Species with a chemical defence, but not chemical offence, live longer.

Evolutionary hypotheses for ageing generally predict that delayed senescence should evolve in organisms that experience lower extrinsic mortality. Thus, one might expect species that are highly toxic or venomous (i.e. chemically protected) will have longer lifespans than related species that are not likewise protected. This remarkable relationship has been suggested to occur in amphibians and snakes. First, we show that chemical protection is highly conserved in several lineages of amphibians and snakes. Therefore, accounting for phylogenetic autocorrelation is critical when conservatively testing evolutionary hypotheses because species may possess similar longevities and defensive attributes simply through shared ancestry. Herein, we compare maximum longevity of chemically protected and nonprotected species, controlling for potential nonindependence of traits among species using recently available phylogenies. Our analyses confirm that longevity is positively correlated with body size in both groups which is consistent with life-history theory. We also show that maximum lifespan was positively associated with chemical protection in amphibian species but not in snakes. Chemical protection is defensive in amphibians, but primarily offensive (involved in prey capture) in snakes. Thus, we find that although chemical defence in amphibians favours long life, there is no evidence that chemical offence in snakes does the same.

A comparative analysis of senescence in adult damselflies and dragonflies (Odonata).

Any population whose members are subject to extrinsic mortality should exhibit an increase in mortality with age. Nevertheless, the prevailing opinion is that populations of adult damselflies and dragonflies do not exhibit such senescence. Here, we challenge this contention by fitting a range of demographic models to the data on which these earlier conclusions were based. We show that a model with an exponential increase in age-related mortality (Gompertz) generally provides a more parsimonious fit than alternative models including age-independent mortality, indicating that many odonates do indeed senesce. Controlling for phylogeny, a comparison of the daily mortality of 35 odonate species indicates that although male and female mortalities are positively correlated, mortality tends to be higher in males of those species that exhibit territoriality. Hence, we show for the first time that territoriality may impose a survivorship cost on males, once the underlying phylogenetic relationships are accounted for.

Empirical evidence of senescence in adult damselflies (Odonata: Zygoptera).

1. Age-dependent increases in mortality have been documented in a variety of species of insect under laboratory conditions. However, while strong statistical evidence has been presented for senescence in vertebrate populations in the wild, we know little about the rate and shape of senescence in wild populations of insects. 2. Odonates (damselflies and dragonflies) provide excellent candidate species for evaluating demographic senescence as they are large enough to be marked individually and they are easily re-sighted without recapture. The prevailing opinion - based entirely on qualitative examination of the declines in log numbers alive with time since marking - is that odonates exhibit age-independent daily survivorship. 3. Here, we examine mark-recapture data on the Azure Damselfly Coenagrion puella over two consecutive seasons. For the first time, we evaluate and compare the fit of quantitative models that not only account for weather-dependent daily variation in daily re-sighting rates, but also age-dependent variation in daily survivorship. 4. Models with age-dependent declines in daily survivorship provide a more parsimonious explanation for the data than similar models without these age-dependent effects. In general, models in which mortality increases in an exponential (Gompertz) fashion explain the mark-recapture sequences more efficiently than a range of alternative models, including those in which mortality increases as a power function (Weibull) or reaches a plateau (logistic). These results are indicative of a general senescent decline in physiological functioning, which is particularly marked after 15 days as a mature adult. 5. Weather (temperature, sun and precipitation) and initial mite load influenced the probability of daily re-sighting. Weather and mite load also influenced daily survivorship, but their effects differed between seasons. 6. Overall, fitting models with age as an explicit covariate demonstrates that odonates do indeed senesce. This contradicts previously held assumptions that Odonata do not exhibit age-dependent survivorship in the wild.

The (Under)Use of Eye-Tracking in Evolutionary Ecology.

To survive and pass on their genes, animals must perform many tasks that affect their fitness, such as mate-choice, foraging, and predator avoidance. The ability to make rapid decisions is dependent on the information that needs to be sampled from the environment and how it is processed. We highlight the need to consider visual attention within sensory ecology and advocate the use of eye-tracking methods to better understand how animals prioritise the sampling of information from their environments prior to making a goal-directed decision. We consider ways in which eye-tracking can be used to determine how animals work within attentional constraints and how environmental pressures may exploit these limitations.

Event-related brain potentials during the visuomotor mental rotation task: The contingent negative variation scales to angle of rotation.

Perceptual judgments about the angular disparity of a character from its standard upright (i.e., mental rotation task) result in a concurrent increase in reaction time (RT) and modulation of the amplitude of the P300 event-related brain potential (ERP). It has therefore been proposed that the P300 represents the neural processes associated with a visual rotation. In turn, the visuomotor mental rotation (VMR) task requires reaching to a location that deviates from a target by a predetermined angle. Although the VMR task exhibits a linear increase in RT with increasing oblique angles of rotation, work has not examined whether the task is supported via a visual rotation analogous to its mental rotation task counterpart. This represents a notable issue because seminal work involving non-human primates has ascribed VMR performance to the motor-related rotation of directionally tuned neurons in the primary motor cortex. Here we examined the concurrent behavioral and ERP characteristics of a standard reaching task and VMR tasks of 35°, 70°, and 105° of rotation. Results showed that the P300 amplitude was larger for the standard compared to each VMR task--an effect independent of the angle of rotation. In turn, the amplitude of the contingent negative variation (CNV)--an ERP related to cognitive and visuomotor integration for movement preparation--was systematically modulated with angle of rotation. Thus, we propose that the CNV represents an ERP correlate related to the cognitive and/or visuomotor transformation demands of increasing the angular separation between a stimulus and a movement goal.

The design of a new group of angiotensin-converting enzyme inhibitors.

Using X-ray and NMR data relating to the conformation of the antihypertensive, angiotensin-converting enzyme inhibitor, captopril, and structure--activity relationships of analogues, it has been possible to postulate with the aid of computer graphics, the orientation of the three functions, the thiol, the terminal carboxyl and the carbonyl group which are involved in binding to the enzyme. Bicyclic mimetics of captopril, with related arrays of these functions, have been designed and synthesized. Compounds with the closest approximation to the array in captopril are the most active inhibitors of angiotensin converting enzyme, in vitro.

A new class of inhibitors of human leucocyte elastase.

Studies of the inhibition of elastases at a molecular level have resulted in the identification of protected dipeptides which are reversible and highly specific inhibitors of human leucocyte elastase (HLE). These have been further developed by increasing their hydrophilicity and potency to give a new family of elastase inhibitors, typically N alpha-(1-adamantanesulphonyl)-N epsilon-(4-carboxybenzoyl)-L-lysyl-L-alanyl-L-valinal. These compounds are active in pharmacological models designed to detect compounds of potential therapeutic value in the treatment of emphysema.

New potent inhibitors of angiotensin converting enzyme.

Using an earlier model of the favoured orientation of binding functions of angiotensin converting enzyme (ACE) inhibitors, it has been possible to postulate a new, 7,6-bicyclic system, based on hexahydropyridazine, which might be expected to have high potency. Some members of this system which have been synthesised have been shown to be very active ACE inhibitors, in vitro and in vivo.

Synthesis and structure-activity relationships of antibacterial phosphonopeptides incorporating (1-aminoethyl)phosphonic acid and (aminomethyl)phosphonic acid.

Phosphonodipeptides and phosphonooligopeptides based on L- and D-(1-aminoethyl)phosphonic acids L-Ala(P) and D-Ala(P) and (aminomethyl)phosphonic acid Gly(P) at the acid terminus have been synthesized and investigated as antibacterial agents, which owe their activity to the inhibition of bacterial cell-wall biosynthesis. A method for large-scale synthesis of the potent antibacterial agent L-Ala-L-Ala(P) (1, Alafosfalin) is described. Structure-activity relationships in the dipeptide series have been studied by systematic variation of structure 1. L stereochemistry is generally required for both components. Changes in the L-Ala(P) moiety mostly lead to loss of antibacterial activity, but the phosphonate analogues of L-phenylalanine, L-Phe(P), and L-serine, L-Ser(P), give rise to weakly active L-Ala-L-Phe(P) and L-Ala-L-Ser(P). Replacement of L-Ala in 1 by common and rare amino acids can give rise to more potent in vitro antibacterials such as L-Nva-L-Ala(P) (45). Synthetic variation of these more potent dipeptides leads to decreased activity. Phosphonooligopeptides such as (L-Ala)2-L-Ala(P) have a broader in vitro antibacterial spectrum than their phosphonodipeptide precursor, but this is not expressed in vivo, presumably due to rapid metabolism to 1. Stabilized compounds such as Sar-L-Nva-L-Nva-L-Ala(P) (46) have been developed that are more potent in vivo and have a broader in vivo antibacterial spectrum than the parent phosphonodipeptide.

Synthesis and antitumor activity of novel 4-demethoxyanthracyclines.

A versatile and efficient synthetic route to 4-demethoxyanthracyclinones has been utilized in the preparation of a number of aglycons having 9-alkyl, 9-(hydroxylalkyl), or 9-carbamoyl substituents. Silver trifluoromethanesulfonate catalyzed coupling of these aglycons with various daunosamine derivatives has yielded a series of novel anthracyclines which have been evaluated as antitumor agents. 9-Alkylanthracyclines 22, 23, 33, and 34 have higher efficacy vs L-1210 leukemia than the parent 4-demethoxydaunorubicin (21), or the natural anthracyclines daunorubicin (1) and doxorubicin (2). 9-(Hydroxyalkyl) derivatives have in most cases high efficacy but are slightly less potent than 21. 9-Methyl analogue 22 has higher efficacy vs P388 leukemia than other anthracyclines tested, while 9-(hydroxymethyl) derivative 37 retains similar efficacy to anthracyclines 1, 2, and 21 but is considerably more potent. The N-substituted 9-carbamoylanthracyclines are devoid of antitumor activity.

Synthesis and antitumor activity of 9-[(carbamoyloxy)alkyl]anthracyclines: a novel class of anthracycline derivatives.

A number of 4-demethoxyanthracyclines having hydroxylalkyl functions at the 9-position have previously been synthesized and shown to have potent antitumor activity. A series of carbamate derivatives of these (hydroxyalkyl)anthracyclines have now been prepared, many of which possess considerably greater efficacy in an L-1210 leukemia test system than do the parent alcohols or the known anthracyclines daunorubicin (1), doxorubicin (2), and 4-demethoxydaunorubicin (3). Phenylcarbamate 8a was more active than methyl analogue 8b, while the 4'-deoxy and 4'-epi phenylcarbamates 17 and 18 showed particularly high efficacy at optimal dose levels similar to that of doxorubicin. Secondary carbamates were more potent, with the 13R isomer 23 having significantly higher efficacy than 13S analogue 24.

Evidence for uptake and synthesis of 5-hydroxytryptamine by a subpopulation of intrinsic neurons in the guinea-pig heart.

Using an indirect immunofluorescence technique, a subpopulation of 5-hydroxytryptamine-like immunoreactive neurons was observed in cell cultures dissociated from the atria and interatrial septum of newborn guinea-pig heart maintained in fetal calf serum-supplemented medium. 5-Hydroxytryptamine has not been demonstrated in intracardiac neurons in situ, and since 5-hydroxytryptamine has been previously shown to be a constituent of fetal calf serum, the 5-hydroxytryptamine-like immunoreactivity seen in culture may have been the result of neuronal uptake of 5-hydroxytryptamine from the growth medium. This was examined by growing the cultures in a serum-free, hormone-supplemented, defined medium. Under these conditions, 5-hydroxytryptamine-like immunoreactive neurons were not present. When cultures were grown in hormone-supplemented, defined medium containing 10(-4) to 10(-6) M 5-hydroxytryptamine, some intracardiac neurons accumulated 5-hydroxytryptamine. This type of neuron also developed 5-hydroxytryptamine-like immunoreactivity after incubation with 5 X 10(-5) M 5-hydroxytryptophan, indicating that the subpopulation of intracardiac neurons which can take up exogenous 5-hydroxytryptamine can also synthesize it from 5-hydroxytryptophan. However, no 5-hydroxytryptamine-like immunoreactive neurons were observed after incubation with L-tryptophan, the other 5-hydroxytryptamine precursor molecule. Under all of the conditions described, some small, 5-hydroxytryptamine-like immunofluorescent cells, very similar to the catecholamine-containing, small intensely fluorescent cells of the heart, were observed in culture. Bright, 5-hydroxytryptamine-like immunoreactive endothelial cells were seen only in cultures maintained in defined medium and loaded with 5-hydroxytryptamine. The present study shows that some intracardiac neurons are amine-handling, and also raises the possibility that 5-hydroxytryptamine is utilized as a neurotransmitter or neuromodulator by these neurons in the mammalian heart. Further, there is evidence to suggest that two populations of small intensely fluorescent cells, one containing 5-hydroxytryptamine, the other a catecholamine, are present in the heart; and to indicate that atrial endothelial cells can take up 5-hydroxytryptamine.

Co-expression of four muscarinic receptor genes by the intrinsic neurons of the rat and guinea-pig heart.

Expression of the messenger RNAs encoding the five different muscarinic acetylcholine receptor subtypes was examined in intracardiac neurons from the rat and guinea-pig heart by in situ hybridization techniques. Newborn guinea-pig intracardiac neurons were studied in dissociated cell culture preparations employing both 35S- and digoxigenin-labelled oligonucleotide probes specific for the m1, m2, m3, m4 or m5 muscarinic receptor messenger RNAs. When 35S-tailed oligonucleotides were used, all intracardiac neurons in culture were found to express m1, m2, m3 and m4, but not m5 messenger RNAs. However after hybridization with digoxigenin-tailed probes, only m1 and m2 transcripts were detected. This may reflect differences in the sensitivity of the two techniques. Further to these experiments, intracardiac ganglia in sections of adult rat heart were studied employing m1-, m2-, m3- or m4-specific, 35S-labelled oligonucleotides, and again, all intracardiac neurons expressed messenger RNA for each of these four muscarinic receptor subtypes. Atrial myocytes in culture were only labelled by [35S]- and digoxigenin-tailed m2 oligonucleotides. No other heart cell type seen expressed messenger RNA for any of the muscarinic receptors. The expression of four different muscarinic receptor transcripts by intrinsic neurons of the heart provides the molecular basis for the diverse muscarinic actions observed in these and other autonomic ganglia.

Heme oxygenase-2 and nitric oxide synthase in guinea-pig intracardiac neurones.

There is increasing evidence that carbon monoxide (CO), like nitric oxide (NO), may be a neuronal messenger molecule. This study investigated the expression of heme oxygenase-2 (HO-2), the enzyme responsible for the synthesis of CO, by intracardiac neurones. Many, if not all newborn guinea-pig intracardiac neurones in culture were HO-2-immunoreactive. Furthermore, double labelling showed that a relatively small subpopulation of these neurones also expressed NO synthase/nicotinamide dinucleotide phosphate (NADPH)-diaphorase (NOS/NADPH-d) activity. These findings suggest that intracardiac neurones can synthesize CO and that CO may be fundamental to their function. Comparison of the proportions of intracardiac neurones that contain HO-2 with those that express NOS/NADPH-d activity also indicates that CO may be more important than NO in the intrinsic neuronal control of the heart.

Expression of NADPH-diaphorase activity by guinea-pig paratracheal neurones.

Pharmacological evidence suggests that nitric oxide (NO) is involved in non-adrenergic, non-cholinergic, nerve mediated responses seen in guinea-pig trachealis muscle. The synthetic enzyme for NO (NO synthase) has recently been shown to be responsible for neuronal NADPH-diaphorase activity. Therefore, to determine whether intrinsic paratracheal neurones could be a source of NO in the trachea, expression of NADPH-diaphorase activity was examined histochemically using whole mount preparations of the tracheal plexus. Many paratracheal neurones were found to express moderate to high levels of NADPH-diaphorase activity and are thus likely to be a source of NO in this tissue. This observation provides further evidence that NO is involved in the regulation of relaxation in airway smooth muscle.

Colocalization of nitric oxide synthase and NADPH-diaphorase in cultured myenteric neurones.

Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity were examined in explant culture preparations of the myenteric plexus from beneath the taenia coli of the guinea-pig caecum. Nitric oxide synthase immunoreactive neurones formed approximately one third of the total neuronal population. NADPH-diaphorase positive neurones, demonstrated histochemically, constituted a similar proportion of the total number of neurones. Immunocytochemistry and NADPH-diaphorase histochemistry performed on the same preparations revealed that all nitric oxide synthase immunoreactive neurones expressed NADPH-diaphorase activity. This histochemical evidence is consistent with the view that nitric oxide may act as a regulatory agent in the guinea-pig caecum.

Intrinsic neurones and associated cells of the guinea-pig heart in culture.

This paper describes a method for dissociation of intrinsic neurones from the atria and interatrial septum of newborn guinea-pig heart and their maintenance in culture. The appearance of the cultured intracardiac neurones, muscle and other non-neuronal cell types also present in the preparation has been observed by phase-contrast microscopy. Some of the neurochemical properties of the intracardiac neurones in culture have been investigated using histochemical methods. All the neurones studied were shown to contain acetylcholinesterase. No catecholamine-containing neurones were found. Using an indirect immunofluorescence technique, 20-50% of clearly identifiable neurones in culture contained neuropeptide Y-like immunoreactivity. Vasoactive intestinal polypeptide-like immunoreactive neurones were found in only one out of 15 culture preparations; no substance P-, neurotensin-, or enkephalin-like immunoreactivity was observed. These findings are consistent with those described for intracardiac neurones studied in situ, suggesting that the neurochemical differentiation of the intrinsic heart neurones is retained in culture. The culture preparation provides an opportunity to study the properties and role of intrinsic neurones of the heart. The characteristics of the intracardiac neurones may be distinguished from those of the extrinsic nerve fibres which degenerate in culture. Further, the intracardiac neurones are more accessible to experimental manipulation in culture than in situ.

Immunocytochemical localisation of neuropeptide Y and 5-hydroxytryptamine in a subpopulation of amine-handling intracardiac neurones that do not contain dopamine beta-hydroxylase in tissue culture.

The colocalisation of neuropeptide Y (NPY)- and 5-hydroxytryptamine (5-HT)-immunoreactivities in intracardiac neurones in dissociated cultures from the atria and interatrial septum of newborn guinea pig heart was demonstrated by the sequential application of specific antisera which were visualised by two different fluorochromes. In this way it was observed that most 5-HT-immunoreactive neurones also contained NPY-immunoreactivity (approximately 40% of identified neurones), some neurones were 5-HT-immunoreactive alone (approximately 10%), while neurones that were NPY-immunoreactive only were rarely seen. No dopamine beta-hydroxilase (DBH)-immunoreactive intracardiac neurones were demonstrated in any of the culture preparations studied, although DBH-immunoreactive neurons could be detected in sections of the newborn guinea pig heart containing intracardiac ganglia. The possible implications of the colocalisation of 5-HT, that has been taken up from the culture medium, with NPY in a population of intracardiac neurones are discussed; and reasons for the loss of expression of DBH by these neurones under the conditions of culture are considered.

Localization of muscarinic receptors on peptide-containing neurones of the guinea pig myenteric plexus in tissue culture.

A combined autoradiographic and immunocytochemical procedure has been used to identify neurochemically the subpopulation of cultured myenteric neurones which expresses muscarinic receptors. Antibodies to substance P (SP), [Met]enkephalin (ENK), vasoactive intestinal polypeptide (VIP) and somatostatin (SOM) were used to immunostain cultures that had previously been labelled with the irreversible muscarinic antagonist, [3H]propylbenzilylcholine mustard. Most neurites which displayed SP-like, ENK-like or SOM-like immunoreactivity did not possess muscarinic receptors. In contrast, many VIP-like immunopositive fibres also possessed muscarinic receptors. The identity of the majority of neurones which express muscarinic receptors, that do not contain VIP, remains to be determined.

Neuropeptide Y-like immunoreactivity in cultured intrinsic neurones of the heart.

A novel culture preparation from the atria of newborn guinea-pig hearts was employed to study the intrinsic innervation of the heart under conditions of unequivocal extrinsic denervation. Using an indirect immunofluorescence technique, a subpopulation of intracardiac neurones grown in dissociated cell culture was demonstrated to contain neuropeptide Y (NPY)-like immunoreactivity. This shows that NPY is not confined to sympathetic nerves. Further, since no endogenous catecholamines could be demonstrated in neurone cell bodies in the culture preparation with fluorescence histochemistry, confirming previous studies in situ, the findings also suggest that NPY does not coexist with catecholamine in these intrinsic heart neurones.