Characterizing the phylogenetic specialism-generalism spectrum of mammal parasites.

The distribution of parasites across mammalian hosts is complex and represents a differential ability or opportunity to infect different host species. Here, we take a macroecological approach to investigate factors influencing why some parasites show a tendency to infect species widely distributed in the host phylogeny (phylogenetic generalism) while others infect only closely related hosts. Using a database on over 1400 parasite species that have been documented to infect up to 69 terrestrial mammal host species, we characterize the phylogenetic generalism of parasites using standard effect sizes for three metrics: mean pairwise phylogenetic distance (PD), maximum PD and phylogenetic aggregation. We identify a trend towards phylogenetic specialism, though statistically host relatedness is most often equivalent to that expected from a random sample of host species. Bacteria and arthropod parasites are typically the most generalist, viruses and helminths exhibit intermediate generalism, and protozoa are on average the most specialist. While viruses and helminths have similar mean pairwise PD on average, the viruses exhibit higher variation as a group. Close-contact transmission is the transmission mode most associated with specialism. Most parasites exhibiting phylogenetic aggregation (associating with discrete groups of species dispersed across the host phylogeny) are helminths and viruses.

Structural complexity in isoprenoid glycerol dialkyl glycerol tetraether lipid cores of Sulfolobus and other archaea revealed by liquid chromatography-tandem mass spectrometry.

Liquid chromatography-tandem mass spectrometry of membrane lipid cores from Sulfolobus species reveals isomeric forms of ring-containing isoprenoid glycerol dialkyl glycerol tetraether components not previously recognised via the use of NMR and liquid chromatography-mass spectrometry techniques. Equivalent isomerism was confirmed for the components in other hyperthermophilic genera and in sediments which contain the lipids of mesophilic archaea. The recognition of the isomeric structures in distinct archaeal clades suggests that profiles of tetraether lipids reported previously may have oversimplified the true lipid complexity in archaeal cultures and natural environments. Accordingly, the extent of variation in tetraether structures revealed by the work should direct more informative interpretations of lipid profiles in the future. Moreover, the results emphasise that tandem mass spectrometry provides a unique capability for assigning the structures of intact tetraether lipid cores for co-eluting species during chromatographic separation.

Parasite-mediated evolution of the functional part of the MHC in primates.

The major histocompatibility complex (MHC) is a key model of genetic polymorphism, but the mechanisms underlying its extreme variability are debated. Most hypotheses for MHC diversity focus on pathogen-driven selection and predict that MHC polymorphism evolves under the pressure of a diverse parasite fauna. Several studies reported that certain alleles offer protection against certain parasites, yet it remains unclear whether variation in parasite pressure more generally covaries with allelic diversity and rates of molecular evolution of MHC across species. We tested this prediction in a comparative study of 41 primate species. We characterized polymorphism of the exon 2 of DRB region of the MHC class II. Our phylogenetic analyses controlled for the potential effects of neutral mutation rate, population size, geographic origin and body mass and revealed that nematode species richness associates positively with nonsynonymous nucleotide substitution rate at the functional part of the molecule. We failed to find evidence for allelic diversity being strongly related to parasite species richness. Continental distribution was a strong predictor of both allelic diversity and substitution rate, with higher values in Malagasy and Neotropical primates. These results indicate that parasite pressure can influence the different estimates of MHC polymorphism, whereas geography plays an independent role in the natural history of MHC.

Sexual dimorphism in primate aerobic capacity: a phylogenetic test.

Male intrasexual competition should favour increased male physical prowess. This should in turn result in greater aerobic capacity in males than in females (i.e. sexual dimorphism) and a correlation between sexual dimorphism in aerobic capacity and the strength of sexual selection among species. However, physiological scaling laws predict that aerobic capacity should be lower per unit body mass in larger than in smaller animals, potentially reducing or reversing the sex difference and its association with measures of sexual selection. We used measures of haematocrit and red blood cell (RBC) counts from 45 species of primates to test four predictions related to sexual selection and body mass: (i) on average, males should have higher aerobic capacity than females, (ii) aerobic capacity should be higher in adult than juvenile males, (iii) aerobic capacity should increase with increasing sexual selection, but also that (iv) measures of aerobic capacity should co-vary negatively with body mass. For the first two predictions, we used a phylogenetic paired t-test developed for this study. We found support for predictions (i) and (ii). For prediction (iii), however, we found a negative correlation between the degree of sexual selection and aerobic capacity, which was opposite to our prediction. Prediction (iv) was generally supported. We also investigated whether substrate use, basal metabolic rate and agility influenced physiological measures of oxygen transport, but we found only weak evidence for a correlation between RBC count and agility.

Sensium: an ultra-low-power wireless body sensor network platform: design & application challenges.

In this paper we present a system-on-chip for wireless body sensor networks, which integrates a transceiver, hardware MAC protocol, microprocessor, IO peripherals, memories, ADC and custom sensor interfaces. Addressing the challenges in the design, this paper will continue to discuss the issues in the applications of this technology to body worn monitoring for real-time measurement of ECG, heart rate, physical activity, respiration and/or skin temperature. Two application challenges are described; the real-time measurement of energy expenditure using the LifePebble, and; the development issues surrounding the 'Digital Patch'.

Examining landscape factors influencing relative distribution of mosquito genera and frequency of virus infection.

Mosquito-borne infections cause some of the most debilitating human diseases, including yellow fever and malaria, yet we lack an understanding of how disease risk scales with human-driven habitat changes. We present an approach to study variation in mosquito distribution and concomitant viral infections on the landscape level. In a pilot study we analyzed mosquito distribution along a 10-km transect of a West African rainforest area, which included primary forest, secondary forest, plantations, and human settlements. Variation was observed in the abundance of Anopheles, Aedes, Culex, and Uranotaenia mosquitoes between the different habitat types. Screening of trapped mosquitoes from the different habitats led to the isolation of five uncharacterized viruses of the families Bunyaviridae, Coronaviridae, Flaviviridae, and Rhabdoviridae, as well as an unclassified virus. Polymerase chain reaction screening for these five viruses in individual mosquitoes indicated a trend toward infection with specific viruses in specific mosquito genera that differed by habitat. Based on these initial analyses, we believe that further work is indicated to investigate the impact of anthropogenic landscape changes on mosquito distribution and accompanying arbovirus infection.

Rapid evolution of social learning.

Culture is widely thought to be beneficial when social learning is less costly than individual learning and thus may explain the enormous ecological success of humans. Rogers (1988. Does biology constrain culture. Am. Anthropol. 90: 819-831) contradicted this common view by showing that the evolution of social learning does not necessarily increase the net benefits of learned behaviours in a variable environment. Using simulation experiments, we re-analysed extensions of Rogers' model after relaxing the assumption that genetic evolution is much slower than cultural evolution. Our results show that this assumption is crucial for Rogers' finding. For many parameter settings, genetic and cultural evolution occur on the same time scale, and feedback effects between genetic and cultural dynamics increase the net benefits. Thus, by avoiding the costs of individual learning, social learning can increase ecological success. Furthermore, we found that rapid evolution can limit the evolution of complex social learning strategies, which have been proposed to be widespread in animals.

Energetic constraints, not predation, influence the evolution of sleep patterning in mammals.

Mammalian sleep is composed of two distinct states - rapid-eye-movement (REM) and non-REM (NREM) sleep - that alternate in cycles over a sleep bout. The duration of these cycles varies extensively across mammalian species. Because the end of a sleep cycle is often followed by brief arousals to waking, a shorter sleep cycle has been proposed to function as an anti-predator strategy. Similarly, higher predation risk could explain why many species exhibit a polyphasic sleep pattern (division of sleep into several bouts per day), as having multiple sleep bouts avoids long periods of unconsciousness, potentially reducing vulnerability.Using phylogenetic comparative methods, we tested these predictions in mammals, and also investigated the relationships among sleep phasing, sleep-cycle length, sleep durations and body mass.Neither sleep-cycle length nor phasing of sleep was significantly associated with three different measures of predation risk, undermining the idea that they represent anti-predator adaptations.Polyphasic sleep was associated with small body size, shorter sleep cycles and longer sleep durations. The correlation with size may reflect energetic constraints: small animals need to feed more frequently, preventing them from consolidating sleep into a single bout. The reduced daily sleep quotas in monophasic species suggests that the consolidation of sleep into one bout per day may deliver the benefits of sleep more efficiently and, since early mammals were small-bodied and polyphasic, a more efficient monophasic sleep pattern could be a hitherto unrecognized advantage of larger size.

Why are some species more commonly afflicted by arthritis than others? A comparative study of spondyloarthropathy in primates and carnivores.

Spondyloarthropathy is a painful arthritic affliction of humans that also occurs in wild mammals. Important questions remain concerning the underlying causes of spondyloarthropathy in mammals, particularly regarding whether it is infectious in origin or driven by genetic predisposition and environmental stressors. Moreover, spondyloarthropathy has negative effects on host fitness, leading to potential conservation concerns if it impacts threatened species. Using a comparative data set on the prevalence of joint disease in 34 primate species and 100 carnivore species, we tested predictions involving the epidemiological correlates of spondyloarthropathy in wild mammals. Analyses revealed that 5.6% of primates and 3.6% of carnivores exhibited signs of spondyloarthropathy, with maximum incidence as high as 22% in great apes and 27% in bears. We tested whether prevalence of spondyloarthropathy increases with population density and group size, greater contact with soil, a slower host life history, increased ranging, dietary factors and body mass. We found general support for an effect of body mass, with larger bodied primates and carnivores exhibiting a higher prevalence of spondyloarthropathy. In addition, more threatened species experienced higher rates of spondyloarthropathy, with this association influenced by body mass and phylogeny. The effect of body mass could reflect that larger animals are exposed to more pathogens through greater consumption of resources, or that joints of larger bodied mammals experience greater biomechanical stresses, resulting in inflammation and activation of local joint infections.

Fish somatostatin sst3 receptor: comparison of radioligand and GTPgammaS binding, adenylate cyclase and phospholipase C activities reveals different agonist-dependent pharmacological signatures.

1 The fish somatostatin receptor 3 (fsst3) is one of the few somatostatin (SRIF) receptors cloned from a non-mammalian species so far. Here we extended our earlier characterization of this receptor by investigating the guanine nucleotide sensitivity of agonist radioligand binding at the fsst3 receptor recombinantly expressed in CCL39 (Chinese hamster lung fibroblast) cells. Further, we measured somatostatin (SRIF) and cortistatin (CST) analogues stimulated GTPgammaS binding, inhibition of forskolin-stimulated adenylate cyclase (FSAC) and stimulation of phospholipase C (PLC) activities. The present transductional data were then compared with previous radioligand binding and/or second messenger features determined for fsst3 and/or human SRIF receptors (hsst2, hsst3 and hsst5). 2 The GTP analogue guanylylimidodiphosphate (GppNHp) inhibited binding of [125I]CGP 23996 and [125I][Tyr3octreotide by 72 and 83% suggesting preferential labelling of G-protein-coupled fsst3 receptors. By contrast, [125I]LTT-SRIF28 and [125I][Tyr10]CST14 binding was rather GppNHp insensitive (42 and 35% inhibition) suggesting labelling of both coupled and non-coupled receptor states. These results might explain the apparent higher receptor densities determined in saturation experiments with [125I]LTT-SRIF28 and [125I][Tyr10]CST14 (4470 and 4030 fmol mg(-1)) compared with [125I]CGP 23996 and [125I][Tyr3]octreotide (3420 and 1520 fmol mg(-1)). 3 SRIF14 (10 microm)-stimulated specific [35S]GTPgammaS binding by three-fold; SRIF28 and octreotide displayed full agonism, whereas most other ligands displayed 60-80% intrinsic activity compared with SRIF14. SRIF14 and SRIF28 inhibited forskolin-stimulated AC (FSAC) activity by 60%; all tested ligands except BIM 23056 inhibited FSAC with comparable high intrinsic activities. SRIF14 stimulated PLC activity five- to six-fold, as determined by measuring total [3H] IP(x) accumulation; it was rather insensitive to pertussis toxin (PTX, 100 ng ml(-1), 21% inhibition), which suggests the G(q)-family proteins couple to PLC activity. SRIF14, SRIF28 and [Tyr10]CST14 showed full agonism at PLC, whereas all other ligands behaved as partial agonists (20-70% intrinsic activity). BIM 23056, which showed weak partial or no agonism, antagonized SRIF14-induced total [3H]-IP(x) production (pK(B) = 6.83), but failed to block competitively agonist-stimulated [35S]GTPgammaS binding or agonist-induced inhibition of FSAC activity. 4 Comparison of the pharmacological profiles of fsst3 receptors established in GTPgammaS binding, FSAC inhibition and PLC stimulation resulted in low correlations (r = 0.410-0.594). Both rank orders of potency and rank orders of relative efficacy varied in the three second messenger experiments. Significant, although variable correlations were obtained comparing GTPgammaS binding and inhibition of FSAC activity with previously reported affinity profiles of [125I]LTT-SRIF28, [125I][Tyr10]CST14, [125I]CGP 23996, [125I][Tyr3]octreotide (r = 0.75-0.83; 0.68-0.89). By contrast, the PLC stimulation and radioligand-binding profiles did not correlate. 5 Comparison of the functional data (GTPgammaS binding, FSAC inhibition, PLC stimulation) of fsst3 receptors with those of human sst2, sst3, sst5 receptors expressed in CCL39 cells resulted in highest correlation with the hsst5 receptor (r = 0.94, 0.97, 0.49) > hsst2 (0.80, 0.50, n.d.) > hsst3 (0.25, 0.19, 0.17). 6 In summary, fsst3 receptors expressed in CCL39 cells are involved in signalling cascades similar to those reported for mammalian SRIF receptors, suggesting SRIF receptors to be highly conserved in evolution. Binding and functional data showed highest similarity of fsst3 receptors with the human sst5 receptor subtype. Different affinities, receptor densities and GppNHp-sensitivities determined with the four radioligands (agonists) are assumed to results from ligand-specific states of the fsst3-ligand complex. The differences in the rank orders of potency and relative efficacy in the various signalling cascades may be explained by agonist-induced receptor trafficking.

SRA880, in vitro characterization of the first non-peptide somatostatin sst(1) receptor antagonist.

This report describes the in vitro features of the first somatostatin sst(1) receptor selective non-peptide antagonist, SRA880 ([3R,4aR,10aR]-1,2,3,4,4a,5,10,10a-Octahydro-6-methoxy-1-methyl-benz[g] quinoline-3-carboxylic-acid-4-(4-nitro-phenyl)-piperazine-amide, hydrogen malonate). SRA was evaluated in a number of in vitro systems of various species, both at native and recombinant receptors, using radioligand binding and second messenger/transduction studies. SRA880 has high affinity for native rat, mouse, monkey and human cerebral cortex somatostatin sst(1) receptors (pK(d) = 7.8-8.6) and for human recombinant sst(1) receptors (pK(d) = 8.0-8.1). SRA880 displayed significantly lower affinity for the other human recombinant somatostatin receptors ( pK(d) < or = 6.0) or a wide range of neurotransmitter receptors, except for the human dopamine D4 receptors. SRA880 was characterized in various transduction assays: somatotropin release inhibiting factor (SRIF) induced inhibition of forskolin-stimulated cAMP accumulation, SRIF stimulated-GTPgammaS binding, and SRIF stimulated luciferase gene expression; in all tests, SRA880 was devoid of intrinsic activity and acted as an apparently surmountable antagonist with pK(B) values of 7.5-7.7. Combined with the data from binding studies, these results suggest that SRA880 acts as a competitive antagonist. Thus, SRA880 is the first non-peptide somatostatin sst(1) receptor antagonist to be reported; SRA880 will be a useful tool for the characterization of somatostatin sst(1) receptor-mediated effects both in vitro and in vivo.

Somatostatin receptor subtypes 2 and 4 affect seizure susceptibility and hippocampal excitatory neurotransmission in mice.

We have investigated the role of somatostatin receptor subtypes sst2 and sst4 in limbic seizures and glutamate-mediated neurotransmission in mouse hippocampus. As compared to wild-type littermates, homozygous mice lacking sst2 receptors showed a 52% reduction in EEG ictal activity induced by intrahippocampal injection of 30 ng kainic acid (P < 0.05). The number of behavioural tonic-clonic seizures was reduced by 50% (P < 0.01) and the time to onset of seizures was doubled on average (P < 0.05). Seizure-associated neurodegeneration was found in the injected hippocampus (CA1, CA3 and hilar interneurons) and sporadically in the ipsilateral latero-dorsal thalamus. This occurred to a similar extent in wild-type and sst2 knock-out mice. Intrahippocampal injection of three selective sst2 receptor agonists in wild-type mice (Octreotide, BIM 23120 and L-779976, 1.5-6.0 nmol) did not affect kainate seizures while the same compounds significantly reduced seizures in rats. L-803087 (5 nmol), a selective sst4 receptor agonist, doubled seizure activity in wild-type mice on average. Interestingly, this effect was blocked by 3 nmol octreotide. It was determined, in both radioligand binding and cAMP accumulation, that octreotide had no direct agonist or antagonist action at mouse sst4 receptors expressed in CCl39 cells, up to micromolar concentrations. In hippocampal slices from wild-type mice, octreotide (2 micro m) did not modify AMPA-mediated synaptic responses while facilitation occurred with L-803087 (2 micro m). Similarly to what was observed in seizures, the effect of L-803087 was reduced by octreotide. In hippocampal slices from sst2 knock-out mice, both octreotide and L-803087 were ineffective on synaptic responses. Our findings show that, unlike in rats, sst2 receptors in mice do not mediate anticonvulsant effects. Moreover, stimulation of sst4 receptors in the hippocampus of wild-type mice induced excitatory effects which appeared to depend on the presence of sst2 subtypes, suggesting these receptors are functionally coupled.

Community thrombolysis in the Coromandel region. Audit of the "Cardiac Events in the Coromandel-Assessment Strategy and Triage" (CE-COAST) pilot program.

AIM: To audit the experience of a pilot program for community thrombolysis undertaken within the Coromandel region. METHODS: Community thrombolysis for patients suffering acute myocardial infarction (MI) was undertaken in areas within the Coromandel peninsula greater than half an hour by road from Thames Hospital. Thrombolytic therapy (Retelapse) was given following a discussion and review of a digitally transmitted ECG with the cardiology registrar. Treatment times and patients demographics were prospectively recorded. Subsequent clinical events were by chart review. Comparison of treatment times were made with an historical cohort for the same population which had received in-hospital thrombolysis between 1993 and 1998. RESULTS: Between July 1998 and December 1999, nineteen patients received thrombolysis in the community. There were no arrhythmic events during transportation and no deaths or reinfarctions during hospital stay. Median time from pain onset to thrombolysis was 135 (mean 175.5 +/- 144.9 SD) minutes which equated to a reduction in median time delay of 135 minutes compared to that experienced by the historical cohort (median 270, mean 316.7 +/- 145.8 SD minutes), p=0.0003. CONCLUSION: Community thrombolysis is logistically feasible within the New Zealand setting and results in major time reductions in the treatment of patients with acute MI.

Factors associated with healthy aging: the cardiovascular health study.

OBJECTIVES: To identify factors associated with remaining healthy in older adults. DESIGN: Longitudinal cohort study. SETTING: Data were collected at the four Cardiovascular Health Study field centers. PARTICIPANTS: 5,888 participants age 65 years and older in the Cardiovascular Health Study. MEASUREMENTS: Presence of chronic disease was assessed at baseline and over a maximum 7-year follow-up period. Participants who were free of chronic disease (no cardiovascular disease (CVD), chronic obstructive pulmonary disease, or self-reported cancer, except nonmelanoma skin cancer) at the baseline examination were then monitored for the onset of incident cancer, cardiovascular disease, and fatal outcomes. RESULTS: A high proportion of these older adults was healthy at the initial examination and remained healthy over the follow-up period. Numerous behavioral factors were associated with continued health, including physical activity, refraining from cigarette smoking, wine consumption (women), higher educational status, and lower waist circumference. A number of CVD risk factors and subclinical disease measures were associated with continued health, including higher high-density lipoprotein (HDL) cholesterol, lack of diabetes, thinner common carotid intimal nmedial thickness, lower blood pressure, lower C-reactive protein, and higher ankle-arm blood pressure ratio. Among the behavioral factors, exercise, not smoking, and not taking aspirin remained significant predictors of health even after controlling for CVD risk factors and subclinical disease in older adults. CONCLUSIONS: These data suggest that a number of modifiable behavioral factors (physical activity, smoking, and obesity) and cardiovascular risk factors (diabetes, HDL cholesterol, and blood pressure) are associated with maintenance of good health in older adults.

Treatment delays for patients with acute myocardial infarction within the Coromandel region of New Zealand.

AIM: To assess treatment delays incurred by Coromandel patients requiring thrombolytic therapy for acute myocardial infarction at Thames Hospital. METHODS: A chart search was undertaken at Thames Hospital to identify all patients admitted from July 1993 to July 1998 with a diagnosis of acute myocardial infarction who were treated with thrombolytic therapy. Times of pain onset, general practitioner (GP) assessment, transportation, hospital arrival and thrombolytic administration were noted. Additional information, when required, was obtained from the patient's GP or the St Johns Ambulance service. RESULTS: 153 patients were thrombolysed at Thames Hospital over this period, mean age 65.1 years, 36.6% in heart failure. The mean time from pain onset to GP contact was 157.2 minutes and varied from 63.2 minutes in Coromandel Township to 272.5 minutes in Pauanui. Delays from GP contact to thrombolysis were longer for patients living in outlying areas versus Thames and its environs, 169.4 +/- 45.9 versus 125.2 +/- 50.4 minutes (mean +/-SD) respectively, p<0.001. This contributed to a total delay from pain onset to thrombolysis of 316.7 +/- 145.8 minutes for patients in outlying areas versus 269.1 +/- 185.8 minutes for local patients (p=0.014). CONCLUSIONS: Delays in providing thrombolytic therapy for acute infarct patients reflect not only transport times but also delays in seeking initial medical assessment and hospital triage times. Transport times become particularly significant for those outside of Thames and its environs. Only with improved patient education and local delivery of thrombolytic therapy will these delays be adequately addressed.

Broad specificity alkaline proteases efficiently reduce the visual scaling associated with soap-induced xerosis.

In xerotic skin, the proteolysis of desmosomes is reduced leading to the accumulation of corneocytes on the surface of the skin. The effect of proteases applied topically to soap-induced xerotic skin was evaluated using a five-point visual scale. The visual scaling associated with soap-induced xerosis could be ameliorated by the topical application of exogenous protease. Bovine pancreatic chymotrypsin, papain, and a bacterial protease from Bacillus licheniformis were all capable of facilitating the reduction in visual scaling in a short time. Alcalase and Optimase, both broad specificity alkaline bacterial proteases, were the most weight-efficient at delivering this clinical effect. The reduction in scaling could be achieved either by occluded application of an aqueous enzyme solution or by a two-step unoccluded application first of an aqueous enzyme solution followed by a commercial moisturizer. Morphological and immunological analysis of bacterial enzyme-treated skin revealed that topically applied protease specifically induced the degradation of the desmosomes thereby promoting desquamation. These results indicate that topical application of protease can significantly and rapidly reduce the visual scaling associated with soap-induced xerosis by promoting desmosome degradation within the corneocyte clumps.

Promiscuity and the primate immune system.

The behavioral and ecological factors involved in immune system evolution remain poorly explored. We present a phylogenetic analysis of white blood cell counts in primates to test three hypotheses related to disease risk: increases in risk are expected with group size or population density, exposure to soil-borne pathogens, and mating promiscuity. White blood cell counts were significantly greater in species where females have more mating partners, indicating that the risk of sexually transmitted disease is likely to be a major factor leading to systematic differences in the primate immune system.

The impact of coronary stenting on immediate procedural complication and long-term clinical restenosis at Waikato Hospital.

AIM: Clinical data on coronary stenting from within New Zealand is scarce and, in particular, the impact of current stent technologies is unknown. We reviewed all angioplasties undertaken at Waikato Hospital over a two year period to determine the clinical effect of coronary stenting on the local population. METHODS: Data from all patients who underwent coronary angioplasty at Waikato Hospital between July 1, 1995 and July 1, 1997 were included. Stents were deployed either to remedy sub-optimal results, or were electively used for saphenous vein grafts or restenotic lesions. Patient follow-up was obtained through a combination of database review, chart search and GP or patient contact. RESULTS: 662 lesions were dilated in 441 patients. 91 lesions were stented, 52.7% for sub-optimal results following balloon angioplasty. 98% of patients were followed up at six months. Whilst procedural success rate was higher in stented patients compared to unstented patients (96.7% vs 87.5% respectively, p=0.009) the in-hospital sub-acute occlusion rate was also increased (6.8% vs 1.9% respectively, p=0.007). At six months, coronary restenosis requiring repeat angioplasty was infrequent (10.9% overall) with no significant difference between the two groups (8.1% vs. 11.2% for stented vs unstented patients respectively, p=NS). CONCLUSIONS: The use of stents appears effective in improving immediate procedural success rates. Despite stented patients being at higher risk initially, their complication and six month clinical restenosis rates were similar.

Cloning, expression, functional coupling and pharmacological characterization of the rat dopamine D4 receptor.

It has been difficult to observe functional coupling of the D4 receptor to second messenger systems and a robust functional assay system for this receptor is still lacking. In the present study, the rat dopamine D4 receptor was cloned from rat retina. Sequence comparison revealed identity with the published sequence of Ashgari and co-workers, including the two amino acid insertions (V-Q) at position 92 which are not present in the published sequence of O'Malley and coworkers. The rat dopamine D4 receptor was stably expressed in Chinese hamster lung fibroblast CCL39 cells. [3H]spiperone saturation binding yielded a Bmax of 2,370+/-546 fmol/mg protein and a pKD of 8.74+/-0.14 (n=4). Forskolin-stimulated cAMP accumulation was inhibited by dopamine (Emax 61+/-1% inhibition of forskolin-stimulated levels, pEC50 7.33+/-0.06, n=23). A similar concentration-dependent inhibition was observed with the dopamine D2-like receptor agonists quinpirole and 7-OH-DPAT which elicited nearly the same Emax as dopamine. By contrast, apomorphine and a number of compounds with reported affinity for human dopamine D4 receptors (PD168077, U-101958, SDZ GLC 756, L-745,870 and NGD 94-1) behaved as partial agonists (Emax ranging between 26% and 56% of that of dopamine). The agonist effect of dopamine was completely blocked by preincubation with pertussis toxin, no further accumulation of cAMP above the forskolin-stimulated levels being observed. Antagonist pKB-values obtained against dopamine in this system were: 8.55+/-0.19 (n=3) for the partial agonist L-745,870, 8.38+/-0.23 (n=5) for spiperone, 7.18+/-0.17 (n=4) for haloperidol, 7.04+/-0.13 (n=4) for clozapine and <6 for raclopride. Other functional assays applicable were stimulation of [35S]GTPgammaS binding, extracellular acidification rate and a serum-responsive element using luciferase expression as a reporter gene. However, the receptor did not couple to phosphatidylinositol turnover or to intracellular Ca2+. Thus, expression of the rat dopamine D4 receptor in CCL39 cells provided several functional assay systems, of which inhibition of cAMP appeared to be the most robust one. These functional models can be used to evaluate the activity of compounds at the rat dopamine D4 receptor.