Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae)

Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine > spermidine > putrescine. Spermidine affected K-0.5 values for Na+ with minor alterations in K-0.5 values for K+ and N-H-4(+), causing a decrease in maximal velocities under saturating Na+, K+ and NH4+ concentrations. Phosphorylation measurements in the presence of 20 mu M ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na+, both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na+, the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na+ at the Na+-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments. (c) 2008 Elsevier Inc. All rights reserved.

Synthesis of naturally occurring diene and trienes by Te/Li exchange on (1Z,3Z)-butyltelluro-4-methoxy-1,3-butadiene

(1Z,3Z)-Butyltelluro-o-4-methoxy-1,3-butadiene 2 was obtained by the hydrotelluration of(Z)-1-methoxy-but-1-en-3-ynes 1. The butadienyllithium 3 obtained by the Te/Li exchange reaction in the (1Z,3Z)-1-butyltelluro-4-methoxy-1.3-butadiene 2 reacted with aldehydes to form the corresponding alcohols 4a-d with total retention of configuration. The alcohols formed undergo hydrolysis, resulting in the alpha,beta,gamma,delta-unsaturated aldehydes of (E,E) configuration, which are precursors of trienes obtained from natural sources. The products of this reaction were employed in the synthesis of methyl-(2E,4E)-decadienoate 7, which is a component of the flavor principles of ripe Bartlett pears. Performing the Wittig reaction of the methyl triphenylphosphorane with the deca-(2E,4E)-dienal 5a, we were able to synthesize the undeca-(1,3E,5E)-triene 6a. This compound is a sex-pheromone component of the marine brown algae Fucus serratus, Dictyopteris plagiograma, and Dictyopteris australis. Performing the Wittig reaction of methyl triphenylphosphorane with the octa-(2E,4E)-dienal 5c, the nona-(1,3E,5E)-triene 6b was synthesized. The compound obtained is a sex-pheromone component of the marine brown alga Sargassum horneri. The octa-( 1,3E,5E)-triene 6c was easily obtained from hepta-(2E,4E)-dienal 5d by the Wittig reaction with methyl triphenylphophorane. This compound is a sex-pheromone component of the marine brown alga Fucus serratus. (C) 2010 Elsevier Ltd. All rights reserved.

Refolding and purification of the human secreted group IID phospholipase A(2) expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A(2) (sPLA(2)s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A(2) (hsPLA(2)-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA(2)-IID in Escherichia coli, the DNA-coding sequence for hsPLA(2)-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA(2)-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A(2). The refolded recombinant hsPLA(2)-IID demonstrated Ca(2+)-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA(2)-IID which will advance our understanding of the structure-function relationship and biological effects of the protein. (C) 2009 Elsevier Inc. All rights reserved.

Hemolymph ionic regulation and adjustments in gill (Na(+), K(+))-ATPase activity during salinity acclimation in the swimming crab Callinectes ornatus (Decapoda, Brachyura)

We evaluate hemolymph osmotic and ionic regulatory abilities and characterize a posterior gill microsomal (Na(+), K(+))-ATPase from the marine swimming crab, Callinectes ornatus, acclimated to 21 parts per thousand or 33 parts per thousand salinity. C ornatus is isosmotic after acclimation to 21 parts per thousand but is hyposmotic at 33 parts per thousand salinity; hemolymph ions do not recover initial levels on acclimation to 21 parts per thousand salinity but are anisoionic compared to ambient concentrations, revealing modest regulatory ability. NH(4)(+) modulates enzyme affinity for K(+), which increases 187-fold in crabs acclimated to 33%. salinity. The (Na(+), K(+))-ATPase redistributes into membrane fractions of different densities, suggesting that altered membrane composition results from salinity acclimation. ATP was hydrolyzed at maximum rates of 182.6 +/- 7.1 nmol Pi min(-1) mg(-1) (21 parts per thousand) and 76.2 +/- 3.5 nmol Pi min(-1) mg(-1) (33 parts per thousand), with little change in K(M) values (approximate to 50 mu mol L(-1)). K(+) together with NH(4)(+) synergistically stimulated activity to maximum rates of approximate to 240 nmol Pi min(-1) mg(-1). K, values for ouabain inhibition (approximate to 110 mu mol L(-1)) decreased to 44.9 +/- 1.0 mu mol L(-1) (21 parts per thousand) and 28.8 +/- 1.3 mu mol L(-1) (33 parts per thousand) in the presence of both K(+) and NH(4)(+). Assays employing various inhibitors suggest the presence of mitochondrial F(0)F(1)- and K(+)- and V-ATPase activities in the gill microsomes. (C) 2009 Elsevier Inc. All rights reserved.

Regulation of conditioned and unconditioned fear in rats by 5-HT1A receptors in the dorsal periaqueductal gray

Studies on the involvement of 5-HT1-mediated mechanisms in the dorsal periaqueductal gray (dPAG) of animals with past stressful experiences have not been conducted so far. We investigated the role of 5-HT1 receptors in the dPAG of rats previously submitted to contextual fear conditioning. Defensive behaviors induced by activation of the dPAG were assessed by measuring the lowest electric current applied to this structure (threshold) able to produce freezing and escape responses during testing sessions of contextual fear conditioning, in which animals were placed in a context previously paired to footshocks. The 5-HT1A function of the dPAG was evaluated by local injections of 8-OH-DPAT (4 and 8 nmol/0.2 mu L) and WAY-100635 (10 nmol/0.2 mu L), selective agonist and antagonist of 5-HT1A receptors, respectively. In accordance with previous studies, 8-OH-DPAT increased aversive thresholds (antiaversive effects) but injections of WAY 100635 into the dPAG did not produce significant effects on the aversive thresholds in naive rats. However, the aversive thresholds of animals exhibiting contextual fear remained unchanged with both treatments. Moreover, 8-OH-DPAT and WAY 100635 did not change the dPAG post-stimulation freezing. The present results suggest that the stressful experience of being fear conditioned has an effect on the role of the 5-HT1A receptors in mediating unconditioned fear. Also, the reduction in the regulation of the defensive behaviors by 5-HT1A-mediated mechanisms in the dPAG of these animals may underlie the stress precipitated psychopathology associated with the neural substrates of aversion of the dPAG. (c) 2007 Elsevier Inc. All rights reserved.

GABAergic REGULATION OF AUDITORY SENSORY GATING IN LOW- AND HIGH-ANXIETY RATS SUBMITTED TO A FEAR CONDITIONING PROCEDURE

The inferior colliculus (IC) is primarily involved in the processing of acoustic stimuli, being in a position to send auditory information to motor centers that participate in behaviors such as prey catching and predators` avoidance The role of the central nucleus of the IC (CIC) on fear and anxiety has been suggested on the basis that rats are able to engage in tasks to decrease the aversiveness of CIC stimulation, increased Fos immunolabeling during diverse aversive states and increased CIC auditory evoked potentials (AEP) induced by conditioned fear stimuli Additionally it was shown that brainstem AEP, represented by wave V, for which the main generator is the IC, is increased during experimentally induced anxiety Rats segregated according to their low or high emotional reactivity have been used as an important tool in the study of fear and anxiety The IC contains a high density of GABA receptors Since the efficacy of an anxiolytic compound is a function of the animal`s anxiety level, it is possible that GABA-benzodiazepine (Bzp) agents affect LA and HA animals differently In this study we investigated the GABA-Bzp influence on the modulation of AEP in rats with low (LA) or high-anxiety (HA) levels, as assessed by the elevated plus maze test (EPM) GABA-Bzp modulation on the unconditioned AEP response was analyzed by using intra CIC injections (0 2 mu l) of the GABA-Bzp agonists muscimol (121 ng) and diazepam (30 mu g) or the GABA inhibitors bicuculline (10 ng) and semicarbazide (7 mu g) In a second experiment, we evaluate the effects of contextual aversive conditioning on AEP using foot shocks as unconditioned stimuli On the unconditioned fear paradigm GABA inhibition in creased AEP in LA rats and decreases this measure in HA counterparts Muscimol was effective in reducing AEP in both LA and HA rats Contextual fear stimuli increased the magnitude of AEP In spite of no effect obtained with diazepam in LA rats the drug inhibited AEP in HA animals The specificity of the regulatory mechanisms mediated by GABA Bzp for the ascending neurocircuits responsible for the acquisition of aversive information in LA and HA animals shed light on the processing of sensory information underlying the generation of defensive reactions (C) 2010 IBRO Published by Elsevier Ltd All rights reserved

Immunoexpression of adrenergic receptors in detrusor from patients with prune belly syndrome: a digital quantification

Introduction: Prune belly syndrome (PBS) presents with large-capacity bladders, high compliance and post-void residual volumes. Operative and conservative treatments are controversial. When histologically compared to normal bladder, bladder outlet obstruction results in an up- or down-regulation of adrenoceptors. Our goal was to study the immunoexpression of adrenoceptors in detrusor from patients with PBS. Materials and methods: Bladder domes from PBS patients (n = 14) were studied (PBG). For normal controls, bladder specimens were obtained at adult surgery (n = 13) (CG1) and at child autopsy (n = 5) (CG2). Staining was performed using antibodies to alpha 1a, alpha 1b, alpha 1d and beta 3 adrenoceptors. Five to 10 images were captured on an optic microscope with a digital camera and analysed with Photoshop(R). The immunocyhistochemical index with arbitrary units was calculated and compared. Results: Mean age was 1.28, 64 and 1.41 years for PBG, CG1 and CG2, respectively. The immunohistochemical index with arbitrary units of alpha 1a receptors was 0.06 in PBG, 0.16 in CG1 and 0.14 in CG2 (p = 0.008); of alpha 1b 0.06, 0.06 and 0.07 (p = 0.781); and of alpha 1d 0.04, 0.04 and 0.05 (p = 0.618). Regarding beta 3 the respective values were 0.07, 0.14 and 0.10 (p = 0.378). Conclusion: Our results show a decrease in ala-adrenoceptor immunostaining intensity in detrusor from children with PBS. Further in vitro studies are needed to determine whether these observations are physiologically significant. (C) 2009 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

Extra-curricular involvement and self-regulation in children

The research investigated the relationship between extra-curricular involvement (ECI) and self-regulated behaviours in 8 to 9 year old children, and identified sex, location, and socio-economic status (SES) differences in their ECI and self-regulatory behaviours. 550 children from 44 schools in Queensland and New South Wales completed the Child Self-Regulatory Process Inventory and questions about their ECI. Nearly 90 percent of students were involved in at least one extra-curricular activity with the mean number of activities being 1.27. Girls and urban children were significantly more involved in school-based extra- curricular activities than their male and rural counterparts; there were no significant differences among SES groups. Urban children and children in the high SES group reported significantly greater involvement for non-school based activities. For the three self- regulation strategies, girls scored significantly higher than boys. Moreover, children in the high ECI group reported significantly greater use of self-regulation strategies than children in both the low and medium ECI groups. Implications of findings are discussed in light of the need for quality extra-curricular programs, especially in terms of emotional climate and self-directed activities.

Ion and urea regulation in elasmobranch fish

In recent years our understanding of the control of ion and urea metabolism in elasmobranch fish has increased with many more species being investigated. This has demonstrated that many species regarded as stenohaline marine are at least, partially euryhaline and may survive in environments less concentrated than full seawater. This presentation will review these recent findings and then compare the osmoregulatory strategies of a partially euryhaline species, Scyliorhinus canicula, with a fully euryhaline migratory species Carcharinus leucas. This will include new data for both species and will generate new models for the control of ion and urea metabolism in elasmobranchs on which to base future research.

Price regulation in Australia: How consistent has it been?

We assemble a database consisting of 52 regulatory decisions made by seven different regulators across five different industries. We examine how the proportion of firms' revenue requirements that were disallowed by the regulator vary by regulator, industry and time. Despite the differences in the implementation of price regulation across industries and across jurisdictions in Australia, outcomes are surprisingly consistent. For example, we show that it is not possible to reject the hypothesis that the regulators outcomes in South Australia, New South Wales, the Australian Capital Territory and Victoria are similar despite the different regulatory approaches undertaken in these jurisdictions.

Regulation of CSF-1 receptor expression

Cells of the mononuclear phagocyte lineage possess receptors for macrophage colony-stimulating factor (CSF-1) encoded by the c-fms protooncogene and respond to CSF-1 with increased survival, growth, differentiation, and reversible changes in function. The c-fms gene is itself a macrophage differentiation marker. In whole mount analyses of mRNA expression in embryos, c-fms is expressed at very high levels on placental trophoblasts. It is detectable on individual cells in the yolk sac around 8.5 to 9 days postcoitus, appears on isolated cells in the head of the embryo around 9.5 dpc, and appears on numerous cells throughout the embryo by day 10.5. The extent of c-fms expression is much greater than for other macrophage-specific genes including lysozyme and a macrophage-specific protein tyrosine phosphatase. Our studies of the cis-acting elements of the c-fms promoter have indicated a key role for collaboration between the macrophage-specific transcription factor, Pu.1, which functions in determining the site of transcription initiation, and other members of the Ets transcription factor family. This is emerging as a common pattern in macrophage-specific promoters. We have shown that two PU box elements alone can function as a macrophage-specific promoter. The activity of both the artifical promoter and the c-fms promoter is activated synergistically by coexpression of Pu.1 and another Ets factor, c-Ets-2. A 3.5kb c-fms exon 2 promoter (but not the 300bp proximal promoter) is also active in a wide diversity of tumor cell lines. The interesting exception is the melanoma cell line K1735, in which the promoter is completely shut down and expression of c-fms causes growth arrest and cell death. The activity of the exon 2 promoter in these nonmacrophages is at least as serum responsive as the classic serum-responsive promoter of the c-fos gene. It is further inducible in nonmacrophages by coexpression of the c-fms product. Unlike other CSF-1/c-fms-responsive promoters, the c-fms promoter is not responsive to activated Ras even when c-Ets-2 is coexpressed. In most lines, production of full length c-fms is prevented by a downstream intronic terminator, but in Lewis lung carcinoma, read-through does occur, and expression of both c-fms and other macrophage-specific genes such as lysozyme and urokinase becomes detectable in conditions of serum deprivation. (C) 1997 Wiley-Liss, Inc.