Adrenomedullin Gene Delivery is Cardio-protective in a Model of Chronic Nitric Oxide Deficiency Combining Pressure Overload, Oxidative Stress and Cardiomyocyte Hypertrophy

BACKGROUND/AIMS:
Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of the vasodilator peptide, adrenomedullin (AM) and its receptors is augmented in cardiomyocytes, indicating that the myocardial AM system may be activated in response to pressure loading and ischemic insult to serve a counter-regulatory, cardio-protective role. The study examined the hypothesis that oxidative stress and hypertrophic remodeling in NO-deficient cardiomyocytes are attenuated by adenoviral vector-mediated delivery of the human adrenomedullin (hAM) gene in vivo.

METHODS:
The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 15mg . kg(-1) . day(-1)) was given to rats for 4 weeks following systemic administration via the tail vein of a single injection of either adenovirus harbouring hAM cDNA under the control of the cytomegalovirus promoter-enhancer (Ad.CMV-hAM-4F2), or for comparison, adenovirus alone (Ad.Null) or saline. Cardiomyocytes were subsequently isolated for assessment of the influence of each intervention on parameters of oxidative stress and hypertrophic remodelling.

RESULTS: Cardiomyocyte expression of the transgene persisted for > or =4 weeks following systemic administration of adenoviral vector. In L-NAME treated rats, relative to Ad.Null or saline administration, Ad.CMV-hAM-4F2 (i) reduced augmented cardiomyocyte membrane protein oxidation and mRNA expression of pro-oxidant (p22phox) and anti-oxidant (SOD-3, GPx) genes; (ii) attenuated increased cardiomyocyte width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP) genes; (iii) did not attenuate hypertension.

CONCLUSIONS: Adenoviral vector mediated delivery of hAM resulted in attenuation of myocardial oxidative stress and hypertrophic remodelling in the absence of blood pressure reduction in this model of chronic NO-deficiency. These findings are consistent with a direct cardio-protective action in the myocardium of locally-derived hAM which is not dependant on NO generation.

Nociception or pain in a decapod crustacean?

Nociception is the ability to perceive a noxious stimulus and react in a re flexive manner and occurs across a wide range of taxa. However, the ability to experience the associated aversive sensation and feeling, known as pain, is not widely accepted to occur in nonvertebrates. We examined the responses of a decapod crustacean, the prawn, Palaemon elegans, to different noxious stimuli applied to one antenna to assess reflex responses (nociception) and longer-term, specifically directed behavioural responses that might indicate pain. We also examined the effects of benzocaine, a local anaesthetic, on these responses. Noxious stimuli elicited an immediate reflex tail flick response, followed by two prolonged activities, grooming of the antenna and rubbing of the antenna against the side of the tank, with both activities directed specifically at the treated antenna. These responses were inhibited by benzocaine; however, benzocaine did not alter general swimming activity and thus the decline in grooming and rubbing is not due to general anaesthesia. Mechanical stimulation by pinching also resulted in prolonged rubbing, but this was not inhibited by benzocaine. These results indicate an awareness of the location of the noxious stimuli, and the prolonged complex responses indicate a central involvement in their organization. The inhibition by a local anaesthetic is similar to observations on vertebrates and is consistent with the idea that these crustaceans can experience pain.

Probing aggressive motivation in a cichlid fish

The duration of startles provides an inverse measure of motivation to resume the previous activity. Here, we use a novel method in which one convict cichlid fish (Amatitlania nigrofasciata) of a competing pair was startled independently of the opponent. Fish were given various opponents and the mean startle duration determined. This mean was negatively correlated with the mean use of highly escalated 'frontal activities' such as biting and frontal display, but not the less escalated lateral displays or tail beating. Thus the startle duration was a reliable surrogate measure of the most escalated components of aggressive interactions. That is, it provided a motivational probe for aggressiveness of individual fish. Fight motivation is often determined in terms of fight duration or physiological costs for losers, who reveal the costs they are prepared to pay. We discuss various potential advantages of the motivational probe over previous measures, particularly with respect to winners and losers and different times during the interactions.

Gender differences in aggressive behaviour in convict cichlids

Males and females of many species engage in agonistic encounters. However, differing selection pressures on each sex are predicted to result in sex differences in aggressive behaviour during contests. Comparing male and female intrasexual contests can yield intriguing differences, shedding light on the forces shaping the use of particular aggressive tactics. We investigated whether fundamental gender-related differences in aggression, not explained by current parental role, are present in convict cichlids, Amatitlania nigrofasciata. Intrasexual agonistic encounters between isolated males and between isolated females not previously paired to a breeding partner were staged. Using this approach we first tested for behavioural differences between the sexes. Second, using a novel startle technique aimed at probing motivation to fight, we tested for gender-related differences in aggressive motivation. Third, we examined whether size, rather than gender, plays a role in determining the tactics used during contests. In addressing these aims we found: (1) females used more frontal display and biting, and spent more time in close proximity to their opponent, whereas males used more lateral display and tail beating than females during agonistic encounters; (2) there was no difference in the response of male or female convict cichlids to a startling stimulus aimed at probing motivation to fight; and (3) the addition of focal weight and length as possible covariates had no significant effect on the analyses. Possible causal and functional reasons for these gender-related differences in fight tactics are discussed. (C) 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Peptide DV-28 amide: An inhibitor of bradykinin-induced arterial smooth muscle relaxation encoded by Bombina orientalis skin kininogen-2

Skin kininogens from bombinid toads encode an array of bradykinin-related peptides and one such kininogen from Bombina maxima also encodes the potent bradykinin B2-receptor antagonist, kinestatin. In order to determine if the skin secretion of the closely-related toad, Bombina orientalis, contained a bradykinin inhibitory peptide related to kinestatin, we screened reverse phase HPLC fractions of defensive skin secretion using a rat tail artery smooth muscle preparation. A fraction was located that inhibited bradykinin-induced relaxation of the preparation and this contained a peptide of 3198.5 Da as determined by MALDI-TOF MS. Automated Edman degradation of this peptide established the identity of a 28-mer as: DMYEIKGFKSAHGRPRVCPPGEQCPIWV, with a disulfide-bridge between Cys18 and Cys24 and an amidated C-terminal Val residue. Peptide DV-28 was found to correspond to residues 133–160 of skin pre-kininogen-2 of B. orientalis that also encodes two copies of (Thr6)-bradykinin. The C-terminal residue, Gly-161, of the precursor open-reading frame, acts as the C-terminal amide donor of mature DV-28. DV-28 amide thus represents a new class of bradykinin inhibitor peptide from amphibian skin secretion.

Peptide DV-28 amide: Prototype of a novel class of bradykinin receptor antagonist isolated from the defensive skin secretion of bombinid toads

Kinestatin, isolated from the skin of the Chinese toad, Bombina maxima, was the first bradykinin B2 receptor antagonist identified in amphibians. Molecular cloning established that it is co-encoded with the bradykinin-related peptide, maximakinin, within one of several skin kininogens. To examine other species within the genus Bombina for the presence of structural homologues of kinestatin, we subjected skin secretion of the toad, Bombina orientalis, to HPLC fractionation with subsequent bioassay of fractions for antagonism of bradykinin activity using an isolated rat tail artery smooth muscle preparation. A single fraction was located that inhibited bradykinin-induced relaxation of rat arterial smooth muscle and MALDI-TOF analysis of this fraction revealed that it contained a single peptide of molecular mass 3198.5 Da. Further primary structural analysis of this peptide showed that it was a 28-mer with an N-terminal Asp (D) residue and a C-terminal Val (V) residue that was amidated. The peptide was named DV-28 amide in accordance with these primary structural attributes. Synthetic DV-28 amide replicated the observed bradykinin antagonistic effect within the smooth muscle bioassay in a dose-dependent manner. In addition, it was observed to inhibit the proliferation of human microvessel endothelial cells (HMECs) as assessed by MTT assay. Bioinformatic analysis revealed that DV-28 amide was, like kinestatin, co-encoded with a bradykinin receptor agonist on one of two skin kininogens identified in B. orientalis. DV-28 amide thus represents a novel class of bradykinin antagonist from skin secretions of bombinid toads that appear to be a rich source of such novel peptides.

Hylambatin and (Thr)11-hylambatin from the skin secretion of the African hyperoliid frog, Kassina maculata: Structural characterisation, precursor cDNA cloning and preliminary pharmacological testing

The tachykinins hylambatin and (Thr)11-hylambatin have been isolated from the defensive skin secretion of the African hyperoliid frog, Kassina maculata,. Hylambatin (DPPDPNRFYGMMamide) is revised in structure from the original sequence by a single site substitution (Asn/Asp at position 6), and (Thr)11-hylambatin, a novel tachykinin, differs in structure from hylambatin by a single Thr/Met substitution. (Thr)11-hylambatin is five- to ten-fold more abundant than hylambatin in secretions. Synthetic replicates of both peptides were active in smooth muscle preparations including the rat tail artery, rat ileum and bovine trachea. While hylambatin displayed activity consistent with an NK1-receptor ligand, (Thr)11-hylambatin was more active than either substance P or neurokinin A in both NK1- and NK-2 receptor rich preparations. Incorporation of a threoninyl residue rather than the canonical leucyl residue at the penultimate position in both substance P and neurokinin A, generated active ligands in both arterial and intestinal smooth muscle preparations. Hylambatin precursor cDNAs, designated HYBN-1 and HYBN-2, respectively, were cloned from a skin library by 3'- and 5'-RACE reactions. Both were highly-homologous containing open-reading frames of 66 amino acids encoding single copies of either hylambatin or (Thr)11-hylambatin. These data reveal a hitherto unrecognized structure/activity attribute of mammalian tachykinin receptors revealed though discovery of a novel amphibian skin-derived, site-substituted peptide ligand.

Helokinestatins: A novel family of bioactive peptides with drug-lead potential fromthe venomof the Gila Monster (Heloderma suspectum)

Venom of the Gila Monster (Heloderma suspectum) has proven to be an unlikely source of lead compounds (exendins) for the development of new injectable peptide therapeutics for the treatment of Type 2 diabetes. However, no systematic searches for new classes of bioactive peptides in lizard venom have appeared until recently. Here we describe the discovery of a new class of peptides – the helokinestatins – from H. suspectum venom, their structural characterisation and that of their biosynthetic precursors from cloned cDNA. In addition, we have subjected members of the family to preliminary pharmacological characterisation. Helokinestatins 1–6 are a family of proline-rich peptides containing 10–15 amino acid residues terminating in a common -Pro-Arg.OH motif. They are encoded in tandem within two virtually identical biosynthetic precursors of 177 and 178 amino acid residues, differing by only a single Pro residue. Each precursor also encodes a single copy of a C-type natriuretic peptide located at the C-terminus. Synthetic replicates of all helokinestatins were shown to be devoid of any direct action on the smooth muscle of rat tail artery but were found to be potent inhibitors of bradykinin-induced relaxation in this preparation in a manner that is suggestive of a non-competitive mechanism. Helokinestatin-3 (VPPPPLQMPLIPR) and helokinestatin-5 (VPPPLQMPLIPR) were found to be most potent in this respect causing almost complete inhibition of bradykinin-induced relaxation. Helokinestatins and BPPs may have a shared evolutionary history but the former do not inhibit ACE. The bradykinin inhibitory potential of helokinestatins may be exploited in the local control of chronic inflammation.

Evidence for a Direct and Functional Interaction between the Regulators of G Protein Signaling-2 and Phosphorylated C Terminus of Cholecystokinin-2 Receptor

Signaling of G protein-coupled receptors (GPCRs) is regulated by different mechanisms. One of these involves regulators of G protein signaling (RGS), which are diverse and multifunctional proteins that bind to active G alpha subunits of G proteins and act as GTPase-activating proteins. Little is known about the molecular mechanisms that govern the selective use of RGS proteins in living cells. We first demonstrated that CCK2R-mediated inositol phosphate production, known to be G(q-)dependent, is more sensitive to RGS2 than to RGS4 and is insensitive to RGS8. Both basal and agonist-stimulated activities of the CCK2R are regulated by RGS2. By combining biochemical functional, and in silico structural approaches, we demonstrate that a direct and functional interaction occurs between RGS2 and agonist-stimulated cholecystokinin receptor-2 (CCK2R) and identified the precise residues involved: phosphorylated Ser434 and Thr439 located in the C-terminal tail of CCK2R and Lys62, Lys63, and Gln67, located in the N-terminal domain of RGS2. These findings confirm previous reports that RGS proteins can interact with GPCRs to modulate their signaling and provide a molecular basis for RGS2 recognition by the CCK2R.

Induction and rejoining of DNA double-strand breaks in bladder tumor cells

The induction and rejoining of radiation-induced double-strand breaks (DSBs) in cells of six bladder tumor cell lines (T24, UM-UC3, TCC-SUP, RT112, J82, HT1376) were measured using the neutral comet assay. Radiation dose-response curves (0-60 Gy) showed damage (measured as mean tail moment) for five of the cell lines in the same rank order as cell survival (measured over 0-10 Gy), with the least damage in the most radioresistant cell line. Damage induction correlated well with clonogenic survival at high doses (SF10) for all six cell lines. At the clinically relevant dose of 2 Gy, correlation was good for four cell lines but poor for two (TCC-SUP and T24), The rejoining process had a fast and slow component for all cell lines. The rate of these two components of DNA repair did not correlate with cell survival. However, the time taken to reduce the amount of DNA damage to preirradiated control levels correlated positively with cell survival at 10 Gy but not 2 Gy; radioresistant cells rejoined the induced DSBs to preirradiation control levels more quickly than the radiosensitive cells. Although the results show good correlation between SF10 and DSBs for all six cell lines, the lack of correlation with SF2 for TCC-SUP and T24 cells would suggest that a predictive test should be carried out at the clinically relevant dose. At present the neutral comet assay cannot achieve this. (C) 2000 by Radiation Research Society.

Aerodynamic performance of a bypass engine with fan nozzle exit area change by warped chevrons

Variation of the bypass nozzle exit area enables optimization of the turbofan engine operating cycle over a wider range of operational conditions resulting in improved thrust and/or fuel consumption. Two mechanisms for varying the nozzle area have been investigated. The first uses an array of chevrons which when closed, form a full body of revolution and when warped/curved, increase the exit area while forming a serrated trailing edge. The second technique incorporates an axially translating section of the nacelle shroud and uses the change in the nozzle boat-tail radial location with the axial location as a means to vary the nozzle exit area. To analyse the effects on a typical rotor/stator stage, computational fluid dynamics simulations of the NASA Rotor 67, Stator 67A stage integrated into a custom-built nacelle were performed. Nozzles with 8, 12, and 16 chevrons were simulated to evaluate the impact of the variation in geometry upon the nacelle wake and local forces. Gross thrust of the nacelle and the turbulent kinetic energy (TKE) variation through the wake is compared. The chevron nozzle attains a nearly 2 per cent maximum thrust improvement over the translating nozzle technique. The chevron nozzle also has significantly lower (nearly 8 per cent) peak TKE levels in the jet plume.

Dynamic Density Forecasts for Multivariate Asset Returns

We propose a simple and flexible framework for forecasting the joint density of asset returns. The multinormal distribution is augmented with a polynomial in (time-varying) non-central co-moments of assets. We estimate the coefficients of the polynomial via the Method of Moments for a carefully selected set of co-moments. In an extensive empirical study, we compare the proposed model with a range of other models widely used in the literature. Employing a recently proposed as well as standard techniques to evaluate multivariate forecasts, we conclude that the augmented joint density provides highly accurate forecasts of the “negative tail” of the joint distribution.

Parasitic foraminifers on a deep-sea chiton (Mollusca, Polyplacophora, Leptochitonidae) from Iceland

Epibiotic foraminifers selectively settle on the most food-rich area of the host substrate, even when the species acts as a facultative ectoparasite in later life stages. In 398 specimens examined of the deep-sea chiton Leptochiton arcticus from Iceland, 46% show evidence of infestation by foraminifers, with many showing extensive shell damage from present and past bioeroding epibionts. Disturbances to the inner layer of the host shell are indicative of parasitism, as evidenced both by wound healing calcification and protrusions of the foraminiferan tubules. The epibionts employ different feeding strategies at different stages of their life cycle, taking advantage of nutrient availability from the posterior respiration currents and excrement of the chitons as juveniles, and feeding parasitically as adults. Epibiont persistence on individual hosts-through successive generations, or long-term continuous bioerosion by epibionts-allow larger adult parasitic foraminifers of Hyrrokkin sarcophaga to penetrate the thick tail valve of a chiton and feed parasitically on the host tissue. The proportion of chitons infested increases with host size, indicating that epibionts are accumulated through a chiton's life, seemingly without major detriment to host survivorship.

Tegumental surface changes in juvenile Fasciola hepatica in response to treatment in vivo with triclabendazole.

Eight indoor-reared crossbred sheep with no pre-exposure to Fasciola hepatica were infected, by oral gavage, with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. Anthelmintic dosing occurred at 4 weeks post-infection with 10 mg/kg triclabendazole. Two treated sheep were euthanized at 48 h, 72 h and 96 h post-treatment with triclabendazole. Two control sheep were euthanized alongside the 48 h triclabendazole-treated sheep. Juvenile flukes were recovered from each of the sheeps’ liver and processed for scanning electron microscopy (SEM).

Flukes were still active 48 h post-treatment and displayed limited morphological disruption. There was some blebbing and sloughing of the tegument around the oral sucker. In several of the specimens, an extra layer had been deposited on the fluke surface, giving it a flattened appearance. At 72 h post-treatment, only one fluke remained alive and the disruption varied in degree. In the majority of flukes, there was severe swelling of the tegument, accompanied by isolated areas of flattening along the lateral margins of the flukes and in the tail region. Limited areas of sloughing occurred in the tail region. In more seriously affected specimens, the syncytium had been stripped away to reveal the basal lamina and some deeper lesions were also observed. By 96 h post-treatment, all the flukes were dead and were grossly disrupted. They were totally devoid of tegument and deep lesions exposed the internal tissues of the fluke.

Jettisoning Ballast or Fuel? Caudal Autotomy and Locomotory Energetics of the Cape Dwarf Gecko Lygodactylus capensis (Gekkonidae)

Many lizard species will shed their tail as a defensive response (e.g., to escape a putative predator or aggressive conspecific). This caudal autotomy incurs a number of costs as a result of loss of the tail itself, loss of resources (i.e., stored in the tail or due to the cost of regeneration), and altered behavior. Few studies have examined the metabolic costs of caudal autotomy. A previous study demonstrated that geckos can move faster after tail loss as a result of reduced weight or friction with the substrate; however, there are no data for the effects of caudal autotomy on locomotory energetics. We examined the effect of tail loss on locomotory costs in the Cape dwarf gecko Lygodactylus capensis (similar to 0.9 g) using a novel method for collecting data on small lizards, a method previously used for arthropods. We measured CO2 production during 5-10 min of exhaustive exercise (in response to stimulus) and during a 45-min recovery period. During exercise, we measured speed (for each meter moved) as well as total distance traveled. Contrary to our expectations, tailless geckos overall expended less effort in escape running, moving both slower and for a shorter distance, compared with when they were intact. Tailless geckos also exhibited lower excess CO2 production (CO2 production in excess of normal resting metabolic rate) during exercising. This may be due to reduced metabolically active tissue (tails represent 8.7% of their initial body mass). An alternative suggestion is that a change in energy substrate use may take place after tail loss. This is an intriguing finding that warrants future biochemical investigation before we can predict the relative costs of tail loss that lizards might experience under natural conditions.