Snake scales record environmental metal(loid) contamination.

Wetland snakes, as top predators, are becoming globally recognised as bioindicators of wetland contamination. Livers are the traditional test organ for contaminant exposure in organisms, but research is moving towards a preference for non-lethal tissue sampling. Snake scales can be used as an indicator of exposure, as many metals bind to the keratin. We used laser ablation with inductively coupled plasma-atomic emission spectroscopy and mass spectrometry (LA-ICP-MS) to quantify the concentrations of 19 metals and metalloids (collectively referred to 'metals' hereafter) in Western tiger snake (Notechis scutatus occidentalis) scales from four wetlands along an urban gradient, and compared them to concentrations measured in captive tiger snake scales. We conducted repeat measures to determine the concentration accuracy of each metal using LA-ICP-MS. Concentrations in wild Western tiger snake scales were significantly higher than in reference tiger snake scales for most metals analysed, suggesting accumulation from environmental exposure. We compared the scale concentrations to sediment concentrations of sampled wetlands, and found inter-site differences between mean concentrations of metals in scales parallel patterns recorded from sediment. Four metals (Mn, As, Se, Sb) had strong positive correlations with liver tissue contents suggesting scale concentrations can be used to infer internal concentrations. By screening for a larger suite of metals than we could using traditional digestive methods, we identified additional metals (Ti, V, Sr, Cs, Tl, Th, U) that may be accumulating to levels of concern in tiger snakes in Perth, Western Australia. This research has progressed the use of LA-ICP-MS for quantifying a suite of metals available in snake scales, and highlights the significance of using wetland snake scales as a non-lethal indicator of environmental contamination.

Identification of protein kinase C phosphorylation sites within the AMPA receptor GluR2 subunit.

Phosphorylation of AMPA receptor subunits is believed to regulate channel function and synaptic plasticity. Extensive biochemical and molecular studies have identified sites of PKA, PKC and CamKII phosphorylation in the C-termini of the GluR1 and 4 subunits. Recent studies have shown GluR1 phosphorylation to be bidirectionally altered during long-term potentiation (LTP) and long-term depression (LTD) in the hippocampus. The majority of AMPA receptors in the brain are believed to contain the GluR2 subunit that also contains potential sites for protein phosphorylation. Here we characterize PKC phosphorylation on the GluR2 subunit using biochemical and molecular techniques. Site-directed mutagenesis confirmed that this phosphorylation occurs on Serine 863 and Serine 880 of the GluR2 subunit C-terminus. Site identification allowed the generation of phosphorylation site-specific antibodies to facilitate the examination of GluR2 modification in primary neuronal culture. These studies confirmed that GluR2 is modified in response to the activation of PKC and suggests that phosphorylation of the ubiquitous GluR2 subunit may be important in the regulation of excitatory synaptic transmission.

The evolutionary history of the coral genus Acropora (Scleractinia, Cnidaria) based on a mitochondrial and a nuclear marker: reticulation, incomplete lineage sorting, or morphological convergence?

This study examines molecular relationships across a wide range of species in the mass spawning scleractinian coral genus Acropora. Molecular phylogenies were obtained for 28 species using DNA sequence analyses of two independent markers, a nuclear intron and the mtDNA putative control region. Although the compositions of the major clades in the phylogenies based on these two markers were similar, there were several important differences. This, in combination with the fact that many species were not monophyletic, suggests either that introgressive hybridization is occurring or that lineage sorting is incomplete. The molecular tree topologies bear little similarity to the results of a recent cladistic analysis based on skeletal morphology and are at odds with the fossil record. We hypothesize that these conflicting results may be due to the same morphology having evolved independently more than once in Acropora and/or the occurrence of extensive interspecific hybridization and introgression in combination with morphology being determined by a small number of genes. Our results indicate that many Acropora species belong to a species complex or syngameon and that morphology has little predictive value with regard to syngameon composition. Morphological species in the genus often do not correspond to genetically distinct evolutionary units. Instead, species that differ in timing of gamete release tend to constitute genetically distinct clades.

GABAA receptor phosphorylation and functional modulation in cortical neurons by a protein kinase C-dependent pathway.

GABA(A) receptors are critical mediators of fast synaptic inhibition in the brain, and the predominant receptor subtype in the central nervous system is believed to be a pentamer composed of alpha, beta, and gamma subunits. Previous studies on recombinant receptors have shown that protein kinase C (PKC) and PKA directly phosphorylate intracellular serine residues within the receptor beta subunit and modulate receptor function. However, the relevance of this regulation for neuronal receptors remains poorly characterized. To address this critical issue, we have studied phosphorylation and functional modulation of GABA(A) receptors in cultured cortical neurons. Here we show that the neuronal beta3 subunit is basally phosphorylated on serine residues by a PKC-dependent pathway. PKC inhibitors abolish basal phosphorylation, increasing receptor activity, whereas activators of PKC enhance beta3 phosphorylation with a concomitant decrease in receptor activity. PKA activators were shown to increase the phosphorylation of the beta3 subunit only in the presence of PKC inhibitors. We also show that the main sites of phosphorylation within the neuronal beta3 subunit are likely to include Ser-408 and Ser-409, residues that are important for the functional modulation of beta3-containing recombinant receptors. Furthermore, PKC activation did not change the total number of GABA(A) receptors in the plasma membrane, suggesting that the effects of PKC activation are on the gating or conductance of the channel. Together, these results illustrate that cell-signaling pathways that activate PKC may have profound effects on the efficacy of synaptic inhibition by directly modulating GABA(A) receptor function.

Inhibition of NADPH-cytochrome P450 reductase and glyceryl trinitrate biotransformation by diphenyleneiodonium sulfate.

We reported previously that the flavoprotein inhibitor diphenyleneiodonium sulfate (DPI) irreversibly inhibited the metabolic activation of glyceryl trinitrate (GTN) in isolated aorta, possibly through inhibition of vascular NADPH-cytochrome P450 reductase (CPR). We report that the content of CPR represents 0.03 to 0.1% of aortic microsomal protein and that DPI caused a concentration- and time-dependent inhibition of purified cDNA-expressed rat liver CPR and of aortic and hepatic microsomal NADPH-cytochrome c reductase activity. Purified CPR incubated with NADPH and GTN under anaerobic, but not aerobic conditions formed the GTN metabolites glyceryl-1,3-dinitrate (1,3-GDN) and glyceryl-1,2-dinitrate (1,2-GDN). GTN biotransformation by purified CPR and by aortic and hepatic microsomes was inhibited > 90% after treatment with DPI and NADPH. DPI treatment also inhibited the production of activators of guanylyl cyclase formed by hepatic microsomes. We also tested the effect of DPI on the hemodynamic-pharmacokinetic properties of GTN in conscious rats. Pretreatment with DPI (2 mg/kg) significantly inhibited the blood pressure lowering effect of GTN and inhibited the initial appearance of 1,2-GDN (1-5 min) and the clearance of 1,3-GDN. These data suggest that the rapid initial formation of 1,2-GDN is related to mechanism-based GTN biotransformation and to enzyme systems sensitive to DPI inhibition. We conclude that vascular CPR is a site of action for the inhibition by DPI of the metabolic activation of GTN, and that vascular CPR is a novel site of GTN biotransformation that should be considered when investigating the mechanism of GTN action in vascular tissue.

Adjacent phosphorylation sites on GABAA receptor beta subunits determine regulation by cAMP-dependent protein kinase.

Activation of cAMP-dependent protein kinase (PKA) can enhance or reduce the function of neuronal GABAA receptors, the major sites of fast synaptic inhibition in the brain. This differential regulation depends on PKA-induced phosphorylation of adjacent conserved sites in the receptor beta subunits. Phosphorylation of beta 3 subunit-containing receptors at S408 and S409 enhanced the GABA-activated response, whereas selectively mutating S408 to alanine converted the potentiation into an inhibition, comparable to that of beta 1 subunits, which are phosphorylated solely on S409. These distinct modes of regulation were interconvertible between beta 1 and beta 3 subunits and depended upon the presence of S408 in either subunit. In contrast, beta 2 subunit-containing receptors were not phosphorylated or affected by PKA. Differential regulation by PKA of postsynaptic GABAA receptors containing different beta subunits may have profound effects on neuronal excitability.

An investigation on the influence of feline flea allergy on the fecundity of the cat flea.

Survival, egg production and egg viability of fleas allowed to feed on eight flea-allergic and six flea-naive cats were compared. Fifty fleas, in feeding cages or free roaming, were allowed to feed on the cats. Flea-allergic cats removed significantly more free-roaming fleas than did flea-naive cats. Female fleas produced fewer eggs on flea-allergic cats than on flea-naive cats regardless of feeding method. There was no significant difference in egg viability between the two groups. The data suggest that flea-allergic cats efficiently removed fleas by grooming and that they also produce unknown factor(s) that affect the fecundity of fleas.

Identification of a Zn2+ binding site on the murine GABAA receptor complex: dependence on the second transmembrane domain of beta subunits.

1. Whole-cell currents were recorded from Xenopus laevis oocytes expressing wild-type and mutant recombinant GABAA receptors to locate a binding site for Zn2+ ions in the beta 3 subunit. 2. The Cl(-)-selective current, spontaneously gated by beta 3 subunit homomers, was enhanced by pentobarbitone and inhibited by picrotoxinin. The potencies of these agents were minimally affected by mutating histidine (H) 292 to alanine (A) in the second transmembrane domain (TM2). 3. Zn2+ inhibited the beta 3 subunit-gated conductance (IC50, 0.31 microM); the inhibition was voltage insensitive. The H292A mutation in beta 3 subunits caused a 1000-fold reduction in Zn2+ potency (IC50, 307 microM). 4. GABA-activated responses recorded from heteromeric alpha 1 beta 3 GABAA receptors were also inhibited by Zn2+ (IC50, 0.11 microM). This inhibition was reduced by mutating H292A in the beta 3 subunit (IC50, 22.8 microM). 5. H292 in TM2 of the beta 3 subunit is an important determinant of a Zn2+ binding site on the GABAA receptor. Its location in the presumed ion channel lining suggests that Zn2+ can penetrate into an anion-selective channel and that the ionic selectivity filter and channel gate are located beyond H292.

Conserved phosphorylation of the intracellular domains of GABA(A) receptor beta2 and beta3 subunits by cAMP-dependent protein kinase, cGMP-dependent protein kinase protein kinase C and Ca2+/calmodulin type II-dependent protein kinase.

All mammalian GABA(A) receptor beta subunits contain a conserved consensus site for phosphorylation by a number of serine/threonine protein kinases. This site corresponds to Serine 410 of the beta2 subunit and Serine 409 of the beta3 subunit, each of which lies within the conserved sequence R-R-R-X-S-L-Q-K, where X = A (beta1, beta2 and beta4) or S (beta3). We have analysed the phosphorylation of the beta2 and beta3 subunits of the murine GABA(A) receptor by expressing the large intracellular domains of these subunits as soluble fusion proteins in E. coli. The intracellular domain of the beta2 subunit was phosphorylated to high stoichiometry by both cAMP- and cGMP-dependent protein kinases, protein kinase C and Ca2+/calmodulin type II-dependent protein kinase in vitro. Site-directed mutagenesis identified Serine 410 as the single site within the beta2 subunit phosphorylated by these four protein kinases. Using similar methodologies, Serine 409 of the beta3 subunit was shown to be a substrate for phosphorylation by these protein kinases. Serine 408 was also seen to be phosphorylated by protein kinase C and Serine 383 was phosphorylated by Ca2+/calmodulin type II-dependent protein kinase. Since beta subunits are believed to be essential for robust GABA(A) receptor expression, these results suggest a critical role for conserved phosphorylated amino acids within the beta subunits in coordinating cellular regulation of GABA(A) receptors via multiple protein kinases.

Assembly and cell surface expression of heteromeric and homomeric gamma-aminobutyric acid type A receptors.

The ability of differing subunit combinations of gamma-aminobutyric acid type A (GABAA) receptors produced from murine alpha 1, beta 2, and gamma 2L subunits to form functional cell surface receptors was analyzed in both A293 cells and Xenopus oocytes using a combination of molecular, electrophysiological, biochemical, and morphological approaches. The results revealed that GABAA receptor assembly occurred within the endoplasmic reticulum and involved the interaction with the chaperone molecules immunoglobulin heavy chain binding protein and calnexin. Despite all three subunits possessing the ability to oligomerize with each other, only alpha 1 beta 2 and alpha 1 beta 2 gamma 2L subunit combinations could produce functional surface expression in a process that was not dependent on N-linked glycosylation. Single subunits and the alpha 1 gamma 2L and beta 2 gamma 2L combinations were retained within the endoplasmic reticulum. These results suggest that receptor assembly occurs by defined pathways, which may serve to limit the diversity of GABAA receptors that exist on the surface of neurons.

Phosphorylation of the predicted major intracellular domains of the rat and chick neuronal nicotinic acetylcholine receptor alpha 7 subunit by cAMP-dependent protein kinase.

The predicted major intracellular domains of the chick and rat neuronal nicotinic acetylcholine receptor alpha 7 subunits were expressed in E. coli as glutathione-S-transferase fusion proteins. These proteins were then purified to near homogeneity by chromatography on immobilized glutathione. The intracellular domains of the alpha 7 subunit from both species were phosphorylated to high stoichiometry by cAMP-dependent protein kinase, but not by protein kinase C, cGMP-dependent protein kinase, or calcium/calmodulin-dependent protein kinase. Phosphorylation occurred on serine residues only within an identical single tryptic peptide for both proteins. This conserved phosphorylation site was identified as Ser 342 utilizing site-directed mutagenesis. These results demonstrate that the intracellular domain of the alpha 7 subunit is a substrate of PKA, and suggest a role for protein phosphorylation in mediating cellular regulation upon neuronal AChRs containing this subunit.

Platelet-derived growth factor receptor is a novel modulator of type A gamma-aminobutyric acid-gated ion channels.

Platelet-derived growth factor (PDGF) and PDGF receptors (PDGFRs) are ubiquitously expressed in the mammalian central nervous system, where they exert trophic actions on both neuronal and glial cells. However, the acute actions of PDGF on synaptic transmission are unknown. We report a novel regulatory action of PDGF/PDGFR. Activation of PDGFRs inhibited the function of native type A gamma-aminobutyric acid (GABAA) receptors (GABAA-RS) in rat hippocampal CA1 pyramidal neurons and mouse brain membrane vesicles. The mechanism of this inhibition was studied with a panel of mutant PDGFRS-beta coexpressed with cloned human GABAA-Rs in Xenopus oocytes. These experiments revealed that phospholipase C-gamma is the protein that relays the inhibitory signal from PDGFRS to GABAA-Rs. Experiments with microinjected EGTA and inositol-1, 3, 4-triphosphate demonstrated that inhibition of GABAA-Rs depended on a phospholipase C-gamma-mediated increase in intracellular Ca(2+)-levels. The PDGFR-induced inhibitory effect was independent of the subunit composition of GABAA-RS. Moreover, GABAA-RS composed of alpha 1 beta 1 S409A subunits, which do not contain any known protein kinase C phosphorylation sites, were inhibited by PDGF to the same extent as wild-type GABAA-RS. Inhibitors of protein kinase C, CA2+/calmodulin-dependent protein kinase II, calcineurin, and tyrosine phosphatases did not affect the modulatory actions of PDGFR. In conclusion, our results suggest that PDGFRs exert potent modulatory actions on GABAA-R-dependent inhibitory synaptic transmission. These regulatory actions of PDGF could play important roles in the function of the mammalian central nervous system during physiological and pathophysiological conditions.

Improving care through a medication administration process action team.

The article discusses the use of a process action team to improve the medication administration system. The interdisciplinary team suggests alternatives after reviewing current practice, systems, and procedures. After suggested alternatives are applied, the medication dispensing and administration system improves and enhances the delivery of medications to patients. This opportunity to improve care explains the use of the process action team in patient care system improvement.

Effect of dexamethasone treatment on the biotransformation of glyceryl trinitrate: cytochrome P450 3A1 mediated activation of rat aortic guanylyl cyclase by glyceryl trinitrate.

It is generally accepted that organic nitrates act via vascular biotransformation to an activator of guanylyl cyclase (presumably NO), resulting in increased cyclic GMP accumulation and vascular smooth muscle relaxation. Previously, we have shown that cytochrome P450 can mediate the biotransformation of glyceryl trinitrate (GTN) and that at least a portion of this biotransformation results in the formation of an activator of guanylyl cyclase. To assess the role of the cytochrome P450 3A subfamily in this phenomenon, we treated male and female rats with dexamethasone (DEX) (150 mg/kg, i.p., daily for 3 days). Under anerobic conditions, hepatic microsomal biotransformation of GTN was increased three-fold in DEX-treated male rats compared with all other treatment groups. Incubation of aortic 100,000 x g supernatant fraction from untreated rats (as a source of guanylyl cyclase) with GTN and hepatic microsomes from all groups resulted in concentration-dependent increases in guanylyl cyclase activation. Microsomes from DEX-treated male and female rats demonstrated a significantly greater activation of guanylyl cyclase compared with microsomes from untreated males and females. Furthermore, GTN-induced guanylyl cyclase activation mediated by microsomes from DEX-treated male and female rats was markedly inhibited by a polyclonal antibody raised to rat CYP3A1. Since CYP3A2 is absent or very low in hepatic microsomes from DEX-treated adult female rats, this identifies CYP3A1 as an isoform capable of biotransforming GTN to an activator of guanylyl cyclase. Similarly, CYP2C11 was identified as an isoform capable of biotransforming GTN to an activator of guanylyl cyclase, since monoclonal antibody to CYP2C11 inhibited GTN-induced activation of guanylyl cyclase mediated by microsomes from control male rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential phosphorylation of intracellular domains of gamma-aminobutyric acid type A receptor subunits by calcium/calmodulin type 2-dependent protein kinase and cGMP-dependent protein kinase.

gamma-Aminobutyric acid type A receptor subunits (GABAA) can be divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cGMP-dependent protein kinase (PKG) and multifunctional calcium/calmodulin-dependent protein kinase (CAM KII). Both PKG and CAM KII phosphorylated a purified beta 1 subunit fusion. Both of these kinases phosphorylated serine 409 within the beta 1 subunit; in addition, CAM KII also phosphorylated serine 384 as determined by site-specific mutagenesis. Fusion proteins of the major intracellular domains of the gamma 2S and gamma 2L subunits were produced. These proteins differ by 8 amino acids (LLRMFSFK). Both the gamma 2L and gamma 2S fusion proteins were excellent substrates of CAM KII. However, the gamma 2L fusion protein was phosphorylated to higher stoichiometry due to the phosphorylation of serine 343 within this 8-amino acid insertion. Both the gamma 2L and gamma 2S subunits were phosphorylated on common residues by CAM KII identified as serine 348 and threonine 350. These results identify specific sites of phosphorylation for CAM KII and PKG within GABAA receptor subunits, suggesting a role for these two kinases in modulating GABAA receptor function in vivo.

Biotransformation of organic nitrates and vascular smooth muscle cell function.

The organic nitrates are interesting examples of drugs that undergo biotransformation at their site of action to generate the active form of the drug. Furthermore, tolerance to the vasodilator effects of organic nitrates is associated with impairment of this metabolic activation process. Despite considerable research effort, the intracellular processes and the chemical reaction pathways by which organic nitrates are converted to their active form are still unresolved. This review by Brian Bennett and colleagues summarizes the characteristics of organic-nitrate biotransformation in vascular smooth muscle, the difficulties encountered when assessing this biotransformation, and the evidence for the role of two identified vascular biotransformation systems (glutathione-S-transferases and the cytochrome P450 system) in the metabolic activation of organic nitrates.

Biotransformation of glyceryl trinitrate by rat aortic cytochrome P450.

Denitration of glyceryl trinitrate (GTN) by the microsomal fraction of rat aorta was found to be NADPH dependent and followed apparent first-order kinetics (T1/2 70.1 min). Biotransformation of GTN was regioselective for glyceryl-1,2-dinitrate formation, and was inhibited by carbon monoxide, SKF-525A, and oxygen. In aortic microsomes prepared from phenobarbital-pretreated rats, biotransformation was increased 7-fold, and was regioselective for glyceryl-1,3-dinitrate formation. These data strongly suggest the involvement of aortic cytochrome P450 in the biotransformation of GTN.

Inhibition of nitrovasodilator- and acetylcholine-induced relaxation and cyclic GMP accumulation by the cytochrome P-450 substrate, 7-ethoxyresorufin.

We examined the effect of the cytochrome P-450 substrate, 7-ethoxyresorufin (7-ER), and its corresponding product, resorufin, on nitrovasodilator- and endothelium-dependent relaxation of isolated rat aorta. The EC50 value for glyceryl trinitrate (GTN) induced relaxation was increased over 100-fold by 7-ER and less than 3-fold by resorufin. The EC50 value for sodium nitroprusside (SNP) induced relaxation was increased approximately 12-fold by 7-ER, acetylcholine (ACh) induced relaxation was abolished, and relaxation induced by isopropylnorepinephrine was not significantly affected. GTN-, SNP-, and ACh-induced increases in cyclic GMP accumulation were inhibited by 7-ER, as were basal cyclic GMP levels in endothelium-intact, but not endothelium-denuded tissues. 7-ER decreased GTN biotransformation in intact aorta and decreased the regioselective formation of glyceryl-1,2-dinitrate. The activation by GTN and SNP of aortic guanylyl cyclase in broken cell preparations was not affected by 7-ER, indicating that the inhibitory effect of 7-ER is probably not due to a direct interaction with guanylyl cyclase. The inhibitory effect of 7-ER on GTN-induced relaxation was not altered by the addition of superoxide dismutase, suggesting that 7-ER does not act by increasing superoxide anion concentration (which would serve to increase the degradation of nitric oxide (NO) formed during vascular GTN biotransformation). Our data provide further evidence for the role of the cytochrome P-450--cytochrome P-450 reductase system in the biotransformation of GTN to an activator (presumably nitric oxide) of guanylyl cyclase. The data are consistent with a mode of action of 7-ER involving either competitive inhibition of vascular cytochrome P-450 or uncoupling of vascular cytochrome P-450 reductase from cytochrome P-450. The data also suggest that the cytochrome P-450 system facilitates NO release from SNP and that 7-ER has an inhibitory effect on endothelial nitric oxide synthase.

Effect of immunization against melatonin on seasonal fleece growth in feral goats.

Four vaccination protocols were utilized to investigate the effects of immunoneutralizing circulating melatonin on the annual cashmere growth cycle and cashmere production in Australian feral goats. A fluctuating anti-melatonin antibody response, achieved by repeated booster vaccinations, resulted in an acceleration of the growth cycle in goats which exhibited a significant immune response, compared to sham-immunized controls. Responding goats showed two cycles of cashmere length growth in the first 16 months and increased annual cashmere production in the first year. However, in the second year, these effects were no longer apparent, suggesting either some form of desensitization to melatonin, or a diminished response due to declining antibody titre. The effects of immunization were observed in both sexes; the effect on cashmere length was greater in wethers than in does. Cashmere fibre growth in response to a continuously declining plane of specific antibody showed increased cycle frequency, albeit with a decreased amplitude; guard hair growth cycles were affected to a much lesser extent. Small transient peaks of specific immunity at the summer or winter solstice were without significant effect on cashmere growth. Immunization to provoke a persistent anti-melatonin antibody response at the winter solstice resulted in significantly increased greasy fleece weight, % cashmere yield, and mass of cashmere produced, but no change in fibre diameter in both sexes. Thus the timing of cashmere growth cycles in goats may be, at least transiently, altered by appropriately timed immunization against melatonin. The mechanism of pineal-mediated regulation of cashmere growth cycles may involve (i) entrainment of an endogenous rhythm by melatonin, or (ii) seasonal alteration of cashmere follicle sensitivity to the effect of melatonin.