Assessing dynamic violence risk: Common language risk levels and recidivism rates for the Violence Risk Scale.

The present study features the development of new risk categories and recidivism estimates for the Violence Risk Scale (VRS), a violence risk assessment and treatment planning tool. We employed a combined North American multisite sample (k = 6, N = 1,338) of adult mostly male offenders, many with violent criminal histories, from correctional or forensic mental health settings that had complete VRS scores from archival or field ratings and outcome data from police records (N = 1,100). There were two key objectives: (a) to identify the rates of violent recidivism associated with VRS scores and (b) to generate updated evidence-based VRS violence risk categories with external validation. To achieve the first objective, logistic regression was applied using VRS pretreatment and change scores on treated samples with a minimum 5-year follow-up (k = 5, N = 472) to model 2-, 3-, and 5-year violent and general recidivism estimates, with the resulting logistic regression algorithms retained to generate a VRS recidivism rates calculator. To achieve the second objective, the Council of State Governments' guidelines were applied to generate five risk levels using the common language framework using percentiles, risk ratios (from Cox regression), and absolute violent and general recidivism estimates (from logistic regression). Construct validity of the five risk levels was examined through group comparisons on measures of risk, need, protection, and psychopathy obtained from the constituent samples. VRS applications to enhance risk communication, treatment planning, and violence prevention in light of the updated recidivism estimates and risk categories are discussed. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Signatures of Post-Glacial Genetic Isolation and Human-Driven Migration in the Dothistroma Needle Blight Pathogen in Western Canada.

Many current tree improvement programs are incorporating assisted gene flow strategies to match reforestation efforts with future climates. This is the case for the lodgepole pine (Pinus contorta var. latifolia), the most extensively planted tree in western Canada. Knowledge of the structure and origin of pathogen populations associated with this tree would help improve the breeding effort. Recent outbreaks of the Dothistroma needle blight (DNB) pathogen Dothistroma septosporum on lodgepole pine in British Columbia and its discovery in Alberta plantations raised questions about the diversity and population structure of this pathogen in western Canada. Using genotyping-by-sequencing on 119 D. septosporum isolates from 16 natural pine populations and plantations from this area, we identified four genetic lineages, all distinct from the other DNB lineages from outside of North America. Modeling of the population history indicated that these lineages diverged between 31.4 and 7.2 thousand years ago, coinciding with the last glacial maximum and the postglacial recolonization of lodgepole pine in western North America. The lineage found in the Kispiox Valley from British Columbia, where an unprecedented DNB epidemic occurred in the 1990s, was close to demographic equilibrium and displayed a high level of haplotypic diversity. Two lineages found in Alberta and Prince George (British Columbia) showed departure from random mating and contemporary gene flow, likely resulting from pine breeding activities and material exchanges in these areas. The increased movement of planting material could have some major consequences by facilitating secondary contact between genetically isolated DNB lineages, possibly resulting in new epidemics.

A comprehensive examination of the psychometric properties of the Hare Psychopathy Checklist-Revised in a Canadian multisite sample of indigenous and non-indigenous offenders.

The present study examined the psychometric properties of Hare Psychopathy Checklist-Revised (PCL-R; Hare, 2003) scores in a multisite sample of 1,163 federally incarcerated Canadian indigenous and non-indigenous offenders from the Prairie Region of the Correctional Service of Canada. The research occurred against the backdrop of the Ewert v. Canada (2015) matter, in which the PCL-R was originally impugned in Federal Court for use with indigenous persons (later overturned in Canada v. Ewert, 2016). Indigenous men scored higher than non-indigenous men on most components of the PCL-R and had higher rates of recidivism, irrespective of follow-up. Discrimination analyses, however, supported the predictive efficacy of PCL-R total, factor, and facet scores for violent and general recidivism across both ancestral groups, with most group differences in area under the curve (AUC) magnitudes being small and nonsignificant. Calibration analyses demonstrated that higher PCL-R scores were associated with higher rates of general and violent recidivism for both ancestral groups, although higher recidivism rates were observed and estimated for indigenous men at specific PCL-R score thresholds. Confirmatory factor analyses supported the 4-factor model of psychopathy and hence, structural invariance, of PCL-R scores across ancestral groups. Structural equation modeling affirmed the predictive efficacy of the 4-factor model for recidivism. We discuss these findings in terms of clinical applications of the PCL-R and the psychopathy construct in general, with male offenders of indigenous ancestry. (PsycINFO Database Record

Cardiac CRFR1 Expression Is Elevated in Human Heart Failure and Modulated by Genetic Variation and Alternative Splicing.

Corticotropin-releasing factor (CRF) and the CRF-related peptides, urocortin (Ucn)-1, Ucn2, and Ucn3 signal through receptors CRFR1 and CRFR2 to restore homeostasis in response to stress. The Ucns exert potent cardioprotective effects and may have clinical utility in heart failure. To explore the activity of this system in the heart, we measured the levels of myocardial gene expression of the CRF/Ucn family of ligands/receptors and investigated genetic variation and alternative splicing of CRFR1 in 110 heart failure patients and 108 heart donors. Using quantitative real-time PCR, we detected CRFR1, CRFR2, CRF, Ucn1, Ucn2, and Ucn3 in all samples. CRFR2α was the most abundant receptor and Ucn3 the most abundant ligand, both in patients and donors. Compared with donors, cardiac expression of CRFR1, CRF, and Ucn3 was higher (P < .001) and CRFR2α lower (P = .012) in patients. In patients and donors, genetic variation within CRFR1, represented by the chromosome 17q21.31 inversion polymorphism, was associated with markedly higher CRFR1 expression (P < .001), making CRFR1 and CRFR2α expression almost equivalent in some patients. A novel, truncated splice variant of CRFR1, designated CRFR1j, was identified and shown to exert a dominant-negative effect on CRFR1 signaling in vitro. The novel variant was expressed in a greater proportion of patients (60%) than donors (3%, P < .001). In summary, cardiac expression of CRFR1, CRF, and Ucn3 genes is elevated in heart failure and may contribute to the activation of the CRF/Ucn system in these patients. A common variant within the CRFR1 gene and a novel CRFR1 splice variant may modulate CRFR1 expression and signaling.

Characterization of Multisubstituted Corticotropin Releasing Factor (CRF) Peptide Antagonists (Astressins).

CRF mediates numerous stress-related endocrine, autonomic, metabolic, and behavioral responses. We present the synthesis and chemical and biological properties of astressin B analogues {cyclo(30-33)[D-Phe(12),Nle(21,38),C(α)MeLeu(27,40),Glu(30),Lys(33)]-acetyl-h/r-CRF(9-41)}. Out of 37 novel peptides, 17 (2, 4, 6-8, 10, 11, 16, 17, 27, 29, 30, 32-36) and 16 (3, 5, 9, 12-15, 18, 19, 22-26, 28, 31) had k(i) to CRF receptors in the high picomolar and low nanomole ranges, respectively. Peptides 1, 2, and 11 inhibited h/rCRF and urocortin 1-induced cAMP release from AtT20 and A7r5 cells. When Astressin C 2 was administered to adrenalectomized rats at 1.0 mg subcutaneously, it inhibited ACTH release for >7 d. Additional rat data based on the inhibitory effect of (2) on h/rCRF-induced stimulation of colonic secretory motor activity and urocortin 2-induced delayed gastric emptying also indicate a safe and long-lasting antagonistic effect. The overall properties of selected analogues may fulfill the criteria expected from clinical candidates.

Characterization of a Pachymedusa dacnicolor-Sauvagine analog as a new high-affinity radioligand for corticotropin-releasing factor receptor studies.

The corticotropin-releasing factor (CRF) peptide family comprises the mammalian peptides CRF and the urocortins as well as frog skin sauvagine and fish urophyseal urotensin. Advances in understanding the roles of the CRF ligand family and associated receptors have often relied on radioreceptor assays using labeled CRF ligands. These assays depend on stable, high-affinity CRF analogs that can be labeled, purified, and chemically characterized. Analogs of several of the native peptides have been used in this context, most prominently including sauvagine from the frog Phyllomedusa sauvageii (PS-Svg). Because each of these affords both advantages and disadvantages, new analogs with superior properties would be welcome. We find that a sauvagine-like peptide recently isolated from a different frog species, Pachymedusa dacnicolor (PD-Svg), is a high-affinity agonist whose radioiodinated analog, [(125)ITyr(0)-Glu(1), Nle(17)]-PD-Svg, exhibits improved biochemical properties over those of earlier iodinated agonists. Specifically, the PD-Svg radioligand binds both CRF receptors with comparably high affinity as its PS-Svg counterpart, but detects a greater number of sites on both type 1 and type 2 receptors. PD-Svg is also ∼10 times more potent at stimulating cAMP accumulation in cells expressing the native receptors. Autoradiographic localization using the PD-Svg radioligand shows robust specific binding to rodent brain and peripheral tissues that identifies consensus CRF receptor-expressing sites in a greater number and/or with greater sensitivity than its PS-Svg counterpart. We suggest that labeled analogs of PD-Svg may be useful tools for biochemical, structural, pharmacological, and anatomic studies of CRF receptors.

A cluster analytic examination and external validation of psychopathic offender subtypes in a multisite sample of Canadian federal offenders.

The present study is a cluster analytic examination and validation of psychopathic offender subtypes from 4 combined samples of Canadian federally incarcerated offenders, most of whom were serving sentences for violent offenses. The men were rated on the Hare Psychopathy Checklist-Revised (PCL-R; Hare, 1991, 2003) on the basis of comprehensive file information and 314 cases were extracted using a PCL-R total cut score of 25. Cluster analysis of the 4 PCL-R facets converged at a 2-cluster solution: a primary subtype characterized by prominent interpersonal and affective features of psychopathy and a secondary subtype characterized by comparatively few interpersonal features and high scores on the remaining facets. Validation analyses found that the vast majority of primary psychopathic offenders (74.1%) were White or of non-Aboriginal descent in contrast to the secondary subtype (47.6%). Secondary psychopathic offenders tended to be actuarially higher risk, have greater criminogenic needs, and to make greater amounts of treatment change on criminogenic targets; however, contrary to expectations, within-treatment changes from a violence reduction program were significantly associated with reductions in violent recidivism for primary, but not secondary, variants. There were few differences in rates of recidivism between the groups overall; secondary variants had higher rates of sexual violence which was largely accounted for by individual differences in baseline static risk. Implications for risk assessment, treatment planning, and the classification and etiology of primary and secondary psychopathy are discussed.

Spore Dispersal by Dothistroma septosporum in Northwest British Columbia.

We studied spore dispersal by Dothistroma septosporum, causal agent of a serious outbreak of red band needle blight in lodgepole pine plantations in northwest British Columbia. Spore abundance was assessed at different distances and heights from inoculum sources and microclimatic factors were recorded during two consecutive years. Conidia were observed on spore traps from June to September during periods of rainfall. It was rare to detect spores more than 2 m away from inoculum sources. The timing and number of conidia dispersed were strongly tied to the climatic variables, particularly rainfall and leaf wetness. Should the trend toward increased spring and summer precipitation in the study area continue, the results suggest that disease spread and intensification will also increase. Increasing the planting distances between lodgepole pine trees through mixed species plantations and overall reduction in use of lodgepole pine for regeneration in wet areas are the best strategies to reduce the spread of the disease and enhance future productivity of plantations in the study area.

Expression and functional characterization of membrane-integrated mammalian corticotropin releasing factor receptors 1 and 2 in Escherichia coli.

Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2ß as well as fragments lacking the N-terminal extracellular domain, were overproduced in E. coli. The characteristics of different CRFR2ß-PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2ß expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of E. coli bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2ß product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2ß were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2ß membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our E. coli expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.

The Violence Risk Scale: predictive validity and linking changes in risk with violent recidivism in a sample of high-risk offenders with psychopathic traits.

The Violence Risk Scale (VRS) uses ratings of static and dynamic risk predictors to assess violence risk, identify targets for treatment, and assess changes in risk following treatment. The VRS was rated pre- and posttreatment on a sample of 150 males, mostly high-risk violent offenders many with psychopathic personality traits. These individuals attended a high-intensity institution-based cognitive-behavioral-oriented violence reduction treatment program in Canada and were then followed up for approximately 5 years postrelease to determine court adjudicated community violent recidivism. VRS scores significantly predicted violent recidivism. Measurements of risk reduction using dynamic VRS predictors were significantly correlated with reduction of violent recidivism after controlling for various potential confounds. The results suggest that, in a high-risk group of offenders with significant psychopathic traits, the VRS demonstrated predictive validity and the dynamic predictors can be used to assess treatment progress, which is linked to a specific criterion variable, thus, fulfilling the criteria for causal dynamic predictors set forth by Kraemer et al.

Risk reduction treatment of high-risk psychopathic offenders: the relationship of psychopathy and treatment change to violent recidivism.

The relationships of psychopathy, therapeutic change, and violent recidivism were examined in a sample of 152 high-risk violent offenders treated in a high-intensity violence reduction program at the Regional Psychiatric Centre (RPC) in Saskatoon, SK. The Violence Risk Scale (VRS; Wong & Gordon, 1999-2003) and Psychopathy Checklist-Revised (PCL-R; Hare, 1991, 2003) were rated on the sample. As an extension on a prior psychometric study of the VRS (Lewis, Olver, & Wong, 2012), the associations of therapeutic change scores, obtained from pre- and posttreatment ratings of VRS dynamic items, and violent recidivism were examined among high-risk psychopathic offenders (mean PCL-R >25) over approximately 5 years' follow-up. Positive therapeutic change correlated negatively with the PCL-R, particularly Factor 1 and the Affective facet, and was significantly associated with reductions in violent recidivism after controlling for psychopathy. The association of change to violent outcome decreased, however, when controlling for the Affective facet. Taken together, the present results suggest that risk-related treatment changes demonstrated by high-risk psychopathic offenders can be predictive of reductions in violent recidivism, and that reliable measurements of therapeutic change may be informative about treatment outcome in a high-risk violent offender group.

Ecosystem, location, and climate effects on foliar secondary metabolites of lodgepole pine populations from central British Columbia.

Lodgepole pines, Pinus contorta Douglas ex Louden var. latifolia Engelm. ex S. Watson, are encountering increased abiotic stress and pest activity due to recent increases in temperature and changes in precipitation throughout their range. This tree species counters these threats by producing secondary metabolites, including phenolics and terpenoids. We examined foliar levels of lignin, soluble phenolics, monoterpenoids, sesquiterpenoids, and diterpenoids in 12 stands in British Columbia, Canada. We used these data to assess associations among foliar secondary metabolite levels and ecosystem, geographic, and climatic variables. Regressions were also performed to observe which combinations of variables best explained secondary metabolite variance. Stands of P. c. latifolia in the Coastal Western Hemlock and Interior Cedar/Hemlock biogeoclimatic zones had consistently greater foliar levels of almost all measured secondary metabolites than did other stands. Lignin was present in greater amounts in Boreal White/Black Spruce ecosystem (i.e., northern) stands than in southern stands, suggesting a role for this metabolite in pine survival in the boreal forest. Attempts to develop regression models with geographic and climatic variables to explain foliar secondary metabolite levels resulted in multiple models with similar predictive capability. Since foliar secondary metabolite levels appeared to vary most between stand ecosystem types and not as much due to geographic and climatic variables, metabolic profiles appeared best matched to the stress levels within local environments. It is unknown if differences in secondary metabolite levels are the result of genetic adaptation or phenotypic plasticity, but results from this and other studies suggest that both are important. These results are interpreted in light of ongoing efforts to assist in the migration of certain populations of P. c. latifolia northward in an effort to counter predicted effects of climate change.

Endogenous betaglycan is essential for high-potency inhibin antagonism in gonadotropes.

Inhibins are endocrine hormones that regulate gametogenesis and reproduction through a negative feedback loop with FSH. Inhibin action involves antagonism of signaling by activin or other TGFbeta family ligands. In transfection assays, antagonism by inhibin can be potentiated by betaglycan, a coreceptor for selected TGFbeta family ligands. We tested whether betaglycan is an obligate inhibin coreceptor through disruption of betaglycan function by RNA interference-mediated knockdown and immunoneutralization. Betaglycan knockdown and anti-betaglycan IgG each independently prevented inhibin-A binding to betaglycan and reversed functional effects of transfected betaglycan. Neither betaglycan immunoneutralization nor knockdown affected activin responsiveness in cell lines or in rat anterior pituitary cultures. Betaglycan knockdown decreased the potency of inhibin antagonism of activin-induced FSH secretion in primary gonadotropes. Similarly, anti-betaglycan IgG decreased the potency of inhibin antagonism in primary gonadotropes in a dose-dependent manner, with a reduction in the sensitivity to inhibin-A of greater than 1000-fold. These data establish that betaglycan is an endogenous inhibin coreceptor required for high-sensitivity inhibin antagonism of activin signaling in rat anterior pituitary gonadotropes.

Characterization and distribution of mating type genes in the dothistroma needle blight pathogens.

ABSTRACT Dothistroma septosporum and D. pini are the two causal agents of Dothistroma needle blight of Pinus spp. in natural forests and plantations. Degenerate primers amplified portions of mating type genes (MAT1-1-1 and MAT1-2) and chromosome walking was applied to obtain the full-length genes in both species. The mating-type-specific primers designed in this study could distinguish between the morphologically similar D. pini and D. septosporum and between the different mating types of these species. Screening of isolates from global collections of D. septosporum showed that only MAT2 isolates are present in Australian and New Zealand collections, where only the asexual form of the fungus has been found. In contrast, both mating types of D. septosporum were present in collections from Canada and Europe, where the sexual state is known. Intriguingly, collections from South Africa and the United Kingdom, where the sexual state of the fungus is unknown, included both mating types. In D. pini, for which no teleomorph is known, both mating types were present in collections from the United States. These results provided new insights into the biology and global distribution of two of the world's most important pine pathogens and should facilitate management of the diseases caused by these fungi.

Development and factor structure of a brief instrument to assess the impact of community programs on positive youth development: The Rochester Evaluation of Asset Development for Youth (READY) tool.

PURPOSE: Positive youth development is increasingly recognized as important to the health and well-being of adolescents. We describe the development of a brief, standardized instrument for measuring outcomes and indicators important for youth development evaluation. Additionally, we describe the psychometric properties of these constructs. METHODS: A coalition of representatives from 12 community organizations, funders, and researchers identified four core outcomes associated with positive youth development likely able to be affected by community-based youth-serving agencies. Items were piloted and field tested to test feasibility and establish face validity with experts and with adolescents. Furthermore, we tested construct reliability using factor analysis to determine how well the items reflected underlying constructs. RESULTS: Items representing four empirical constructs were used to survey 389 adolescents. Those who participated in the pilot took an average of 11 minutes to complete the survey. Overall, 24 items loaded on to six discrete factors representing the outcomes of basic social skills, caring adult relationships, and decision-making. When participants under age 13 years were eliminated from the analysis, items were more cohesive, resulting in a five-factor solution with all items loading at .40 or higher. CONCLUSIONS: The four youth development outcomes identified by community-based youth-serving organizations factor into reliable constructs with acceptable alpha coefficients for adolescents over age 13. A major strength of this youth development outcome measurement is that it has an easy-to-administer format, which allows community-based programs to receive feedback for program improvement, and to track the effectiveness of their programs for funders.

Identification of distinct inhibin and transforming growth factor beta-binding sites on betaglycan: functional separation of betaglycan co-receptor actions.

Betaglycan is a co-receptor that mediates signaling by transforming growth factor beta (TGFbeta) superfamily members, including the distinct and often opposed actions of TGFbetas and inhibins. Loss of betaglycan expression, or abrogation of betaglycan function, is implicated in several human and animal diseases, although both betaglycan actions and the ligands involved in these disease states remain unclear. Here we identify a domain spanning amino acids 591-700 of the betaglycan extracellular domain as the only inhibin-binding region in betaglycan. This binding site is within the betaglycan ZP domain, but inhibin binding is not integral to the ZP motif of other proteins. We show that the inhibin and TGFbeta-binding residues of this domain overlap and identify individual amino acids essential for binding of each ligand. Mutation of Val614 to Tyr abolishes both inhibin and TGFbeta binding to this domain. Full-length betaglycan V614Y, and other mutations, retain TGFbeta binding activity via a distinct site, but are unable to bind inhibin-A. These betaglycan mutants fail to mediate inhibin antagonism of activin signaling but can present TGFbeta to TbetaRII. Separating the co-receptor actions of betaglycan toward inhibin and TGFbeta will allow the clarification of the role of betaglycan in disease states such as renal cell carcinoma and endometrial adenocarcinoma.

Outcomes of clinical examination and genetic testing of 500 individuals with hearing loss evaluated through a genetics of hearing loss clinic.

Hearing loss (HL) occurs in approximately 2 out of every 1,000 births and is genetic in origin in approximately 50% of cases. This high incidence coupled with the increasing number of genes implicated in HL and the trend toward universal newborn screening led to the establishment of the Genetics of Hearing Loss Clinic at The Children's Hospital of Philadelphia to manage the diagnosis, genetic screening, and counseling of families with an affected child. To date 500 individuals have been evaluated from 1999 to 2004. To determine the cause of their HL and screen for syndromic forms of HL, individuals were offered a panel of tests. Depending on the type and severity of the HL, recommendations included GJB2 mutation analysis, renal and thyroid function studies, a CT scan of the temporal bones, an ophthalmology evaluation, an EKG, and, at times, additional genetic tests. Of the 500 patients evaluated 70 (14%) had a syndromic etiology for their HL. Twenty-eight different syndromic etiologies were identified. Enlarged vestibular aqueducts (EVAs) and/or Mondini malformations were seen in 18% of individuals with HL who had a CT or MRI of the temporal bones. Genetic testing of the GJB2 gene was completed for 310 of the 377 patients with bilateral sensorineural HL (82.2%). Nineteen different variants were identified in the GJB2 gene. Through GJB2 mutational analysis, clinical examination, and laboratory testing, a definitive etiologic diagnosis was established in 110/500 (22%) of patients.

A soluble mouse brain splice variant of type 2alpha corticotropin-releasing factor (CRF) receptor binds ligands and modulates their activity.

Peptides of the corticotropin-releasing factor (CRF) family signal through the activation of two receptors, CRF receptor type 1 (CRFR1) and type 2 (CRFR2), both of which exist as multiple splice variants. We have identified a cDNA from mouse brain encoding a splice variant, soluble CRFR2alpha (sCRFR2alpha), in which exon 6 is deleted from the gene encoding CRFR2alpha. Translation of this isoform produces a predicted 143-aa soluble protein. The translated protein includes a majority of the first extracellular domain of the CRFR2alpha followed by a unique 38-aa hydrophilic C terminus resulting from a frame shift produced by deletion of exon 6. By using RT-PCR and Southern hybridization, the relative mRNA expression levels of full-length (seven transmembrane domains) CRFR2alpha and the soluble form (sCRFR2alpha) in the mouse brain were measured with a single reaction. The results demonstrate high levels of expression of sCRFR2alpha in the olfactory bulb, cortex, and midbrain regions. A rabbit antiserum raised against a synthetic peptide fragment encoding the unique C terminus revealed specific sCRFR2alpha immunoreactivity in mouse brain slices by immunohistochemistry and in extracts of brain regions by RIA. Interestingly, the sCRFR2alpha immunoreactivity distribution closely approximated that of CRFR1 expression in rodent brain. A protein corresponding to sCRFR2alpha, expressed and purified from either mammalian or bacterial cell systems, binds several CRF family ligands with low nanomolar affinities. Furthermore, the purified sCRFR2alpha protein inhibits cellular responses to CRF and urocortin 1. These data support a potential role of the sCRFR2alpha protein as a possible biological modulator of CRF family ligands.

Mouse corticotropin-releasing factor receptor type 2alpha gene: isolation, distribution, pharmacological characterization and regulation by stress and glucocorticoids.

Effects of the corticotropin-releasing factor (CRF) family of peptides are mediated through activation of two receptors, CRF receptor (CRFR) 1 and CRFR2. Based on the homology between known mammalian CRFR genes, we have isolated a cDNA encoding the mouse CRFR2alpha (mCRFR2alpha) ortholog from brain. The isolated cDNA encodes a 411-amino acid protein with high identity to the rat (approximately 97%) and human (approximately 93%) receptors. Central and peripheral expression of mCRFR2alpha, determined by RT-PCR followed by Southern hybridization, revealed that mCRFR2alpha is restricted mainly to brain structures, with highest levels in the hypothalamus and olfactory bulb. In situ hybridization showed mCRFR2alpha localization in discrete brain regions, including the lateral septum and the ventromedial hypothalamus, whereas mCRFR2beta is found only in the choroid plexus. Binding and signaling of CRF-related ligands was studied using COS-M6 or HEK293T cells transiently transfected with mCRFR2alpha. Urocortins (Ucns) show different affinities for binding to mCRFR2alpha: Ucn 3 binds mCRFR2alpha with approximately 11-fold lower affinity than Ucn 2, which displays an affinity similar to Ucn 1 (approximately 1 nm). Cyclase activation, determined by intracellular cAMP accumulation and cAMP response element-luciferase activity, showed no differences between CRFR2alpha and CRFR2beta in response to stimulation by Ucn 1, Ucn 2, and Ucn 3. Interestingly, Ucn 3 was less efficacious than Ucn 1 or Ucn 2 in activating MAPK (ERK1/2-p44/p42) via CRFR2alpha, but all three Ucns showed equivalent efficacy for activating MAPK through mCRFR2beta. We found a significant reduction in hypothalamic mCRFR2alpha mRNA levels after acute and chronic restraint stress in mice. Hypothalamic mCRFR2alpha gene transcription in mice was inhibited by glucocorticoid administration and elevated by adrenalectomy. In addition, we demonstrated that the mCRFR2alpha gene is increased in the hypothalamus of the CRFR1-null compared with wild type mice. The predicted mCRFR2alpha promoter region was isolated and fused to a luciferase reporter gene and found to be decreased by glucocorticoids in a dose and time-dependent manner when transfected into CATH.a cells. Computer analysis revealed the presence of 23 putative half-palindromic glucocorticoid response element sequences within 2.4 kb of the mCRFR2alpha 5' flanking region. Elucidation of the structure and processing of the mCRFR2 gene and examination of the mCRFR2alpha gene regulation in various conditions will enable better understanding of the involvement of this receptor in the central response to stress in normal and transgenic mice models.