RESUMEN
RATIONALE: Not all family members with BMPR2 mutations develop pulmonary arterial hypertension (PAH), implying that additional modifier genes or proteins are necessary for full expression of the disease. OBJECTIVES: To determine whether protein expression is altered in patients with familial PAH (FPAH) compared with obligate carriers and nondiseased control subjects. METHODS: Protein extracts from transformed blood lymphocytes from four patients with FPAH, three obligate carriers, and three married-in control subjects from one family with a known BMPR2 mutation (exon 3 T354G) were labeled with either Cy3 or Cy5. Cy3/5 pairs were separated by standard two-dimensional differential gel electrophoresis using a Cy2-labeled internal standard of all patient samples. Log volume ratios were analyzed using a linear mixed-effects model. Proteins were identified by matrix-assisted laser desorption ionization, time-of-flight mass spectrometry (MALDI-TOF MS) and tandem TOF/TOF MS/MS. MEASUREMENTS AND MAIN RESULTS: Hierarchical clustering, heat-map, and principal components analysis revealed marked changes in protein expression in patients with FPAH when compared with obligate carriers. Significant changes were apparent in expression of 16 proteins (P < 0.05) when affected patients were compared with obligates: nine showed a significant increase and seven showed a significant reduction. CONCLUSIONS: A series of novel proteins with altered expression were found that could distinguish affected patients from obligate carriers and married-in controls in a single family with a BMPR2 mutation. These differences provide new information highlighting proteins that may be involved in the mechanism(s) that differentiates those individuals with a BMPR2 mutation who develop FPAH from those who do not.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Hipertensión Pulmonar/genética , Activación de Linfocitos/genética , Proteómica , Adulto , Anciano , Electroforesis en Gel Bidimensional , Exones/genética , Femenino , Expresión Génica/fisiología , Tamización de Portadores Genéticos , Predisposición Genética a la Enfermedad/genética , Humanos , Masculino , Persona de Mediana Edad , Valores de Referencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
Anxiety is differentially expressed across a continuum of stressful/fearful intensity, influenced by endocannabinoid systems and receptors. The hippocampus plays important roles in the regulation of affective behavior, emotion, and anxiety, as well as memory. Location of Cb1/Cb2 receptor action could be important in determining emotional valence, because while the dorsal hippocampus is involved in spatial memory and cognition, the ventral hippocampus has projections to the PFC, BNST, amygdala, and HPA axis, and is important for emotional responses to stress. During repeated social defeat in a Stress-Alternatives Model arena (SAM; an oval open field with escape portals only large enough for smaller mice), smaller C57BL6/N mice are subject to fear conditioning (tone=CS), and attacked by novel larger aggressive CD1 mice (US) over four daily (5min) trials. Each SAM trial presents an opportunity for escape or submission, with stable behavioral responses established by the second day of interaction. Additional groups had access to a running wheel. Social aggression plus fear conditioning stimulates enhanced Cb2 receptor gene expression in the dorsal CA1, dorsal and ventral dentate gyrus subregions in animals displaying a submissive behavioral phenotype. Escape behavior is associated with reduced Cb2 expression in the dorsal CA1 region, with freezing and escape latency correlated with mRNA levels. Escaping and submitting animals with access to running wheels had increased Cb2 mRNA in dorsal DG/CA1. These results suggest that the Cb2 receptor system is rapidly induced during anxiogenic social interactions plus fear conditioning or exercise; with responses potentially adaptive for coping mechanisms.
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Regulación de la Expresión Génica/fisiología , Hipocampo/metabolismo , Receptor Cannabinoide CB2/genética , Receptor Cannabinoide CB2/metabolismo , Estrés Psicológico/patología , Estimulación Acústica/efectos adversos , Animales , Condicionamiento Psicológico/fisiología , Modelos Animales de Enfermedad , Reacción de Fuga/fisiología , Miedo/psicología , Relaciones Interpersonales , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Condicionamiento Físico Animal/fisiología , ARN Mensajero/metabolismo , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , Estrés Psicológico/fisiopatologíaRESUMEN
Escalation of anxious behavior while environmentally and socially relevant contextual events amplify the intensity of emotional response produces a testable gradient of anxiety shaped by integrative circuitries. Apprehension of the Stress-Alternatives Model apparatus (SAM) oval open field (OF) is measured by the active latency to escape, and is delayed by unfamiliarity with the passageway. Familiar OF escape is the least anxious behavior along the continuum, which can be reduced by anxiolytics such as icv neuropeptide S (NPS). Social aggression increases anxiousness in the SAM, reducing the number of mice willing to escape by 50%. The apprehension accompanying escape during social aggression is diminished by anxiolytics, such as exercise and corticotropin releasing-factor receptor 1 (CRF1) antagonism, but exacerbated by anxiogenic treatment, like antagonism of α2-adrenoreceptors. What is more, the anxiolytic CRF1 and anxiogenic α2-adrenoreceptor antagonists also modify behavioral phenotypes, with CRF1 antagonism allowing escape by previously submissive animals, and α2-adrenoreceptor antagonism hindering escape in mice that previously engaged in it. Gene expression of NPS and brain-derived neurotrophic factor (BDNF) in the central amygdala (CeA), as well as corticosterone secretion, increased concomitantly with the escalating anxious content of the mouse-specific anxiety continuum. The general trend of CeA NPS and BDNF expression suggested that NPS production was promoted by increasing anxiousness, and that BDNF synthesis was associated with learning about ever-more anxious conditions. The intensity gradient for anxious behavior resulting from varying contextual conditions may yield an improved conceptualization of the complexity of mechanisms producing the natural continuum of human anxious conditions, and potential therapies that arise therefrom.
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Ansiedad/psicología , Red Nerviosa/fisiopatología , Estrés Psicológico/psicología , Agresión/efectos de los fármacos , Agresión/psicología , Animales , Ansiolíticos/farmacología , Ansiedad/patología , Ansiedad/fisiopatología , Conducta Animal/efectos de los fármacos , Reacción de Fuga/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Red Nerviosa/efectos de los fármacos , Red Nerviosa/metabolismo , Condicionamiento Físico Animal/fisiología , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Índice de Severidad de la Enfermedad , Estrés Psicológico/patología , Estrés Psicológico/fisiopatologíaRESUMEN
The benefits of testing on later memory performance are well documented; however, the manner in which testing harms memory performance is less well understood. This research is concerned with the finding that accuracy decreases over the course of testing, a phenomena termed "output interference" (OI). OI has primarily been investigated with episodic memory, but there is limited research investigating OI in measures of semantic memory (i.e., knowledge). In the current study, participants were twice tested for their knowledge of factual questions; they received corrective feedback during the first test. No OI was observed during the first test, when participants presumably searched semantic memory to answer the general-knowledge questions. During the second test, OI was observed. Conditional analyses of Test 2 performance revealed that OI was largely isolated to questions answered incorrectly during Test 1. These were questions for which participants needed to rely on recent experience (i.e., the feedback in episodic memory) to respond correctly. One possible explanation is that episodic memory is more susceptible to the sort of interference generated during testing (e.g., gradual changes in context, encoding/updating of items) relative to semantic memory. Alternative explanations are considered.
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Memoria Episódica , Recuerdo Mental/fisiología , Adulto , Retroalimentación Psicológica , Humanos , Conocimiento , SemánticaRESUMEN
By creating the Visible Burrow System (VBS) Bob Blanchard found a way to study the interaction of genetics, physiology, environment, and adaptive significance in a model with broad validity. The VBS changed the way we think about anxiety and affective disorders by allowing the mechanisms which control them to be observed in a dynamic setting. Critically, Blanchard used the VBS and other models to show how behavioral systems like defense are dependent upon context and behavioral elements unique to the individual. Inspired by the VBS, we developed a Stress Alternatives Model (SAM) to further explore the multifaceted dynamics of the stress response with a dichotomous choice condition. Like the VBS, the SAM is a naturalistic model built upon risk assessment and defensive behavior, but with a choice of response: escape or submission to a large conspecific aggressor. The anxiety of novelty during the first escape must be weighed against fear of the aggressor, and a decision must be made. Both outcomes are adaptively significant, evidenced by a 50/50 split in outcome across several study systems. By manipulating the variables of the SAM, we show that a gradient of anxiety exists that spans the contextual settings of escaping an open field, escaping from aggression, and submitting to aggression. These findings correspond with increasing levels of corticosterone and increasing levels of NPS and BDNF in the central amygdala as the context changes.Whereas some anxiolytics were able to reduce the latency to escape for some animals, only with the potent anxiolytic drug antalarmin (CRF1R-blocker) and the anxiogenic drug yohimbine (α2 antagonist) were we able to reverse the outcome for a substantial proportion of individuals. Our findings promote a novel method for modeling anxiety, offering a distinction between low-and-high levels, and accounting for individual variability. The translational value of the VBS is immeasurable, and it guided us and many other researchers to seek potential clinical solutions through a deeper understanding of regional neurochemistry and gene expression in concert with an ecological behavioral model.
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Ansiedad/fisiopatología , Modelos Animales de Enfermedad , Conducta Social , Estrés Psicológico/fisiopatología , Animales , Humanos , Ratas , TruchaRESUMEN
Familial isolated GH deficiency type II (IGHD II) is caused, in some cases, by heterogeneous IVS3 mutations that affect GH mRNA splicing. We report here our finding an A-->G transition of the fifth base of exon 3 (E3+ 5 A-->G) in affected individuals from an IGHD II family. This mutation disrupts a (GAA)(n) exon splice enhancer (ESE) motif immediately following the weak IVS2 3' splice site. The mutation also destroys an MboII site used to demonstrate heterozygosity in all affected family members. To determine the effect of ESE mutations on GH mRNA processing, GH(3) cells were transfected with expression constructs containing the normal ESE, +5 A-->G, or other ESE mutations, and cDNAs derived from the resulting GH mRNAs were sequenced. All ESE mutations studied reduced activation of the IVS2 3' splice site and caused either partial E3 skipping, due to activation of an E3+ 45 cryptic 3' splice site, or complete E3 skipping. Partial or complete E3 skipping led to loss of the codons for amino acids 32-46 or 32-71, respectively, of the mature GH protein. Our data indicate that the E3+ 5 A-->G mutation causes IGHD II because it perturbs an ESE required for GH splicing.
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Elementos de Facilitación Genéticos/genética , Exones/genética , Genes Dominantes , Hormona de Crecimiento Humana/deficiencia , Mutación Puntual/fisiología , Empalme del ARN , Adulto , Anciano , Alelos , Secuencia de Aminoácidos/genética , Enzimas de Restricción del ADN , Femenino , Humanos , Lactante , Masculino , Enfermedades Metabólicas/genética , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , ARN Mensajero/genéticaRESUMEN
RATIONALE: Previous studies have shown that approximately 55% of patients with familial pulmonary arterial hypertension (FPAH) have BMPR2 coding sequence mutations. However, direct sequencing does not detect other types of heterozygous mutations, such as exonic deletions/duplications. OBJECTIVE: To estimate the frequency of BMPR2 exonic deletions/duplications in FPAH. METHODS: BMPR2 mRNA from lymphoblastoid cell lines of 30 families with PAH and 14 patients with idiopathic PAH (IPAH) was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing. Sequencing of genomic DNA was used to identify point mutations in splice donor/acceptor sites. Multiplex ligation-dependent probe amplification (MLPA) was used to detect exonic deletions/duplications with verification by real-time PCR. MEASUREMENTS AND MAIN RESULTS: Eleven (37%) patients with FPAH had abnormally sized RT-PCR products. Four of the 11 patients were found to have splice-site mutations resulting in aberrant splicing, and exonic deletions/duplications were detected in the remaining seven patients. MLPA identified three deletions/duplications that were not detectable by RT-PCR. Coding sequence point mutations were identified in 11 of 30 (37%) patients. Mutations were identified in 21 of 30 (70%) patients with FPAH, with 10 of 21 mutations (48%) being exonic deletions/duplications. Two of 14 (14%) patients with IPAH exhibited BMPR2 point mutations, whereas none showed exonic deletions/duplications. CONCLUSIONS: Our study indicates that BMPR2 exonic deletions/duplications in patients with FPAH account for a significant proportion of mutations (48%) that until now have not been screened for. Because the complementary approach used in this study is rapid and cost effective, screening for BMPR2 deletions/duplications by MLPA and real-time PCR should accompany direct sequencing in all PAH testing.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Exones/genética , Hipertensión Pulmonar/genética , Mutación/genética , Estudios de Casos y Controles , Humanos , Técnicas de Amplificación de Ácido Nucleico , Sitios de Empalme de ARN/genética , Análisis de SecuenciaRESUMEN
RATIONALE: Serotonin is a pulmonary vasoconstrictor and smooth muscle cell mitogen. The serotonin transporter (SERT) is abundant in pulmonary vascular smooth muscle. Compared with the short (S) allele, the long (L) SERT promoter allele is associated with increased SERT transcription and more severe pulmonary hypertension in a cohort of patients with chronic obstructive pulmonary disease, and was more prevalent in a cohort with idiopathic pulmonary arterial hypertension (IPAH), compared with control subjects. OBJECTIVE: We hypothesized that the SERT L allele would associate with an earlier age at diagnosis and/or shorter survival interval in pulmonary arterial hypertension (PAH) than the S allele. METHODS: SERT promoters from 166 familial PAH (FPAH), 83 IPAH, and 125 control subjects were sequenced. One hundred twenty-seven of the patients with FPAH had a known mutation in bone morphogenetic protein receptor 2 (BMPR2). RESULTS: The mean age at diagnosis was 35.8 yr in patients with FPAH and 41.1 yr in patients with IPAH (p = 0.02). There were no significant differences in distribution of the LL, LS, or SS genotypes in IPAH, FPAH, or unaffected BMPR2 mutation carriers. In FPAH, the LL genotype was associated with an earlier age at diagnosis (p < 0.02). CONCLUSIONS: In patients with IPAH, these SERT genotypes do not correlate with age at diagnosis or survival interval. In patients with FPAH, the LL genotype correlates with an earlier age at diagnosis than SL or SS, although survival among the groups was similar. The correlation of the SERT promoter polymorphism with age at diagnosis in FPAH suggests a possible relationship between the SERT and BMPR2.