Systematic random sampling of the comet assay

The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory ‘tail’ DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the ‘randomness’ of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

Modelling the comet assay

The single-cell gel electrophoresis technique or comet assay is widely regarded as a quick and reliable method of analysing DNA damage in individual cells. It has a proven track record from the fields of biomonitoring to nutritional studies. The assay operates by subjecting cells that are fixed in agarose to high salt and detergent lysis, thus removing all the cellular content except the DNA. By relaxing the DNA in an alkaline buffer, strands containing breaks are released from supercoiling. Upon electrophoresis, these strands are pulled out into the agarose, forming a tail which, when stained with a fluorescent dye, can be analysed by fluorescence microscopy. The intensity of this tail reflects the amount of DNA damage sustained. Despite being such an established and widely used assay, there are still many aspects of the comet assay which are not fully understood. The present review looks at how the comet assay is being used, and highlights some of its limitations. The protocol itself varies among laboratories, so results from similar studies may vary. Given such discrepancies, it would be attractive to break the assay into components to generate a mathematical model to investigate specific parameters.

Rho kinase and protein kinase C involvement in vascular smooth muscle myofilament calcium sensitization in arteries from diabetic rats.

BACKGROUND AND PURPOSE: Diabetes mellitus (DM) causes multiple dysfunctions including circulatory disorders such as cardiomyopathy, angiopathy, atherosclerosis and arterial hypertension. Rho kinase (ROCK) and protein kinase C (PKC) regulate vascular smooth muscle (VSM) Ca(2+) sensitivity, thus enhancing VSM contraction, and up-regulation of both enzymes in DM is well known. We postulated that in DM, Ca(2+) sensitization occurs in diabetic arteries due to increased ROCK and/or PKC activity. EXPERIMENTAL APPROACH: Rats were rendered hyperglycaemic by i.p. injection of streptozotocin. Age-matched control tissues were used for comparison. Contractile responses to phenylephrine (Phe) and different Ca(2+) concentrations were recorded, respectively, from intact and chemically permeabilized vascular rings from aorta, tail and mesenteric arteries. KEY RESULTS: Diabetic tail and mesenteric arteries demonstrated markedly enhanced sensitivity to Phe while these changes were not observed in aorta. The ROCK inhibitor HA1077, but not the PKC inhibitor chelerythrine, caused significant reduction in sensitivity to agonist in diabetic vessels. Similar changes were observed for myofilament Ca(2+) sensitivity, which was again enhanced in DM in tail and mesenteric arteries, but not in aorta, and could be reduced by both the ROCK and PKC blockers. CONCLUSIONS AND IMPLICATIONS: We conclude that in DM enhanced myofilament Ca(2+) sensitivity is mainly manifested in muscular-type blood vessels and thus likely to contribute to the development of hypertension. Both PKC and, in particular, ROCK are involved in this phenomenon. This highlights their potential usefulness as drug targets in the pharmacological management of DM-associated vascular dysfunction.

Induction of Distinct Neurologic Disease Manifestations during Relapsing Fever Requires T Lymphocytes.

Relapsing fever borreliosis is a multisystemic infection characterized primarily by bacteremia but can extend to the CNS. The incidence of CNS disease manifestations in humans depends on the infecting relapsing fever Borrelia species. In the murine model of Borrelia hermsii infection we found high incidence of distinct signs of CNS disease that ranged from a flaccid tail to complete paralysis of hind limbs. Infiltration of large number of T cells into the spinal cord of B. hermsii-infected mice and the upregulation of MHC class II and CD80 on infiltrating macrophages and on microglial cells suggested a role for T cell and Ag-presenting cell interactions in this pathogenesis. Indeed, B. hermsii infection did not induce CNS disease manifestations in T cell-deficient mice (TCR-ß × d-/-), although it resulted in bacteremia comparable to wild-type (Wt) level. Moreover, the infiltration of immune cells into the spinal cord of TCR-ß × d-/- mice was reduced and the resident microglial cells were not activated. Histopathological analysis of lumbar sections of the spinal cord confirmed severe inflammation in Wt but not in TCR-ß × d-/- mice. Induction of CNS disease was dependent on the B. hermsii strain as well as on the ability of the host to control bacteremia. Mice that are impaired in controlling B. hermsii, such as CD14-/- mice, exhibited more severe CNS disease than Wt mice. This study demonstrates that distinct neurologic disease manifestations develop during relapsing fever and that T cells play a critical role in the induction of neuropathogenesis.

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.