Effect of pre-contraction on β-adrenoceptor-mediated relaxation of rat urinary bladder

Purpose The human physiological bladder contraction is largely mediated by acetylcholine acting on muscarinic receptors, but in pathophysiological settings the relative role of non-cholinergic stimuli gains importance. beta-Adrenoceptor agonists are currently in clinical development as treatments for the overactive bladder syndrome. Therefore, we have explored the ability of the beta-adrenoceptor agonist isoprenaline to induce rat isolated bladder strip relaxation on pre-contraction with the muscarinic agonist carbachol as compared to bladder tone induced by several non-cholinergic stimuli. Methods Bladder tone was induced by passive tension, receptor independently by KCl, carbachol, bradykinin or serotonin. Concentration-response curves were generated for relaxation by isoprenaline, and a single concentration of the receptor-independent relaxant forskolin was also tested. Results The various contractile stimuli induced different degrees of bladder tone, but the ability of isoprenaline or forskolin to relax rat bladder was not correlated with the degree of tone. Isoprenaline was significantly less potent and effective in causing relaxation against carbachol-induced tone than against any other stimulus, whereas no such relationship was observed for forskolin. Conclusions We conclude that beta-adrenoceptor agonists can induce rat bladder relaxation against a wide range of contractile stimuli and are more potent and/or effective against non-cholinergic stimuli than against muscarinic agonism. This profile appears desirable for agents intended for the treatment of overactive bladde

Allomorphy as a mechanism of post-translational control of enzyme activity

Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. β-phosphoglucomutase (βPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of βPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In βPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate β-glucose 1,6-bisphosphate, whose concentration depends on the β-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites

Basic mechanisms of urgency: roles and benefits of pharmacotherapy

Introduction Since urgency is key to the overactive bladder syndrome, we have reviewed the mechanisms underlying how bladder filling and urgency are sensed, what causes urgency and how this relates to medical therapy. Materials and methods Review of published literature. Results As urgency can only be assessed in cognitively intact humans, mechanistic studies of urgency often rely on proxy or surrogate parameters, such as detrusor overactivity, but these may not necessarily be reliable. There is an increasing evidence base to suggest that the sensation of ‘urgency’ differs from the normal physiological urge to void upon bladder filling. While the relative roles of alterations in afferent processes, central nervous processing, efferent mechanisms and in intrinsic bladder smooth muscle function remain unclear, and not necessarily mutually exclusive, several lines of evidence support an important role for the latter. Conclusions A better understanding of urgency and its causes may help to develop more effective treatments for voiding dysfunction

Effectiveness of an online group course for adolescents and young adults with depressive symptoms: study protocol for a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Depression is a common condition whose first onset is usually in late adolescence or early adulthood. Internet-based interventions are an effective treatment approach to depression. The aim of this study is to investigate the effectiveness of a Dutch online cognitive-behavioural group course known as Master Your Mood (<it>Grip op Je Dip</it>) for young people reporting depressive symptoms. Secondary research questions involve maintenance of effect at 6 months, mediators, and predictors of better outcomes.</p> <p>Methods</p> <p>We will conduct a randomised controlled trial (RCT) in which 244 young people aged 16-25 are randomly allocated to the Grip op Je Dip (GOJD) online group course or to a waiting list control group. The participants will be recruited from the general population. The primary outcome measure will be the severity of depressive symptoms according to the Center for Epidemiological Studies Depression Scale (CES-D). Other outcomes will include anxiety (Hospital Anxiety and Depression Scale-Anxiety, HADS) and mastery (Mastery Scale). Assessments will take place in both groups at baseline and three months later. Effect maintenance will be studied in the GOJD group six months after baseline, with missing data imputed using the expectation-maximisation method. Mediators and predictors of better outcomes will also be identified.</p> <p>Discussion</p> <p>The trial should add to the body of knowledge on the effectiveness of Internet-based interventions for depression. To our knowledge, this will be the first RCT on an online group intervention in this field.</p> <p>Trial registration</p> <p><a href="http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=NTR1694">NTR1694</a></p

Selective and Irreversible Inhibitors of Mosquito Acetylcholinesterases for Controlling Malaria and Other Mosquito-Borne Diseases

New insecticides are urgently needed because resistance to current insecticides allows resurgence of disease-transmitting mosquitoes while concerns for human toxicity from current compounds are growing. We previously reported the finding of a free cysteine (Cys) residue at the entrance of the active site of acetylcholinesterase (AChE) in some insects but not in mammals, birds, and fish. These insects have two AChE genes (AP and AO), and only AP-AChE carries the Cys residue. Most of these insects are disease vectors such as the African malaria mosquito (Anopheles gambiae sensu stricto) or crop pests such as aphids. Recently we reported a Cys-targeting small molecule that irreversibly inhibited all AChE activity extracted from aphids while an identical exposure caused no effect on the human AChE. Full inhibition of AChE in aphids indicates that AP-AChE contributes most of the enzymatic activity and suggests that the Cys residue might serve as a target for developing better aphicides. It is therefore worth investigating whether the Cys-targeting strategy is applicable to mosquitocides. Herein, we report that, under conditions that spare the human AChE, a methanethiosulfonate-containing molecule at 6 µM irreversibly inhibited 95% of the AChE activity extracted from An. gambiae s. str. and >80% of the activity from the yellow fever mosquito (Aedes aegypti L.) or the northern house mosquito (Culex pipiens L.) that is a vector of St. Louis encephalitis. This type of inhibition is fast (∼30 min) and due to conjugation of the inhibitor to the active-site Cys of mosquito AP-AChE, according to our observed reactivation of the methanethiosulfonate-inhibited AChE by 2-mercaptoethanol. We also note that our sulfhydryl agents partially and irreversibly inhibited the human AChE after prolonged exposure (>4 hr). This slow inhibition is due to partial enzyme denaturation by the inhibitor and/or micelles of the inhibitor, according to our studies using atomic force microscopy, circular dichroism spectroscopy, X-ray crystallography, time-resolved fluorescence spectroscopy, and liquid chromatography triple quadrupole mass spectrometry. These results support our view that the mosquito-specific Cys is a viable target for developing new mosquitocides to control disease vectors and to alleviate resistance problems with reduced toxicity toward non-target species

Prevention of depression and anxiety in adolescents: A randomized controlled trial testing the efficacy and mechanisms of Internet-based self-help problem-solving therapy

<p>Abstract</p> <p>Background</p> <p>Even though depression and anxiety are highly prevalent in adolescence, youngsters are not inclined to seek help in regular healthcare. Therapy through the Internet, however, has been found to appeal strongly to young people. The main aim of the present study is to examine the efficacy of preventive Internet-based guided self-help problem-solving therapy with adolescents reporting depressive and anxiety symptoms. A secondary objective is to test potential mediating and moderating variables in order to gain insight into how the intervention works and for whom it works best.</p> <p>Methods/design</p> <p>This study is a randomized controlled trial with an intervention condition group and a wait-list control group. The intervention condition group receives Internet-based self-help problem-solving therapy. Support is provided by a professional and delivered through email. Participants in the wait-list control group receive the intervention four months later. The study population consists of adolescents (12-18-year-olds) from the general population who report mild to moderate depressive and/or anxiety symptoms and are willing to complete a self-help course. Primary outcomes are symptoms of depression and anxiety. Secondary outcomes are quality of life, social anxiety, and cost-effectiveness. The following variables are examined for their moderating role: demographics, motivation, treatment credibility and expectancy, externalizing behaviour, perceived social support from parents and friends, substance use, the experience of important life events, physical activity, the quality of the therapeutic alliance, and satisfaction. Mediator variables include problem-solving skills, worrying, mastery, and self-esteem. Data are collected at baseline and at 3 weeks, 5 weeks, 4 months, 8 months, and 12 months after baseline. Both intention-to-treat and completer analyses will be conducted.</p> <p>Discussion</p> <p>This study evaluates the efficacy and mechanisms of Internet-based problem-solving therapy for adolescents. If Internet-based problem-solving therapy is shown to reduce depressive and anxiety symptoms in adolescents, the implication is to implement the intervention in clinical practice. Strengths and limitations of the study are discussed.</p> <p>Trial registration</p> <p>Netherlands Trial Register NTR1322</p

Characterization and Expression of Glutamate Dehydrogenase in Response to Acute Salinity Stress in the Chinese Mitten Crab, Eriocheir sinensis

Glutamate dehydrogenase (GDH) is a key enzyme for the synthesis and catabolism of glutamic acid, proline and alanine, which are important osmolytes in aquatic animals. However, the response of GDH gene expression to salinity alterations has not yet been determined in macro-crustacean species.GDH cDNA was isolated from Eriocheir sinensis. Then, GDH gene expression was analyzed in different tissues from normal crabs and the muscle of crabs following transfer from freshwater (control) directly to water with salinities of 16‰ and 30‰, respectively. Full-length GDH cDNA is 2,349 bp, consisting of a 76 bp 5'- untranslated region, a 1,695 bp open reading frame encoding 564 amino acids and a 578 bp 3'- untranslated region. E. sinensis GDH showed 64-90% identity with protein sequences of mammalian and crustacean species. Muscle was the dominant expression source among all tissues tested. Compared with the control, GDH expression significantly increased at 6 h in crabs transferred to 16‰ and 30‰ salinity, and GDH expression peaked at 48 h and 12 h, respectively, with levels approximately 7.9 and 8.5 fold higher than the control. The free amino acid (FAA) changes in muscle, under acute salinity stress (16‰ and 30‰ salinities), correlated with GDH expression levels. Total FAA content in the muscle, which was based on specific changes in arginine, proline, glycine, alanine, taurine, serine and glutamic acid, tended to increase in crabs following transfer to salt water. Among these, arginine, proline and alanine increased significantly during salinity acclimation and accounted for the highest proportion of total FAA.E. sinensis GDH is a conserved protein that serves important functions in controlling osmoregulation. We observed that higher GDH expression after ambient salinity increase led to higher FAA metabolism, especially the synthesis of glutamic acid, which increased the synthesis of proline and alanine to meet the demand of osmoregulation at hyperosmotic conditions

Genetic Basis of Growth Adaptation of Escherichia coli after Deletion of pgi, a Major Metabolic Gene

Bacterial survival requires adaptation to different environmental perturbations such as exposure to antibiotics, changes in temperature or oxygen levels, DNA damage, and alternative nutrient sources. During adaptation, bacteria often develop beneficial mutations that confer increased fitness in the new environment. Adaptation to the loss of a major non-essential gene product that cripples growth, however, has not been studied at the whole-genome level. We investigated the ability of Escherichia coli K-12 MG1655 to overcome the loss of phosphoglucose isomerase (pgi) by adaptively evolving ten replicates of E. coli lacking pgi for 50 days in glucose M9 minimal medium and by characterizing endpoint clones through whole-genome re-sequencing and phenotype profiling. We found that 1) the growth rates for all ten endpoint clones increased approximately 3-fold over the 50-day period; 2) two to five mutations arose during adaptation, most frequently in the NADH/NADPH transhydrogenases udhA and pntAB and in the stress-associated sigma factor rpoS; and 3) despite similar growth rates, at least three distinct endpoint phenotypes developed as defined by different rates of acetate and formate secretion. These results demonstrate that E. coli can adapt to the loss of a major metabolic gene product with only a handful of mutations and that adaptation can result in multiple, alternative phenotypes