The dendritic spine morphogenic effects of repeated cocaine use occur through the regulation of serum response factor signaling.

The nucleus accumbens (NAc) is a primary brain reward region composed predominantly of medium spiny neurons (MSNs). In response to early withdrawal from repeated cocaine administration, de novo dendritic spine formation occurs in NAc MSNs. Much evidence indicates that this new spine formation facilitates the rewarding properties of cocaine. Early withdrawal from repeated cocaine also produces dramatic alterations in the transcriptome of NAc MSNs, but how such alterations influence cocaine's effects on dendritic spine formation remain unclear. Studies in non-neuronal cells indicate that actin cytoskeletal regulatory pathways in nuclei have a direct role in the regulation of gene transcription in part by controlling the access of co-activators to their transcription factor partners. In particular, actin state dictates the interaction between the serum response factor (SRF) transcription factor and one of its principal co-activators, MAL. Here we show that cocaine induces alterations in nuclear F-actin signaling pathways in the NAc with associated changes in the nuclear subcellular localization of SRF and MAL. Using in vivo optogenetics, the brain region-specific inputs to the NAc that mediate these nuclear changes are investigated. Finally, we demonstrate that regulated SRF expression, in turn, is critical for the effects of cocaine on dendritic spine formation and for cocaine-mediated behavioral sensitization. Collectively, these findings reveal a mechanism by which nuclear-based changes influence the structure of NAc MSNs in response to cocaine.

Next-to-Next-to-Leading-Order QCD Corrections to the Transverse Momentum Distribution of Weak Gauge Bosons.

The transverse momentum spectra of weak gauge bosons and their ratios probe the underlying dynamics and are crucial in testing our understanding of the standard model. They are an essential ingredient in precision measurements, such as the W boson mass extraction. To fully exploit the potential of the LHC data, we compute the second-order [next-to-next-to-leading-order (NNLO)] QCD corrections to the inclusive-p_{T}^{W} spectrum as well as to the ratios of spectra for W^{-}/W^{+} and Z/W. We find that the inclusion of NNLO QCD corrections considerably improves the theoretical description of the experimental CMS data and results in a substantial reduction of the residual scale uncertainties.

Fungal Planet description sheets: 128-153.

Novel species of microfungi described in the present study include the following from Australia: Catenulostroma corymbiae from Corymbia, Devriesia stirlingiae from Stirlingia, Penidiella carpentariae from Carpentaria, Phaeococcomyces eucalypti from Eucalyptus, Phialophora livistonae from Livistona, Phyllosticta aristolochiicola from Aristolochia, Clitopilus austroprunulus on sclerophyll forest litter of Eucalyptus regnans and Toxicocladosporium posoqueriae from Posoqueria. Several species are also described from South Africa, namely: Ceramothyrium podocarpi from Podocarpus, Cercospora chrysanthemoides from Chrysanthemoides, Devriesia shakazului from Aloe, Penidiella drakensbergensis from Protea, Strelitziana cliviae from Clivia and Zasmidium syzygii from Syzygium. Other species include Bipolaris microstegii from Microstegium and Synchaetomella acerina from Acer (USA), Brunneiapiospora austropalmicola from Rhopalostylis (New Zealand), Calonectria pentaseptata from Eucalyptus and Macadamia (Vietnam), Ceramothyrium melastoma from Melastoma (Indonesia), Collembolispora aristata from stream foam (Czech Republic), Devriesia imbrexigena from glazed decorative tiles (Portugal), Microcyclospora rhoicola from Rhus (Canada), Seiridium phylicae from Phylica (Tristan de Cunha, Inaccessible Island), Passalora lobeliae-fistulosis from Lobelia (Brazil) and Zymoseptoria verkleyi from Poa (The Netherlands). Valsalnicola represents a new ascomycete genus from Alnus (Austria) and Parapenidiella a new hyphomycete genus from Eucalyptus (Australia). Morphological and culture characteristics along with ITS DNA barcodes are also provided.

Level and pattern of neuropsychological functioning in early-onset psychoses.

The present study aimed to compare the level and pattern of cognitive deficits in patients with early-onset psychoses with an age, gender and IQ matched control group. In order to ensure a representative sample of patients with psychoses, participants with an IQ of less than 70 were included. Forty-eight patients with an onset of psychoses before the age of 16, and 47 age, gender and IQ matched controls without psychoses were recruited. Psychotic symptomatology was assessed using the Schedules for Clinical Assessment to obtain DSM-IV and ICD-10 diagnoses. Positive and negative symptoms were assessed using the Positive and Negative Symptoms Scale. Levels of cognitive function were measured using a comprehensive neuropsychological battery. A pattern of specific impairments was not found, with few significant differences between the two cohorts. However both cohort groups performed lower than age derived norms. Therefore it appears that there are global cognitive deficits rather than specific deficits in early-onset psychoses when compared to normative data, in-line with conclusions of early-onset schizophrenia research, but patients did not have greater deficit than controls when matched on IQ, gender and age, even after excluding participants with IQ less than 70.

d3-GHR genotype does not explain heterogeneity in GH responsiveness in hypopituitary adults.

OBJECTIVE: Heterogeneity in growth hormone (GH) responsiveness in adult hypopituitary patients receiving recombinant human GH (rhGH) is poorly understood; doses vary up to fourfold between individuals. Deletion of exon 3 in the GH receptor (d3-GHR) has been linked to enhanced rhGH responsiveness in children. We investigated the role of the d3-GHR polymorphism in determining adult rhGH responsiveness. METHODS: One hundred and ninety-four patients treated with an identical rhGH dosing protocol in a single centre were genotyped for the d3-GHR, and the results correlated with changes in serum IGF-I and clinical parameters of GH responsiveness after 6 and 12 months of GH replacement therapy. RESULTS: Allele frequencies for homozygous full length (fl/fl), heterozygous d3 (fl/d3) and homozygous d3 (d3/d3) were 52%, 38.7% and 9.3%, respectively, and were in Hardy-Weinberg equilibrium. Baseline IGF-I and DeltaIGF-I at 6 months were comparable between groups. DeltaIGF-I at 12 months was significantly greater in the d3/d3 group (P = 0.028). No difference was detected between fl/d3 and fl/fl groups. Regression analyses of DeltaIGF-I at 12 months and DeltaIGF-I/rhGH dose confirmed a significant relationship of d3/d3 genotype on rhGH response. There was no difference between groups in maintenance rhGH dose between genotypes. CONCLUSION: Homozygosity for d3-GHR confers a marginal increase in GH responsiveness at 12 months but without a detectable change in maintenance rhGH dose required. Both d3 alleles are required to achieve this response; given that only 10% of the population are d3 homozygotes, the d3GHR does not explain the marked heterogeneity of GH responsiveness in hypopituitary adults.

The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via alpha-actinin: receptor positioning in microvilli does not require interaction with alpha-actinin.

The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L-selectin interacts directly with the cytoplasmic actin-binding protein alpha-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified alpha-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of alpha-actinin to the L-selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha-actinin binds directly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo to link actin filaments to the L-selectin cytoplasmic domain. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L-selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of alpha-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L-selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L-selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton.

Vector boson production in association with a jet at forward rapidities.

Final states with a vector boson and a hadronic jet allow one to infer the Born-level kinematics of the underlying hard scattering process, thereby probing the partonic structure of the colliding protons. At forward rapidities, the parton collisions are highly asymmetric and resolve the parton distributions at very large or very small momentum fractions, where they are less well constrained by other processes. Using theory predictions accurate to next-to-next-to-leading order (NNLO) in QCD for both W ± and Z production in association with a jet at large rapidities at the LHC, we perform a detailed phenomenological analysis of recent LHC measurements. The increased theory precision allows us to clearly identify specific kinematical regions where the description of the data is insufficient. By constructing ratios and asymmetries of these cross sections, we aim to identify possible origins of the deviations, and highlight the potential impact of the data on improved determinations of parton distributions.

Radiometric pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in low-shedder cattle.

OBJECTIVE: To identify the optimum pooling rate for pooled faecal culture (PFC) as a diagnostic tool in bovine Johne's disease control, for detection of cattle shedding low concentrations of Mycobacterium avium subsp paratuberculosis (Map). METHOD: Thirteen target animals were selected by delayed growth of Map from initial individual radiometric faecal cultures (first growth index at 5 weeks or later). A procedure based on radiometric culture and IS900 polymerase chain reaction and restriction endonuclease analysis confirmation was then used for PFC. RESULTS: Eight samples (stored for up to 17 months at -80 degrees C) yielded Map on subsequent culture, either from undiluted faeces or those mixed with normal cattle faeces at dilution rates from 1 in 5 to 1 in 50. From a regression equation, culture-positive animals were considered to be shedding relatively low levels of Map (< 6 x 10(4)/g of faeces). Pooling dilutions of more than 1 in 5 reduced PFC sensitivity. A minimum incubation period of 10 weeks at a dilution of 1 in 5 is recommended to detect such infected cattle. This pooling rate in radiometric culture is probably capable of detecting cattle shedding < or = 5 x 10(3) Map organisms/g of faeces, representing an estimated inoculum per culture vial of fewer than 20 viable organisms. CONCLUSION: Map was detected in more than 50% of the stored faecal samples from cattle shedding low concentrations of the organism. A pooling rate of 5 samples per pool is required to reliably detect infected low-shedder cattle using PFC based on radiometric culture.

Dynamics of social behaviour at parturition in a gregarious ungulate.

Group living is the behavioural response that results when individuals assess the costs vs benefits of sociality, and these trade-offs vary across an animal's life. Here we quantitatively assess how periparturient condition (mother/non-mother) and births affect the dynamics of social interactions of a gregarious ungulate, and how such can help to explain evolutionary hypotheses of the mother-offspring bond. To achieve this we used data of the individual movement of a group of Scottish blackface sheep (Ovis aries) marked with GPS collars and properties of mathematical graphs (networks). Euclidean pair-wise distance between sheep were threshold at different percentiles to determine network links, and these thresholds have a profound effect on the connectivity of the resulting network. Births increased the average pair-wise distance between mothers, and between mothers and non-mothers, with less effect on the distance between non-mothers. Mothers occupied peripheral positions within the flock, more evident following births. Associations between individuals (i.e. network community change) were highly dynamic, though mothers were less likely to change community than non-mothers, especially after births. Births hampered individual communication within the flock (assessed via network closeness centrality), especially in mothers. Overall leadership (lead positioning relative to flock movement) was not associated to reproductive condition, and individual leadership rank was not affected by births. A ten minute GPS acquisition time was adequate to capture complex social dynamics in sheep movement. The results on mother's isolation behaviour support the hypotheses of selection for maternal imprint facilitation, reducing risks to nursing alien offspring, and group/multilevel selection on group formation.

Editor's Choice - Frailty and the management of patients with acute cardiovascular disease: A position paper from the Acute Cardiovascular Care Association.

Frailty is increasingly seen among patients with acute cardiovascular disease. A combination of an ageing population, improved disease survival, treatable long-term conditions as well as a greater recognition of the syndrome has accelerated the prevalence of frailty in the modern world. Yet, this has not been matched by an expansion of research. National and international bodies have identified acute cardiovascular disease in the frail as a priority area for care and an entity that requires careful clinical decisions, but there remains a paucity of guidance on treatment efficacy and safety, and how to manage this complex group. This position paper from the Acute Cardiovascular Care Association presents the latest evidence about frailty and the management of frail patients with acute cardiovascular disease, and suggests avenues for future research.

Characterization of MTMR3. an inositol lipid 3-phosphatase with novel substrate specificity.

Inositol lipids play key roles in many fundamental cellular processes that include growth, cell survival, motility, and membrane trafficking. Recent studies on the PTEN and Myotubularin proteins have underscored the importance of inositol lipid 3-phosphatases in cell function. Inactivating mutations in the genes encoding PTEN and Myotubularin are key steps in the progression of some cancers and in the onset of X-linked myotubular myopathy, respectively. Myotubularin-related protein 3 (MTMR3) shows extensive homology to Myotubularin, including the catalytic domain, but additionally possesses a C-terminal extension that includes a FYVE domain. We show that MTMR3 is an inositol lipid 3-phosphatase, with a so-far-unique substrate specificity. It is able to hydrolyze PtdIns3P and PtdIns3,5P2, both in vitro and when heterologously expressed in S. cerevisiae, and to thereby provide the first clearly defined route for the cellular production of PtdIns5P. Overexpression of a catalytically dead MTMR3 (C413S) in mammalian cells induces a striking formation of vacuolar compartments that enclose membranous structures that are highly concentrated in mutant proteins.

Pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in goats.

OBJECTIVE: To evaluate pooled faecal culture for herd diagnosis of caprine Johne's disease and relate these findings to faecal shedding rates of Mycobacterium avium subsp paratuberculosis (Map). DESIGN: Radiometric broth culture was applied to several pooling dilutions, and shedding rates were estimated from a regression equation based on bacterial growth rates and known processing losses during radiometric culture. PROCEDURE: Sixteen faecal samples from goats naturally infected with sheep (n = 3) or cattle (n = 13) strains of Map, were diluted in normal goat faeces from 1 in 5 to 1 in 50. Cultures were confirmed by IS900 polymerase chain reaction and restriction endonuclease analysis, and mycobactin dependency. The numbers of viable Map in the culture inocula were determined by endpoint titration (most probable number) of nine samples and related to a cumulative growth index. RESULTS: A pooling dilution of 1 in 25 with an incubation period of 10 weeks detected 13 of 16 culture positive goats, all shedding > or = 2 x 10(4) Map per gram of faeces. Two samples containing very low numbers of Map (< 2 x 10(3)/g) were only culture positive from undiluted faeces. Thirteen of 16 goats were considered to be shedding low to moderate concentrations of Map (< 2 x 10(5)/g faeces). CONCLUSIONS: These data support a pooling dilution of 1 in 25 for application of pooled faecal culture as a diagnostic tool in caprine Johne's disease control. A test based on this dilution would reduce laboratory costs of whole herd testing in goats by approximately 40% relative to serology and 75 to 90% relative to individual faecal culture.

Individual placement and support (IPS) for patients with offending histories: the IPSOH feasibility cluster randomised trial protocol.

INTRODUCTION: People with involvement in forensic psychiatric services face many obstacles to employment, arising from their offending, as well as their mental health problems. This study aims to assess the feasibility of conducting a randomised controlled trial (RCT) to evaluate the effectiveness of individual placement and support (IPS), in improving employment rates and associated psychosocial outcomes in forensic psychiatric populations. IPS has been found consistently to achieve employment rates above 50% in psychiatric patients without a history of involvement in criminal justice services. METHODS/DESIGN: This is a single-centre feasibility cluster RCT. Clusters will be defined according to clinical services in the community forensic services of Nottinghamshire Healthcare NHS Foundation Trust (NHCT). IPS will be implemented into 2 of the randomly assigned intervention clusters in the community forensic services of NHCT. A feasibility cluster RCT will estimate the parameters required to design a full RCT. The primary outcome is the proportion of people in open employment at 12-month follow-up. Secondary outcome measures will include employment, educational activities, psychosocial and economic outcomes, as well as reoffending rates. Outcome measures will be recorded at baseline, 6 months and 12 months. In accordance with the UK Medical Research Council guidelines on the evaluation of complex interventions, a process evaluation will be carried out; qualitative interviews with patients and staff will explore general views of IPS as well as barriers and facilitators to implementation. Fidelity reviews will assess the extent to which the services follow the principles of IPS prior, during and at the end of the trial. ETHICS AND DISSEMINATION: Ethical approval was obtained from the East Midlands Research Ethics Committee-Nottingham 1 (REC reference number 15/EM/0253). Final and interim reports will be prepared for project funders, the study sponsor and clinical research network. Findings will be disseminated through peer-reviewed journals, conferences and event presentations. TRIAL REGISTRATION NUMBER: NCT02442193; Pre-results.

A case of aggressive multiple metachronous central giant cell granulomas of the jaws: differential diagnosis and management options.

We describe multiple metachronous central giant cell granulomas in a 62-year-old man who has a first degree relative with a history of a solitary central giant cell granulomas. The patient presented in 1997 with a large central giant cell granuloma of the right maxilla which was treated with a partial maxillectomy. A small recurrence was then identified and the successful management of this is described. The patient has also a histologically confirmed central giant cell granuloma previously removed from the right body of the mandible and the left angle of the mandible. The differential diagnosis of multiple central giant cell granulomas of the jaw is considered. It is possible that the present case may indeed represent a new syndrome or subtype of multiple central giant cell granulomas. The problem of treating such aggressive sub-types of giant cell granulomas is also addressed in the context of recent advances of surgical and medical management.

A single five minute period of rapid atrial pacing fails to limit infarct size in the in situ rabbit heart.

OBJECTIVE: Rapid pacing has been shown to precondition the dog heart against ischaemic dysrhythmia. The aim of this study was to determine whether rapid pacing could also limit infarct size. METHODS: Rabbits (n = 5) were rapidly paced via the left atrium at 420-480 beats.min-1. Five min of rapid pacing and 10 min of recovery in sinus rhythm were followed by 45 min of regional ischaemia and 120 min of reperfusion. Control rabbits (n = 9) were treated identically without prior rapid pacing. Infarct size was determined in both groups using tetrazolium and expressed as a percentage of the area at risk demarcated by fluorescent microspheres. In a separate series of experiments, rapidly paced Langendorff perfused rabbit hearts (n = 9) were used to determine coronary flow under perfusion conditions designed to simulate the in vivo situation during rapid pacing. RESULTS: Rapid pacing caused a fall in systolic pressure from 91.4(SEM 4.5) to 47.0(5.9) mm Hg (p < 0.01) and diastolic pressure from 67.2(2.9) to 23.6(3.2) mm Hg (p < 0.01). Both recovered within 30 s of cessation of pacing. During rapid pacing the action potential duration shortened from 192(13) to 128(5) ms (p = 0.01) and developed electrical alternans (n = 4). Following rapid pacing the ECG showed either ST depression or T wave inversion (n = 4). Despite these profound changes, rapid pacing did not reduce infarct size v control [52.7(4.6)% v 60.8(9.1)% of the area at risk, respectively]. The in vitro experiments estimated that rapid pacing would result in a reduction in coronary flow to 44% of that in sinus rhythm without a significant rise in lactate efflux. CONCLUSIONS: In our model, pretreatment with rapid pacing fails to reduce infarct size. The most likely reason for this is that rapid pacing at a rate of 480 beats.min-1 does not cause myocardial ischaemia of sufficient severity to trigger the preconditioning response.

Heat stress limits infarct size in the isolated perfused rabbit heart.

OBJECTIVE: Heat stress, with the expression of heat stress proteins, has been shown to protect the rabbit heart in vitro against global ischaemia/reperfusion injury, though no benefit is apparent in an in vivo rabbit model of infarct size. The aim of this study was therefore to investigate this discrepancy and to discover whether heating itself has any effect which could negate the protection derived from myocardial stress protein synthesis. METHODS: (1) To ascertain whether heat stress could limit infarct size in the absence of blood, isolated buffer perfused hearts, with or without prior heat stress, were subjected to 45 min of regional ischaemia and 120 min reperfusion, and the resulting infarct size was expressed as a percentage of the risk area (I/R%). (2) The observations were repeated in an isolated blood perfused heart model in which a support rabbit (heat stressed or control) was used to perfuse the isolated heart. RESULTS: In the buffer perfused heart, prior heat stress reduced I/R from 70.8(SEM 4.4)%, n = 10, in controls to 51.5(5.7)%, n = 12 (p < 0.05). In hearts perfused by support rabbits, prior heat stress reduced I/R [from 34.7(3.7)%, n = 16, to 23.5(3.3)%, n = 15 (p < 0.05)] only when the perfusing rabbit was a control (not heat stressed). If the perfusing rabbit had been heated, I/R was greater in both heat stressed and control hearts [51.9(7.0)% and 44.9(3.3)%, p < 0.05 v control support rabbit]. CONCLUSIONS: Heat stress limits infarct size in this rabbit model. However it appears to have additional adverse effects, probably on the blood, which may override any benefit associated with myocardial stress protein synthesis.

The clonal organization of the squamous epithelium of the tongue.

Knowledge of the kinetics and stem cell localization of the mouse lingual epithelium is largely based on studies using DNA labelling techniques. We have adopted a different approach, using histochemistry for the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD). We have deduced clone size and morphology from studies of patch size and distribution in mice heterozygous for G6PD deficiency and from the identification of clonal enzyme loss induced in normal mice by application of a mutagen. Lingual epithelium of female mice (CBA X GPDX) heterozygous for G6PD deficiency showed multiple clearly defined patches of strong or weak enzyme activity, corresponding in intensity to the strong staining uniformly present in the normal parental strain (CBA) or to the weak staining uniformly present in the G6PD deficient parental strain (GPDX). This pattern results from the random suppression of either the paternal or the maternal X chromosome in each cell early in embryonic development, and the subsequent inheritance of X inactivation in daughter cells, giving rise to phenotypic patches each composed of one or more clones. The patch borders intersected the base of the lingual epithelium at small indentations or at the apices of connective tissue papillae; the surface intersection in some cases bisected filiform papillae. Patch width measured in tissue sections at the mid rete ridge level, showed a clear mode close to 40 microns, corresponding very closely to the mode for rete ridge width (i.e. distance between connective tissue papillae). Further evidence for clonal organization was obtained by inducing mutations in the lingual epithelium of CBA mice by topical mutagen application. A few clearly defined patches of enzyme loss were found with a mean diameter of 36 microns. Their morphology was very similar to that of patches in the heterozygous animals. We interpret these patches as clones derived from stem cells with induced somatic G6PD mutations. We conclude that the mouse lingual epithelium is a stem cell epithelium composed of clonal units of about 40 microns diameter, based on the rete ridge structure and that both connective tissue papillae and filiform papillae occur at the junction of two or more epithelial clones.

Class-specific antibodies to Streptococcus mutans in human serum, saliva and breast milk.

Previous techniques used for the detection and quantitation of antibodies in body fluids may be inappropriate where only small volumes are available, or may not be sensitive enough to detect low levels of specific antibodies. An indirect ELISA technique has successfully been employed to estimate class-specific antibody levels to Streptococcus mutans in serum and secretions in a group of mothers and their neonates, and an attempt has been made to relate such levels to the presence or absence of active caries in the mothers. A high maternal serum IgG antibody level appears to exert a protective effect against dental caries. Antibody levels in maternal saliva and colostrum/breast milk showed no differences between the 2 groups. The presence of active caries in mothers was associated with an elevated IgA antibody level in neonatal saliva. Although ELISA permitted the detection of low levels of antibody in the small volumes of neonatal saliva collected, a further increase in sensitivity and specificity of the assay would be advantageous.

Design of a potent combined pseudopeptide endothelin-A/endothelin-B receptor antagonist, Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21 (PD 156252): examination of its pharmacokinetic and spectral properties.

The endothelins (ETs) are a family of bicyclic 21-amino acid peptides that are potent and prolonged vasoconstrictors. It has been shown that highly potent combined ETA/ETB receptor antagonists can be developed from the C-terminal hexapeptide of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21), such as Ac-(D)Dip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) and Ac-DBhg16-Leu-Asp-Ile-Ile-Trp21 (PD 145065). However, these compounds are relatively unstable to enzymatic proteolysis as determined in an in vitro rat intestinal perfusate assay. This instability is thought to be due to carboxypeptidase activity. In fact, incubation of PD 145065 with carboxypeptidase inhibitors greatly increased its half-life in rat intestinal perfusate. By performing a reduced amide bond and N-methyl amino acid scan, it was discovered that N-methylation of Ile-20 resulted in a compound (Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21, PD 156252) that retained full receptor affinity at both endothelin receptor subtypes along with enhanced proteolytic stability and cellular permeability. Interestingly, N-methylation of this bond allows the cis configuration to be readily accessible which greatly alters the preferred structure of the entire molecule and may be responsible for the observed enhanced metabolic stability.

Structure-activity relationships of the potent combined endothelin-A/endothelin-B receptor antagonist Ac-DDip16-Leu-Asp-Ile-Ile-Trp21: development of endothelin-B receptor selective antagonists.

The endothelins (ETs) are a family of bicyclic 21-amino acid-containing peptides that are highly potent and prolonged vasoconstrictors. The discovery of potent ET antagonists will facilitate the understanding of the physiological and/or pathophysiological role of ET. Structure-activity studies have revealed the importance of the C-terminal hexapeptide (residues 16-21) of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21) to the development of potent antagonists at both receptor subtypes (ETA and ETB). In particular, it has been shown that Ac-DDip16-Leu-Asp-Ile-Ile-Trp21 (Dip = 3,3-diphenylalanine) has low nanomolar affinity for the two endothelin receptor subtypes and is a functional antagonist of ET activity, both in vitro and in vivo at both receptors. Herein, we will describe the structure-activity relationships of Ac-DDip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) with a particular emphasis on modifications that lead to enhanced receptor affinity and/or individual receptor subtype selectivity. In particular, we will demonstrate how we utilized PD 142893 to develop ETB receptor selective ligands and the pharmacological differences that exist between species ETB receptors with respect to their affinity for C-terminal hexapeptide antagonists.