Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ? 98 %). All samples were analyzed following injection (100 ?l) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 ?m, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2-30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min(-1) flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients

A cardinal role for cathepsin D in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci

The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP) and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D-/- hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function

Airway smooth muscle relaxation results from a reduction in the frequency of Ca(2+ )oscillations induced by a cAMP-mediated inhibition of the IP(3 )receptor

BACKGROUND: It has been shown that the contractile state of airway smooth muscle cells (SMCs) in response to agonists is determined by the frequency of Ca(2+ )oscillations occurring within the SMCs. Therefore, we hypothesized that the relaxation of airway SMCs induced by agents that increase cAMP results from the down-regulation or slowing of the frequency of the Ca(2+ )oscillations. METHODS: The effects of isoproterenol (ISO), forskolin (FSK) and 8-bromo-cAMP on the relaxation and Ca(2+ )signaling of airway SMCs contracted with methacholine (MCh) was investigated in murine lung slices with phase-contrast and laser scanning microscopy. RESULTS: All three cAMP-elevating agents simultaneously induced a reduction in the frequency of Ca(2+ )oscillations within the SMCs and the relaxation of contracted airways. The decrease in the Ca(2+ )oscillation frequency correlated with the extent of airway relaxation and was concentration-dependent. The mechanism by which cAMP reduced the frequency of the Ca(2+ )oscillations was investigated. Elevated cAMP did not affect the re-filling rate of the internal Ca(2+ )stores after emptying by repetitive exposure to 20 mM caffeine. Neither did elevated cAMP limit the Ca(2+ )available to stimulate contraction because an elevation of intracellular Ca(2+ )concentration induced by exposure to a Ca(2+ )ionophore (ionomycin) or by photolysis of caged-Ca(2+ )did not reverse the effect of cAMP. Similar results were obtained with iberiotoxin, a blocker of Ca(2+)-activated K(+ )channels, which would be expected to increase Ca(2+ )influx and contraction. By contrast, the photolysis of caged-IP(3 )in the presence of agonist, to further elevate the intracellular IP(3 )concentration, reversed the slowing of the frequency of the Ca(2+ )oscillations and relaxation of the airway induced by FSK. This result implied that the sensitivity of the IP(3)R to IP(3 )was reduced by FSK and this was supported by the reduced ability of IP(3 )to release Ca(2+ )in SMCs in the presence of FSK. CONCLUSION: These results indicate that the relaxant effect of cAMP-elevating agents on airway SMCs is achieved by decreasing the Ca(2+ )oscillation frequency by reducing internal Ca(2+ )release through IP(3 )receptors

Beyond the public and private divide: Remapping transnational climate governance in de 21th century

This article provides a first step towards a better theoretical and empirical knowledge of the emerging arena of transnational climate governance. The need for such a re-conceptualization emerges from the increasing relevance of non-state and transnational approaches towards climate change mitigation at a time when the intergovernmental negotiation process has to overcome substantial stalemate and the international arena becomes increasingly fragmented. Based on a brief discussion of the increasing trend towards transnationalization and functional segmentation of the global climate governance arena, we argue that a remapping of climate governance is necessary and needs to take into account different spheres of authority beyond the public and international. Hence, we provide a brief analysis of how the public/private divide has been conceptualized in Political Science and International Relations. Subsequently, we analyse the emerging transnational climate governance arena. Analytically, we distinguish between different manifestations of transnational climate governance on a continuum ranging from delegated and shared public-private authority to fully non-state and private responses to the climate problem. We suggest that our remapping exercise presented in this article can be a useful starting point for future research on the role and relevance of transnational approaches to the global climate crisis

The role of dietary fibre in pig production, with a particular emphasis on reproduction

Abstract Fibres from a variety of sources are a common constituent of pig feeds. They provide a means to utilise locally-produced plant materials which are often a by-product of the food or drink industry. The value of a high fibre diet in terms of producing satiety has long been recognised. However the addition of fibre can reduce feed intake, which is clearly detrimental during stages of the production cycle when nutrient needs are high, for example in growing piglets and during lactation. More recently, fibre has been found to promote novel benefits to pig production systems, particularly given the reduction in antimicrobial use world-wide, concern for the welfare of animals fed a restricted diet and the need to ensure that such systems are more environmentally friendly. For example, inclusion of dietary fibre can alter the gut microbiota in ways that could reduce the need for antibiotics, while controlled addition of certain fibre types may reduce nitrogen losses into the environment and so reduce the environmental cost of pig production. Of particular potential value is the opportunity to use crude fibre concentrates as ‘functional’ feed additives to improve young pig growth and welfare. Perhaps the greatest opportunity for the use of high fibre diets is to improve the reproductive efficiency of pigs. Increased dietary fibre before mating improves oocyte maturation, prenatal survival and litter size; providing a consumer-acceptable means of increasing the amount of saleable meat produced per sow. The mechanisms responsible for these beneficial effects remain to be elucidated. However, changes in plasma and follicular fluid concentrations of key hormones and metabolites, as well as effects of the hypothalamic satiety centre on gonadotrophin secretion and epigenetic effects are strong candidates

Psychopharmacological and Other Treatments in Preschool Children with Attention-Deficit/Hyperactivity Disorder: Current Evidence and Practice

Objective: This article reviews rational approaches to treating attention-deficit/hyperactivity disorder (ADHD) in preschool children, including pharmacological and nonpharmacological treatments. Implications for clinical practice are discussed. Data Sources: We searched MEDLINE, PsychINFO, Cumulative Index to Nursing & Allied Health, Educational Resources Information Center, Cochrane Database of Systematic Reviews and Database of Abstracts of Reviews of Effects for relevant literature published in English from 1967 to 2007 on preschool ADHD. We also reviewed the references cited in identified reports. Study Selection: Studies were reviewed if the sample included at least some children younger than 6 years of age or attending kindergarten, the study participants had a diagnosis of ADHD or equivalent symptoms, received intervention aimed at ADHD symptoms, and included a relevant outcome measure. Data Extraction: Studies were reviewed for type of intervention and outcome relevant to ADHD and were rated for the level of evidence for adequacy of the data to inform clinical practice. Conclusions: The current level of evidence for adequacy of empirical data to inform clinical practice for shortterm treatment of ADHD in preschool children is Level A for methylphenidate and Level B for parent behavior training, child training, and additive-free elimination diet

Systemic and Mucosal Immune Responses to Sublingual or Intramuscular Human Papilloma Virus Antigens in Healthy Female Volunteers

The sublingual route has been proposed as a needle-free option to induce systemic and mucosal immune protection against viral infections. In a translational study of systemic and mucosal humoral immune responses to sublingual or systemically administered viral antigens, eighteen healthy female volunteers aged 19–31 years received three immunizations with a quadravalent Human Papilloma Virus vaccine at 0, 4 and 16 weeks as sublingual drops (SL, n = 12) or intramuscular injection (IM, n = 6). IM antigen delivery induced or boosted HPV-specific serum IgG and pseudovirus-neutralizing antibodies, HPV-specific cervical and vaginal IgG, and elicited circulating IgG and IgA antibody secreting cells. SL antigens induced ∼38-fold lower serum and ∼2-fold lower cervical/vaginal IgG than IM delivery, and induced or boosted serum virus neutralizing antibody in only 3/12 subjects. Neither route reproducibly induced HPV-specific mucosal IgA. Alternative delivery systems and adjuvants will be required to enhance and evaluate immune responses following sublingual immunization in humans

Perioperative and long-term operative outcomes after surgery for trigeminal neuralgia: microvascular decompression vs percutaneous balloon ablation

<p>Abstract</p> <p>Objectives</p> <p>Numerous medical and surgical therapies have been utilized to treat the symptoms of trigeminal neuralgia (TN). This retrospective study compares patients undergoing either microvascular decompression or balloon ablation of the trigeminal ganglion and determines which produces the best long-term outcomes.</p> <p>Methods</p> <p>A 10-year retrospective chart review was performed on patients who underwent microvascular decompression (MVD) or percutaneous balloon ablation (BA) surgery for TN. Demographic data, intraoperative variables, length of hospitalization and symptom improvement were assessed along with complications and recurrences of symptoms after surgery. Appropriate statistical comparisons were utilized to assess differences between the two surgical techniques.</p> <p>Results</p> <p>MVD patients were younger but were otherwise similar to BA patients. Intraoperatively, twice as many BA patients developed bradycardia compared to MVD patients. 75% of BA patients with bradycardia had an improvement of symptoms. Hospital stay was shorter in BA patients but overall improvement of symptoms was better with MVD. Postoperative complication rates were similar (21% vs 26%) between the BA and MVD groups.</p> <p>Discussion</p> <p>MVD produced better overall outcomes compared to BA and may be the procedure of choice for surgery to treat TN.</p