Use of fluorescence polarization detection for the measurement of fluopeptidetm binding to G protein-coupled receptors.

G protein-coupled receptors (GPCRs) represent the single largest molecular target of therapeutic drugs currently on the market, and are also the most common target in high throughput screening assays designed to identify potential new drug candidates. A large percentage of these assays are now formatted as radioligand binding assays. Fluorescence polarization ligand binding assays can offer a non-rad alternative to radioligand binding assays. In addition, fluorescence polarization assays are a homogenous format that is easy to automate for high throughput screening. We have developed a series of peptide ligands labeled with the fluorescent dye BODIPY TMR whose binding to GPCRs can be detected using fluorescence polarization methodology. BODIPY TMR has advantages over the more commonly used fluorescein dye in high throughput screening (HTS) assays due to the fact that its excitation and emission spectra are red-shifted approximately 50 nm relative to fluorescein. Assays based on BODIPY TMR ligands are therefore less susceptible to interference from tissue auto-fluorescence in the assay matrix, or the effects of colored or fluorescent compounds in the screening libraries. A series of BODIPY TMR labeled peptides have been prepared that bind to a range of GPCRs including melanin concentrating hormone, bradykinin, and melanocortin receptors. Conditions have been optimized in order to utilize a comparable amount of receptor membrane preparation as is used in a radioligand binding assay. The assays are formatted in 384-well microplates with a standard volume of 40 microL. We have compared the assays across the different fluorescence polarization (FP) readers available to determine the parameters for each instrument necessary to achieve the required precision.

Efficacy of nurse telehealth care and peer support in augmenting treatment of depression in primary care.

BACKGROUND: Primary care treatment of depression needs improvement. OBJECTIVE: To evaluate the efficacy of 2 augmentations to antidepressant drug treatment. DESIGN: Randomized trial comparing usual care, telehealth care, and telehealth care plus peer support; assessments were conducted at baseline, 6 weeks, and 6 months. SETTING: Two managed care adult primary care clinics. PARTICIPANTS: A total of 302 patients starting antidepressant drug therapy. INTERVENTIONS: For telehealth care: emotional support and focused behavioral interventions in ten 6-minute calls during 4 months by primary care nurses; and for peer support: telephone and in-person supportive contacts by trained health plan members recovered from depression. MAIN OUTCOME MEASURES: For depression: the Hamilton Depression Rating Scale and the Beck Depression Inventory; and for mental and physical functioning: the SF-12 Mental and Physical Composite Scales and treatment satisfaction. RESULTS: Nurse-based telehealth patients with or without peer support more often experienced 50% improvement on the Hamilton Depression Rating Scale at 6 weeks (50% vs 37%; P =.01) and 6 months (57% vs 38%; P =.003) and on the Beck Depression Inventory at 6 months (48% vs 37%; P =. 05) and greater quantitative reduction in symptom scores on the Hamilton scale at 6 months (10.38 vs 8.12; P =.006). Telehealth care improved mental functioning at 6 weeks (47.07 vs 42.64; P =.004) and treatment satisfaction at 6 weeks (4.41 vs 4.17; P =.004) and 6 months (4.20 vs 3.94; P =.001). Adding peer support to telehealth care did not improve the primary outcomes. CONCLUSION: Nurse telehealth care improves clinical outcomes of antidepressant drug treatment and patient satisfaction and fits well within busy primary care settings.

The organizational context of non-lethal workplace violence: its interpersonal, temporal, and spatial correlates.

This article examines 993 violent incidents involving faculty, students, and staff that occurred at a highly ranked teaching and research university and its affiliated medical center. Violent incidents were included in the sample if they involved faculty, students, or staff, regardless of their specific location or context (i.e., whether they occurred on campus and/or off-campus and whether they occurred within the context of work or some other activity). The theoretical goal of the project was to compare work-related incidents with non-work-related incidents of interpersonal violence occurring in a single, multifunctioning, and professionally hierarchical organization. Data were collected over a 7-year period from three police departments (city, county, and university), university records, and criminal history records obtained from the state police. The coding protocol was developed to capture crime-scene information pertinent to each of the incidents. This included information about the victim, the perpetrator, the relationship between the victim and perpetrator, and the violent incident. The data were examined using nonparametric statistics and logistic regression to model predictive differences between the workplace and non-workplace incidents. The results suggest that the workplace incidents of violence differ from the non-workplace incidents according to their time, victim age, degree of victim injury, and whether the workplace is a medical location. The authors conclude that these differences are better explained by the movement of people in and out of the workplace who bring societal violence with them, rather than by a category or type of workplace violence.

Characterization of a new AMPA receptor radioligand, [3H]2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid.

(RS)-2-Amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), which is a potent and selective agonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, has previously been shown to desensitize AMPA receptors to a much lower degree than AMPA itself. We now report the synthesis of [3H]ACPA (32.5 Ci/mmol), the neurochemical and pharmacological characterization of [3H]ACPA binding, and a comparison of the distribution of [3H]ACPA, [3H]AMPA, and [3H](S)-5-fluorowillardiine binding sites in rat brain. Under equilibrium conditions, [3H]ACPA was shown to bind to a single population of receptor sites on rat brain membranes. [3H]ACPA was shown to bind with single and similar affinities (15-45 nM) to cloned AMPA receptor subunits (GluR1-4), expressed in insect cells, whereas a K(D) value of 330 nM was determined for the binding of [3H]ACPA to cloned kainic acid preferring GluR5 subunits. Whereas Bmax and K(D) values for [3H]ACPA binding, determined using filtration techniques, were different from such obtained in centrifugation assays, Bmax and K(D) values as well as association and dissociation constants were not significantly affected by the addition of the chaotropic agent KSCN. K(D) values, determined under equilibrium conditions, were, however, markedly different from K(D) values derived from kinetic data. Furthermore, the results of analyses of these kinetic data were consistent with the existence of two different populations of [3H]ACPA binding sites. The pharmacology of [3H]ACPA binding sites was characterized using a series of AMPA receptor agonists and antagonists. Whereas addition of KSCN had little effect on the affinities of AMPA receptor agonists for [3H]ACPA binding, this chaotropic agent reduced the affinities of AMPA receptor antagonists structurally related to AMPA. Based on these and previously reported data, the AMPA receptor agonists, ACPA, AMPA and (S)-5-fluorowillardiine, seem to bind to and activate AMPA receptors in a nonidentical fashion, and these three agonists together may be useful tools for studies of AMPA receptor mechanisms.

Families of the lesbian baby boom: children's contact with grandparents and other adults.

In an exploratory study of 37 lesbian-mother families, the frequency of children's contact with adults in their extended family and friendship networks was found to counter stereotypes of such children as isolated from parents' families of origin. Children were more likely to have regular contact with relatives of the biological than nonbiological mother. Mothers rated those in regular contact with grandparents as having fewer behavior problems, and those in more regular contact with unrelated adults rated themselves more positively on general well-being.

The relationship between alexithymia, depression, and axis II psychopathology in eating disorder inpatients.

OBJECTIVE: The major purpose of this study was to examine alexithymia in relationship to depression and Axis II psychopathology in eating disorder patients. METHOD: Fifty-three female inpatients representing three DSM-IV eating disorder diagnostic groups and 14 control subjects completed the Toronto Alexithymia Scale (TAS), the Eating Disorder Inventory-2, and the Beck Depression Inventory within the first week of their hospital admission and shortly before discharge. Structured Clinical Interviews for DSM-III-R (SCID) I and II were also conducted. Multiple regression analyses were used to determine the contribution of mood, diagnostic, and personality variables in predicting the alexithymia score. RESULTS AND DISCUSSION: After controlling for depression, only the TAS factor, "difficulty expressing feelings," remained significantly different between groups, with the anorexia nervosa-restrictors (AN-R) having significantly higher scores than controls and bulimia nervosa patients. This factor appears to be a relatively stable personality characteristic in AN-R. The level of depression and the presence of avoidant personality disorder were the most predictable variables for the alexithymia total score.

[3H]RY 80: A high-affinity, selective ligand for gamma-aminobutyric acidA receptors containing alpha-5 subunits.

The radiochemical synthesis and pharmacological properties are described of [3H]RY 80 (ethyl-8-acetylene-5, 6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1, 4]benzodiazepine-3-carboxylate, [ethyl-3H]). This compound is one of a series of 8-substituted imidazobenzodiazepines that exhibits both high affinity and selectivity for gamma-aminobutyric acid (GABA)A receptors containing alpha-5 subunits. Saturable, high-affinity (Kd approximately 0.7 nM) binding of [3H]RY 80 was observed in hippocampal membranes. The maximum number (Bmax) of [3H]RY 80 binding sites was approximately 18% of that obtained with [3H]flunitrazepam, a radioligand that labels all "diazepam-sensitive" GABAA receptors. This value is consistent with previous estimates (10-20%) of the proportion of rat hippocampal GABAA receptors containing alpha-5 subunits determined by immunoprecipitation with selective antibodies and competition experiments using an alpha-5-selective ligand. In recombinant GABAA receptors composed of alpha-5 beta-3 gamma-2 subunits, the Kd of [3H]RY 80 (approximately 0.5 nM) was consistent with the value obtained in hippocampus, whereas the Bmax value was not significantly different from that obtained with [3H]flunitrazepam. The potencies of several benzodiazepine site ligands to inhibit [3H]RY 80 binding to hippocampal membranes were in agreement with the values obtained in recombinant (alpha-5 beta-3 gamma-2) GABAA receptors. [3H]RY 80 was used both in a "GABA shift" assay to correctly predict the in vivo actions of a novel, alpha-5-selective ligand and to characterize a population of GABAA receptors containing alpha-5 subunits in neonatal rat cortex. These findings demonstrate that [3H]RY 80 can be used as a radioligand to examine the properties of GABAA receptors containing alpha-5 subunits.

A comparison of psychopathology in eating disorder patients from France and the United States.

OBJECTIVE: This study compares Minnesota Multiphasic Personality Inventory (MMPI) profiles of subtypes of eating disorder patients in France and the United States. METHOD: The patients were hospitalized in psychiatric hospitals in France and the United States. Diagnoses were made by independent clinicians who reviewed the clinical material. The 550-item version of the MMPI was administered to the US subjects; and a 357-item version to the French. RESULTS AND DISCUSSION: In both the US and French subjects, more psychopathology was found in the groups diagnosed with both anorexia nervosa and bulimia nervosa than in those with either anorexia nervosa or bulimia alone, consistent with previous research. The US subjects had generally more psychopathology than the French, except in the anorexia-restrictor subgroup.

New 5-HT1A receptor antagonist: [3H]p-MPPF.

A new 5-HT1A receptor antagonist ligand, [3H]p-MPPF, 4-(2'-methoxy-)-phenyl-1-[2'-(N-2"-pyridyl)-p-fluorobenzamido] ethyl-piperazine, was prepared and characterized. It demonstrated high affinity and selectivity toward 5-HT1A receptors (Kd = 0.34 +/- 0.12 nM and Bmax = 145 +/- 35 fmol/mg protein in rat hippocampal membrane homogenates). The binding is not sensitive to 100 microM Gpp(NH)p. Initial autoradiography studies of rat brain sections exhibit regional localization consistent with the known 5-HT1A receptor distribution. This potential 5-HT1A antagonist ligand may provide a powerful tool for 5-HT1A receptor pharmacology studies in the central nervous system.

Autoradiographic analysis of 5-HT receptor subtypes labeled by [3H]5-CT ([3H]5-carboxamidotryptamine).

This work examines the autoradiographic distribution of serotonin (5-HT) receptor subtypes in rat, guinea pig and human brain, using [3H]5-HT and [3H]5-CT as ligands. Different displacers were used to mask radioligand binding to 5-HT1A, 5-HT1B/1D and 5-HT2C receptors, in an attempt to visualize other receptor populations, which presumably would correspond to 5-HT1E and 5-HT1F sites. Brain areas enriched in 5-HTnon1A/1B/1D sites in guinea pig were the hilus, dentate gyrus, striatum, claustrum, substantia nigra and superior colliculus, among others. In humans, however, the claustrum, a structure supposed to contain 5-HT1E sites, showed significant densities of [3H]5-CT binding. An interesting finding was that blockade [3H]5-CT binding to 5-HT1A receptors by 8-OH-DPAT could only be achieved at very high concentrations of the displacer. This could be due to differences in the affinity of ligands in intact tissue sections compared to membrane homogenates or cell lines. Another possibility would be that [3H]5-CT labels 5-HT1A receptors in the low-affinity state. These hypotheses remain to be investigated.

Suicidal children grow up: ego functions associated with suicide attempts.

OBJECTIVE: To evaluate the relations between suicidal behavior in children and ego functions including impulse control, reality testing, and ego mechanisms of defense. METHOD: One hundred thirty-three children were assessed initially and at a 6- to 8-year follow-up for levels of reality testing and impulse control and frequency of use of several ego mechanisms of defense. Associations between suicidal ideation and suicide attempts at the initial assessment and at follow-up were analyzed with regard to ego functions. RESULTS: Specific ego functions, such as impulsivity, poor reality testing, and ego mechanisms of defense such as projection, regression, compensation, and reaction formation were positively associated with suicide attempts. Repression was a protective factor to prevent suicide attempts in the follow-up period. CONCLUSIONS: The results suggest that ego functions are related to behavior of consequence and are useful in the identification of children at risk for suicidal behavior.

The genetic toxicology of ethylenethiourea: a case study concerning the evaluation of a chemical's genotoxic potential.

Ethylenethiourea (ETU) is a metabolite, environmental degradation product and minor technical impurity of the ethylenebisdithiocarbamate (EBDC) class of fungicides. The genetic toxicology of ETU is important given that ETU causes thyroid tumors in rodents and liver tumors in mice. Although it is clear that ETU induces thyroid tumors via a non-genotoxic, threshold mechanism, the role ETU plays in inducing mouse liver tumors remains to be fully elucidated. Recently, Dearfield (Mutation Res., 317, 111-132, 1994) reviewed the genetic toxicology of ETU, and concluded that, although ETU is not a potent genotoxic agent, it is weakly genotoxic. This view stands in contrast to reports from several independent authorities that have generally concurred that ETU is not a mammalian genotoxin (IARC, 1987; MAFF, 1990; NTP, 1992; FAO/WHO, 1994). These conflicting reports highlight a generic problem in genotoxicity safety assessment: although individual test results typically yield either a positive or negative response, the overall evaluation of an extensive battery of tests for a particular chemical rarely yields an unambiguous conclusion. Recently, Mendelsohn et al. (Mutation Res., 266, 43-60, 1992) showed that the response of a chemical to a battery of genotoxicity tests is not a dichotomous (i.e., either positive or negative) property, but rather, appears to be a continuous property that ranges from strongly negative to strongly positive. We have used these data, together with a four-step weight of the evidence procedure, to evaluate ETU. Our analysis indicates that ETU is not genotoxic in mammalian systems and suggests that ETU likely induces mouse liver tumors by a non-genotoxic mechanism.

The Tridimensional Personality Questionnaire: an exploration of personality traits in eating disorders.

The Tridimensional Personality Questionnaire (TPQ) was tested in four subgroups of eating-disorder patients: anorectic-restrictors (AN-R), anorectic-bulimics (AN-B), normal weight bulimics (BN), and bulimics with a past history of anorexia (B-AN). Normal controls and patients were matched for gender and age. All subjects completed the Beck Depression Inventory (BDI) in addition to the TPQ. AN-Rs scored lower on the Novelty Seeking scale than the bulimic groups and controls, and the two normal weight bulimic groups had higher Novelty Seeking scores than the controls. On the Harm Avoidance scale, all eating disorder groups scored significantly higher than the control group. In addition, the AN-Rs scored lower than the AN-Bs and B-ANs. The Harm Avoidance scale and depression scores were positively correlated while the Reward Dependence scale and depression scores were negatively correlated. Differences between diagnostic groups on the Novelty Seeking and Persistence scales remained clearly significant when depression was partialled out. These results are discussed in terms of the Tridimensional Personality Questionnaire as a stable measure of traits with eating disorder subjects.

Culture shock and synergy. Academic/managed care/corporate alliances in outcomes management.

The Behavioral Health Outcomes Study is a partnership in conducting outcomes measurement involving a corporate healthcare purchaser, five managed behavioral healthcare organizations and academic researchers. The goals of this study are to: evaluate the feasibility of incorporating patient self-reported data in outcomes research; identify factors that may be predictors of outcome; and evaluate the effectiveness of an employee-sponsored aftercare program. The differing perspectives and needs of the three partners have created a number of challenges in the areas of goals, confidentiality, proprietary vs. open access issues and methodology. However, after the study's first year, it is clear not only that outcomes research can be conducted under such a partnership, but that the partnership generates a kind of synergy in problem-solving.

Suicidal children grow up: suicidal episodes and effects of treatment during follow-up.

OBJECTIVES: This paper describes risk for first recurrent suicidal episodes in follow-up of suicidal child psychiatric inpatients. It identifies relations between suicide attempts in follow-up and psychosocial and psychopharmacological treatments. METHODS: First suicidal episodes involving either suicidal ideation or a suicide attempt in a 6 to 8 year follow-up period were rated for 69 child psychiatric inpatients and 64 children selected from the community. Psychiatric treatments were determined from reports from multiple sources. RESULTS: Forty-five percent of 133 subjects reported a suicidal episode during follow-up. Children who reported suicidal ideation or a suicide attempt were greater than twice as likely to report a suicidal episode in follow-up than were children from the community. Children treated with antidepressants in follow-up were more likely to attempt suicide than were those not treated with antidepressants. CONCLUSIONS: Close follow-up of suicidal children is warranted to identity risk and to intervene to prevent suicidal episodes. Lack of efficacy of naturalistic treatments implies that controlled treatment studies are needed to determine effective intervention for suicidal children.

[3H]1-(cyclopropylmethyl)-4-(2-(4-fluorophenyl)-2-oxoethyl) piperidine HBr (DuP 734). A selective ligand for sigma receptors in mouse brain in vivo.

1-(Cyclopropylmethyl)-4-(2-(4-fluorophenyl)-2-oxoethyl) piperidine HBr (DuP 734) is a novel sigma receptor ligand which exhibits promise in preclinical animal models as an antipsychotic agent without motor side effects. In vitro and in vivo receptor binding profiles of DuP 734 in mouse brain using [3H]DuP 734 and [3H]N-allylnormetazocine ((+)-SKF 10,047) were studied. The pharmacology and stereospecificity of [3H]DuP 734-labeled sites in mouse brain, both in vitro and in vivo, was consistent with sigma receptor pharmacology. Specific in vivo binding of [3H]DuP 734 in brain peaked 1 hr after i.v. injection and this level of binding was maintained up to 4 hr. On the other hand, plasma concentration of [3H]DuP 734 decreased rapidly within 20 min after injection, indicating different pharmacokinetics between brain and plasma levels. Nonspecific binding, defined using 1 mg/kg (2.66 mumol/kg) of haloperidol, was approximately 30% of total binding 1 hr after injection of radiotracer. Administration of DuP 734 potently antagonized the binding of [3H]DuP 734 and [3H](+)-SKF 10,047 to brain sigma receptors in vivo with ID50 values of 0.02 and 0.07 mg/kg (0.07 and 0.25 mumol/kg), respectively. However, (+)-SKF 10,047, which possess a high affinity (IC50 = 22.5 nM) for [3H]DuP 734 binding in vitro, failed to displace [3H]DuP 734 binding in vivo. Thus in vitro receptor binding data may not predict in vivo receptor occupancy. Overall, the data suggest [3H] DuP 734 is a good ligand for in vivo imaging of sigma receptors.

Psychometric validation of the Tridimensional Personality Questionnaire: application to subgroups of eating disorders.

Eating disorder patients show extremes of the personality characteristics measured by the Tridimensional Personality Questionnaire (TPQ). For this reason, the TPQ was tested in four subgroups of eating disorder patients. Patients completed the TPQ and their responses were compared with a normative sample of women. Results indicated that the TPQ is an internally consistent and valid instrument to use with eating disorder patients.