions that improve health and prevent disease. Indeed, the results of population genomics research could be useful for public health and national pandemic plans. Yet, firm scientific evidence originating from such research and the indicators of the role of health determinants, gene-gene and gene-environment interaction remain to be assessed and validated before being integrated into pandemic plans or public health programmes. It is not clear what is the role of the State in research on the elucidation of the determinants of gene-gene and gene-environment interactions and how, when, and if such data can be accessed and used for such planning. Over a period of 3 years, we sought to address these questions by gathering data and literature relevant to research in public health genomics, preparing issues papers and, finally, consulting with stakeholders on a provisional 'points to consider' document at various times. Examining in turn the issues of privacy, State powers, stakeholder perceptions, and public participation, we propose in this article, for each of these themes, a series of recommendations aiming to provide guidance on the role of the State in the use of genomic information for public health research, prevention and planning.

Glucocorticoids of bison bulls in relation to social status.

A primary response to stress is an increase in circulating adrenal glucocorticoids (GC) such as cortisol. Two hypotheses propose differential stress responses to agonistic and aggressive interactions in social groups. If subordinate animals are subjected to social and psychological stressors leading to chronic GC elevation, the 'stress of subordination' hypothesis predicts that GCs will be higher in subordinates than dominants. Alternatively, if dominant animals are subject to physiological stressors (e.g., fight at higher rates than subordinates) or hierarchies are unstable, the 'stress of domination' hypothesis predicts higher GCs in dominant individuals. Both models predict that GC levels will peak during the breeding season. We tested these predictions in bison bulls (Bison bison) using fecal steroid analysis to characterize GC concentration and behavioral observations to determine dominance rank, copulatory success, and tending status of bulls. Fecal samples were collected during 2003 from adult bison bulls during pre-rut (June), rut (July-August), and post-rut (September). Matched sample data indicated that mean GC levels (ng/g feces) of bulls strongly peaked during the 4-week rut, doubling from pre-rut to rut and then declining again during post-rut. High ranked dominant bulls maintained higher GC levels than lower ranked subordinate bulls. Dominance rank was positively correlated with copulatory success and age, and dominant bulls were more likely to tend (guard) cows as they approached estrus. There was a positive correlation between GC level and copulatory success, with prime-aged bulls (> or =7 years) obtaining the most copulations. GC levels were positively correlated with bull androgen levels determined in a previous study. These results support the 'stress of domination' hypothesis, indicating that dominant bison bulls pay a significant physiological price for high social status and the opportunity to mate.

Elimination of human enteric viruses during conventional waste water treatment by activated sludge.

The present study was undertaken to determine if viruses were selectively eliminated during waste water treatment. Human enteric viruses were detected at all steps of treatment in a conventional activated sludge waste water treatment plant. Liquid overlays and large volume sampling with multiple passages on BGM cells permitted the detection of poliovirus (serotypes 1, 2, and 3), coxsackievirus B (serotypes 1, 2, 3, 4, and 5), and echovirus (serotypes 3, 14, and 22), as well as reoviruses. The mean virus concentration was 95.1 most probable number of infectious units per litre (mpniu/L) in raw sewage, 23.3 in settled water, 1.4 in effluent after activated sludge treatment, and 40.3 mpniu/L in sludge samples. All samples of raw sewage and settled water, 79% of effluent water, and 94% of sludge samples contained viruses. The mean reduction was 75% after settling and 98% after activated sludge treatment. Poliovirus type 3 was rarely isolated after the activated sludge treatment, but was still detected in about one-third of the sludge samples. Reoviruses and coxsackieviruses were detected at similar rates from all samples and appear to be more resistant to the activated sludge treatment than poliovirus type 3. Poliovirus types 1 and 2 were present in almost every sample of raw sewage and settled water and still found in about half of the effluent and sludge samples, indicating a level of resistance similar to that of reoviruses and coxsackieviruses.

Posttranslational modifications of proopiomelanocortin in rat intermediate lobe cells.

Proopiomelanocortin (POMC), the common precursor to beta-endorphin and alpha-melanocyte-stimulating hormone synthesized in rat intermediate lobe cells, exhibits both charge and size heterogeneity on two-dimensional gels. Pulse-labeling and pulse-chase studies revealed that this heterogeneity is due to co- and post-translational modifications of a single common polypeptide. Short 5-min-pulse incubation with [3H]phenylalanine allowed the preferential labeling of two major forms characterized by an identical isoelectric point (8.2), but slightly different apparent molecular weights (MW = 34,000 and 36,000). These peptides could be labeled with [3H]mannose and the analysis of their tryptic fragments by high-pressure liquid chromatography revealed that they correspond to polypeptides bearing one or two N-linked carbohydrate side chains. Accumulation of more acidic forms was observed during subsequent chase incubations in the absence of phenylalanine. These acidic forms were shown to incorporate sulfate and (or) phosphate groups. Sulfation and phosphorylation occurred on POMC within 5 min after its synthesis and were concomitant with the processing of the N-linked carbohydrates from the high mannose to the complex structure. Finally, partial digestion of the phosphorylated and nonphosphorylated analogs of POMC with either Staphylococcus aureus (V8 strain) protease or chymotrypsin suggests that the presence of a phosphate group may alter POMC sensitivity to exogenously added proteases.

Preparations, characterizations, and structures of (biimidazole)dihalobis(triphenylphosphine)rhenium(III) salts: strong ion-pairing and acid-base properties.

Two-electron reduction occurs when the Re(V) precursors ReOX3(PPh3)2 and ReO(OEt)X2(PPh3)2 are reacted with biimidazole (biimH2) in boiling chloroform, affording rhenium(III) cationic complexes of the type cis,trans-[ReX2(PPh3)2(biimH2)]X with X = Cl, Br, and I. Crystal structures are determined for the compounds with the three halogens, as well as for the [ReCl2(PPh3)2(biimH2)](benzoate) salt. In all cases, the counterion is attached to the complex cation via hydrogen bonding with the N-H groups of coordinated biimidazole. Variable-temperature 1H NMR spectroscopy shows that a mixture of [ReCl2(PPh3)2(biimH2)](benzoate) and [ReCl2(PPh3)2(biimH2)]Cl is in slow exchange below -50 degrees C in CD2Cl2, indicating that ion pairing is retained in solution. Both N-H groups can be deprotonated with sodium methoxide, and their acidities are evaluated from UV-visible spectra. Competition between monodeprotonated [ReCl2(PPh3)2(biimH)] and various carboxylic acids reveals that the acidity of the first N-H proton corresponds to that of acetic acid (pKa(aq) approximately 4.8). By a similar competitive reaction between bis-deprotonated [ReCl2(PPh3)2(biim)]- and phenols, the second acidity is estimated to be close to that of phenol (pKa(aq) approximately 9.8).

Preparation and characterization of oxorhenium(V) complexes with 2,2'-biimidazole: the strong affinity of coordinated biimidazole for chloride ions via N-H...Cl- hydrogen bonding.

N,N'-Dimethylbiimidazole and bipyridine (N-N) react with ReOCl3(OPPh3)(Me2S) to give mer-ReOCl3(N-N) compounds. Nonmethylated biimidazole forms a trans-O,O [ReOCl2(OPPh3)(biimH2)]+ cation, which is tightly associated with the Cl- counterion via N-H...Cl- hydrogen bonding. Hydrolysis of ReOCl3(biimMe2) in wet acetone (5% water) leads to the linear oxo-bridged dinuclear species [(OReCl2(biimMe2)2(mu-O)] containing chelated biimMe2. Acetone solutions containing only 1% water yield the bent oxo-bridged dinuclear species [(OReCl2)2(mu-O)(mu-biimMe2)2], where each Re center retains the ReO2Cl2N2 coordination but the biimMe2 ligands are bridging. The linear oxo-bridged [(OReCl2(biimH2)2(mu-O)] complex obtained with nonmethylated biimidazole includes two Cl- ions held via N-H...Cl- hydrogen bonds, leading to a dianionic [(OReCl2(biimH2...Cl)2(mu-O)]2- unit in the crystals of the PPh4+ salt. The compounds are characterized by IR and NMR spectroscopies, and the structures of [ReOCl2(OPPh3)(biimH2)]Cl, [(OReCl2(biimH2)2(mu-O)](PPh4Cl)(2).2H2O, and [(OReCl2)2(mu-O)(mu-biimMe2)2].acetone are determined by X-ray diffraction.