RESUMEN
Gousiekte, which can be translated literally as "quick disease", is one of the six most important plant toxicoses that affect livestock in South Africa. It is a plant-induced cardiomyopathy of domestic ruminants characterised by the sudden death of animals within a period of 4-8weeks after the initial ingestion of the toxic plant. The main ultrastructural change in sheep hearts is degradation of myofibres. In this study, fluorescent probes were used to investigate subcellular changes induced by pavetamine, the toxic compound that causes gousiekte, in H9c2 cells. The sarcoplasmic reticula (SR) and mitochondria showed abnormalities that were not present in the control cells. The lysosomes of treated cells were more abundant and enlarged than those of the control cells. There was increased activity of cytosolic hexosaminidase and acid phosphatase, indicating increased lysosomal membrane permeability. Lysosomes play an important role in both necrosis and apoptosis. The degradation of the myofibres may be a consequence of the increased lysosomal membrane permeability. Pavetamine was also found to cause alterations in the organisation of F-actin. F-actin in the nucleus is a transcription regulator and can therefore influence protein synthesis. Actin filament organisation also regulates the cardiac L-type Ca(2+) channels. Fluorescent staining demonstrated that pavetamine may damage a number of organelles, all of which can influence the proper functioning of the heart.
Asunto(s)
Poliaminas/toxicidad , Animales , Línea Celular , Colorantes Fluorescentes , Lisosomas/efectos de los fármacos , Lisosomas/ultraestructura , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Ratas , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/ultraestructura , Pruebas de ToxicidadRESUMEN
Pavetamine, a cationic polyamine, is a cardiotoxin that affects ruminants. The animals die of heart failure after a period of four to eight weeks following ingestion of the plants that contain pavetamine. This immunofluorescent study was undertaken in rat neonatal cardiomyocytes (RNCM) to label some of the contractile and cytoskeleton proteins after exposure to pavetamine for 48 h. Myosin and titin were degraded in the RNCM treated with pavetamine and the morphology of alpha-actin was altered, when compared to the untreated cells, while those of beta-tubulin seemed to be unaffected. F-actin was degraded, or even absent, in some of the treated cells. On an ultrastructural level, the sarcomeres were disorganized or disengaged from the Z-lines. Thus, all three contractile proteins of the rat heart were affected by pavetamine treatment, as well as the F-actin of the cytoskeleton. It is possible that these proteins are being degraded by proteases like the calpains and/or cathepsins. The consequence of pavetamine exposure is literally a "broken heart".
Asunto(s)
Proteínas del Citoesqueleto/efectos de los fármacos , Proteínas Musculares/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Poliaminas/toxicidad , Actinas/efectos de los fármacos , Actinas/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Conectina , Proteínas del Citoesqueleto/metabolismo , Microscopía Electrónica de Transmisión , Proteínas Musculares/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/ultraestructura , Miosinas/efectos de los fármacos , Miosinas/metabolismo , Proteínas Quinasas/efectos de los fármacos , Proteínas Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Sarcómeros/efectos de los fármacos , Sarcómeros/ultraestructura , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/metabolismoRESUMEN
Intake of pavetamine, a novel polyamine, synthesized by certain rubiaceous plants, is the cause of gousiekte ("Quick disease") in ruminants. The disease is characterized by a latent period of 4-8 weeks, followed by heart failure. The aim of this study was to firstly investigate the cytotoxicity in H9c2(2-1) cells using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) and LDH (lactate dehydrogenase) release assays. Maximum cell death occurred after pavetamine exposure of cells for 72h at a concentration of 200muM (55%+/-9.84), as measured by the MTT assay. LDH release was only observed after 72h exposure to pavetamine. Secondly, the ultrastructural changes induced by pavetamine in H9c2(2-1) cells were investigated. Changes in the mitochondria and sarcoplasmic reticula were observed. The nucleus was not affected during the first 48h exposure of cells to pavetamine and no chromatin condensation occurred. However, after 72h exposure to pavetamine, the nucleus became fragmented and membrane blebbing occurred. It was concluded that the ultimate cell death of H9c2(2-1) cells treated with pavetamine, was through necrosis and not apoptosis. Thirdly, the effect of pavetamine on the mitochondrial membrane potential (DeltaPsi) was evaluated by using the JC-1 (5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide) and TMRM (tetramethylrhodamine methyl ester perchlorate) probes. Pavetamine treatment led to significant hyperpolarization of the mitochondrial membrane potential. Cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore, did not reduce the cytotoxicity of pavetamine significantly, indicating that the MPTP (mitochondrial permeability transition pore) plays no role in the cytotoxicity of pavetamine.
Asunto(s)
Citotoxinas/toxicidad , Mitocondrias Musculares/efectos de los fármacos , Poliaminas/toxicidad , Retículo Sarcoplasmático/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microscopía Electrónica de Transmisión , Mitocondrias Musculares/ultraestructura , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Poro de Transición de la Permeabilidad Mitocondrial , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/ultraestructura , Necrosis/inducido químicamente , Hojas de la Planta/química , Poliaminas/aislamiento & purificación , Ratas , Rubiaceae/química , Retículo Sarcoplasmático/ultraestructura , Factores de TiempoRESUMEN
BACKGROUND: HER2-positive metastatic breast cancer (MBC) relapsing after trastuzumab-based therapy may require continued HER2 receptor inhibition to control the disease and preserve the patients' quality-of-life. Efficacy and safety of lapatinib monotherapy was evaluated in Japanese breast cancer patients after trastuzumab-based therapies. METHODS: In studies, EGF100642 and EGF104911 evaluated the efficacy and safety of oral lapatinib given 1500 mg once daily in patients with advanced or MBC. All patients progressed on anthracyclines and taxanes; HER2-positive patients had also progressed on trastuzumab. RESULTS: For HER2-positive tumours (n=100), objective response rate was 19.0% (95% confidence interval (CI): 11.8-28.1) and clinical benefit rate (CBR) was 25.0% (95% CI: 16.9-34.7). One out of 22 HER2-negative tumour was documented as complete response (n=22). The median time-to-progression (TTP) in the HER2-positive and HER2-negative groups was 13.0 and 8.0 weeks (P=0.007); median overall survival was 58.3 and 40.0 weeks, respectively. The most frequent adverse event was diarrhoea. TTP and CBR were significantly associated with HER2 expression. Patients with tumours harbouring an H1047R PIK3CA mutation or low expression of PTEN derived clinical benefit from lapatinib. CONCLUSION: Lapatinib monotherapy had shown anti-tumour activity in Japanese patients with HER2-positive MBC that relapsed after trastuzumab-based therapy, including those with brain metastases. Patients benefiting from lapatinib may have biomarker profiles differing from that reported for trastuzumab.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Quinazolinas/uso terapéutico , Adulto , Anciano , Antineoplásicos/efectos adversos , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/metabolismo , Progresión de la Enfermedad , Femenino , Humanos , Japón , Estimación de Kaplan-Meier , Lapatinib , Persona de Mediana Edad , Quinazolinas/efectos adversos , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/genética , Adulto JovenRESUMEN
The glycoprotein (GP) and nucleocapsid (NC) genes of Rift Valley fever virus (RVFV) were expressed in different expression systems and were evaluated for their ability to protect mice from virulent challenge using a prime-boost regime. Mice vaccinated with a lumpy skin disease virus-vectored recombinant vaccine (rLSDV-RVFV) expressing the two RVFV glycoproteins (G1 and G2) developed neutralising antibodies and were fully protected when challenged, as were those vaccinated with a crude extract of truncated G2 glycoprotein (tG2). By contrast mice vaccinated with a DNA vaccine expressing G1 and G2 did not sero-convert with only 20% of them surviving challenge. Mice vaccinated with the DNA vaccine and boosted with rLSDV-RVFV also failed to sero-convert but 40% survived challenge. Surprisingly, although none of the mice immunised with the purified NC protein sero-converted, 60% of them survived virulent challenge. The rLSDV-RVFV construct was then further evaluated in sheep for its dual protective abilities against RVFV and sheeppox virus (SPV). Vaccinated sheep sero-converted for both viruses and were protected against RVFV challenge, however, neither the immunised or negative control animals showed any significant reactions to the virulent SPV challenge.
Asunto(s)
Fiebre del Valle del Rift/prevención & control , Fiebre del Valle del Rift/veterinaria , Virus de la Fiebre del Valle del Rift/inmunología , Vacunas Virales/uso terapéutico , Animales , Antígenos Virales/biosíntesis , Antígenos Virales/aislamiento & purificación , Temperatura Corporal/fisiología , Sistemas de Liberación de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Femenino , Esquemas de Inmunización , Inmunización Secundaria , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Viral/inmunología , Fiebre del Valle del Rift/inmunología , Ovinos/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/uso terapéutico , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/uso terapéutico , Vacunas Virales/administración & dosificaciónRESUMEN
An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of extracellular leukotoxin (LKT) produced in chemostat culture of Mannheimia haemolytica in a serum-free culture medium. Leukotoxin purified with preparative SDS-PAGE was used for the production of chicken polyclonal antibodies (PAb) that served as the primary detecting antibody. Excising the LKT protein from an analytical SDS-PAGE gel proved an efficient technique for the purification of the toxin. Consequently, the 102 kDa LKT polypeptide purified in this manner served as reference toxin and the resulting calibration curve was modelled using a four parameter logistic fit to relate absorbance to LKT protein concentration. The lower detection limit corresponded to an LKT concentration of 14.5 ng ml(-1). The presence of SDS, serum albumin and the coating pH had a distinct effect on the absorbance values of the indirect ELISA.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/veterinaria , Exotoxinas/aislamiento & purificación , Mannheimia haemolytica/metabolismo , Animales , Anticuerpos Monoclonales , Bovinos , Pollos , Medio de Cultivo Libre de Suero , Citotoxinas/biosíntesis , Citotoxinas/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Exotoxinas/biosíntesis , Mannheimia haemolytica/crecimiento & desarrollo , Infecciones por Pasteurella/diagnóstico , Infecciones por Pasteurella/microbiología , Infecciones por Pasteurella/veterinaria , Valores de Referencia , Sensibilidad y Especificidad , OvinosRESUMEN
alpha-Synuclein (alpha-Syn) is implicated in the pathogenesis of Parkinson's Disease, genetically through missense mutations linked to early onset disease and pathologically through its presence in Lewy bodies. alpha-Syn is phosphorylated on serine residues; however, tyrosine phosphorylation of alpha-Syn has not been established (, ). A comparison of the protein sequence between Synuclein family members revealed that all four tyrosine residues of alpha-Syn are conserved in all orthologs and beta-Syn paralogs described to date, suggesting that these residues may be of functional importance (). For this reason, experiments were performed to determine whether alpha-Syn could be phosphorylated on tyrosine residue(s) in human cells. Indeed, alpha-Syn is phosphorylated within 2 min of pervanadate treatment in alpha-Syn-transfected cells. Tyrosine phosphorylation occurs primarily on tyrosine 125 and was inhibited by PP2, a selective inhibitor of Src protein-tyrosine kinase (PTK) family members at concentrations consistent with inhibition of Src function (). Finally, we demonstrate that alpha-Syn can be phosphorylated directly both in cotransfection experiments using c-Src and Fyn expression vectors and in in vitro kinase assays with purified kinases. These data suggest that alpha-Syn can be a target for phosphorylation by the Src family of PTKs.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Familia-src Quinasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Cartilla de ADN , Datos de Secuencia Molecular , Monofenol Monooxigenasa/metabolismo , Proteínas del Tejido Nervioso/química , Fosforilación , Sinucleínas , alfa-SinucleínaRESUMEN
The cellular mechanisms underlying the physiological effects of the orexins are poorly understood. Therefore, the pharmacology of the recombinant human orexin receptors was studied using FLIPR. Intracellular calcium ([Ca2+]i) was monitored in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX1) or orexin-2 (OX2) receptors using Fluo-3AM. Orexin-A and orexin-B increased [Ca2+]i in a concentration dependent manner in CHO-OX1 (pEC50=8.03+/-0.08 and 7. 30+/-0.08 respectively, n=5) and CHO-OX2 (pEC50=8.18+/-0.10 and 8. 43+/-0.09 respectively, n=5) cells. This response was typified as a rapid peak in [Ca2+]i (maximal at 6 - 8 s), followed by a gradually declining secondary phase. Thapsigargin (3 microM) or U73122 (3 microM) abolished the response. In calcium-free conditions the peak response was unaffected but the secondary phase was shortened, returning to basal values within 90 s. Calcium (1.5 mM) replacement restored the secondary phase. In conclusion, orexins cause a phospholipase C-mediated release of calcium from intracellular stores, with subsequent calcium influx.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras/farmacología , Péptidos y Proteínas de Señalización Intracelular , Neuropéptidos/farmacología , Receptores de Neuropéptido/metabolismo , Compuestos de Anilina , Animales , Células CHO , Calcio/antagonistas & inhibidores , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Proteínas Portadoras/antagonistas & inhibidores , Cricetinae , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes , Humanos , Neuropéptidos/antagonistas & inhibidores , Receptores de Orexina , Orexinas , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfolipasa D/antagonistas & inhibidores , Fosfolipasa D/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tapsigargina/farmacología , Factores de Tiempo , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo , XantenosRESUMEN
Urotensin-II (U-II) is a vasoactive 'somatostatin-like' cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.
Asunto(s)
Proteínas de Unión al GTP/agonistas , Proteínas de Unión al GTP/metabolismo , Receptores de Superficie Celular/agonistas , Receptores Acoplados a Proteínas G , Urotensinas/farmacología , Vasoconstrictores/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/metabolismo , Línea Celular , Clonación Molecular , ADN Complementario , Proteínas de Unión al GTP/genética , Humanos , Macaca fascicularis , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular , Urotensinas/metabolismo , Vasoconstrictores/metabolismoRESUMEN
Reagents were prepared for use in ELISAs to determine the concentration of the antigenic components of Clostridium botulinum type C and D. The results obtained were compared with the L+dose assay and a good correlation was found between the two assays for measurement of the C and D neurotoxin concentration. These ELISAs were also used to determine the concentration of the neurotoxins in toxoid form. The relationship between the C neurotoxin dose, in toxoid form, and the immune response in guinea pigs could be deduced from the data obtained. The relationship for the D neurotoxin was not that clear, as the same concentration of the antigen resulted in variable potency values. However, these ELISAs can be used to formulate the concentration of the C and D components in the final bivalent vaccine. Replacement of the preliminary potency assay on the monovalent components after production with the in vitro assays will shorten the total production time of the vaccine by about 60 days. The economical and ethical implications are the reduction in the use of animals to evaluate the vaccine.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Toxinas Botulínicas/inmunología , Clostridium botulinum/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Estudios de Evaluación como Asunto , Cobayas , ConejosRESUMEN
Two human beta1-adrenergic receptor cDNAs, approximately 2.75 kb and approximately 3 kb in length, were isolated from a placenta phage library. Both transcripts are collinear with the previously isolated genomic sequence. Additionally, all clones begin between -274 and -236 bp relative to the translational start site, which is consistent with the previously identified transcriptional start site at -263. Furthermore, the 2.75 and 3 kb transcripts utilize conserved polyadenylation consensus sites at +2469 and +2751, respectively. Thus, this is the first report of the identification of full-length human beta1-AR cDNA clones. Both transcripts are expressed in placenta, heart, cerebral cortex, and lung with the 3 kb transcript more highly expressed than the 2.75 kb transcript in all tissues. RNase protection analysis utilizing left ventricular heart RNA isolated from patients with idiopathic dilated cardiomyopathy demonstrates 2.1-fold and 2.7-fold decreases of the 2. 75 and 3 kb transcripts, respectively, as compared with nonfailing controls. Thus, in heart failure patients the 3 kb transcript decreases to a significantly greater extent than the 2.75 kb transcript. This preferential reduction may be the result of differences in mRNA stability mediated by putative AU-rich elements specific to the 3'-untranslated region of the larger transcript.
Asunto(s)
Cardiomiopatía Dilatada/genética , ARN Mensajero/genética , Receptores Adrenérgicos beta 1/genética , Secuencia de Bases , Estudios de Casos y Controles , Clonación Molecular , Cartilla de ADN , ADN Complementario , Humanos , Datos de Secuencia MolecularRESUMEN
The optimal production of P. haemolytica leukotoxin in the culture supernatant of a fluid medium is dependent on a number of factors. The leukotoxin has to be produced by using a strain that is known for its ability to produce high quantities of leukotoxin, inoculated into the most suitable type of medium at the correct culture density containing the necessary supplements and harvested after a certain growth period. The volume in which it is produced may also have an influence. Two different procedures are described to produce the leukotoxin in 5 to 15-l quantities in RPMI 1640 medium. The first method used to produce leukotoxin is one that has been repeatedly described since the presence of the leukotoxin was first established in 1978. Using this method seven batches of leukotoxin were produced in litre quantities with leukotoxin activity ranging from 23-67 u/ml. The seed culture inoculum is prepared in brain heart infusion broth, which is centrifuged before the organisms are inoculated into RPMI 1640 medium containing 3.5% foetal calf serum and incubated for only 1 h in a fermenter, after, which the leukotoxin is harvested. An improved alternative method was devised which yielded higher levels of leukotoxin activity by utilising the ability of the P. haemolytica organisms to grow and produce leukotoxin during the logarithmic growth phase in a fermenter. A seed culture harvested in the log phase was prepared in brain heart infusion broth by means of a series of cultures and inoculated into RPMI 1640 containing 3.5% foetal calf serum. Three hours of active growth were allowed during which the leukotoxin was measured by its biological activity and an ELISA assay, and the increase in cell mass by means of the optical density every 30 min. The average leukotoxin biological activity measured 260 u/ml and by means of the ELISA test the leukotoxin concentration measured 315 u/l which is a substantial increase in leukotoxin production. In comparison the average optical density only measured 0.469 at 650 nm. Previous findings were substantiated that the highest cell density was not reflected in the highest leukotoxin activity. It is possible to induce high levels of leukotoxin secretion in submerged cultures with RPMI 1640 medium containing foetal calf serum in the controlled environment of a fermenter in large enough quantities for use as a vaccine by the improved preparation of the seed culture inoculum.
Asunto(s)
Toxinas Bacterianas/biosíntesis , Citotoxinas/biosíntesis , Exotoxinas/biosíntesis , Mannheimia haemolytica/metabolismo , Absorción , Animales , Toxinas Bacterianas/aislamiento & purificación , Toxinas Bacterianas/farmacocinética , Vacunas Bacterianas/aislamiento & purificación , Técnicas Bacteriológicas , Reactores Biológicos/microbiología , Bovinos , Medios de Cultivo/análisis , Citotoxinas/aislamiento & purificación , Citotoxinas/farmacocinética , Exotoxinas/aislamiento & purificación , Exotoxinas/farmacocinética , Mannheimia haemolytica/crecimiento & desarrolloRESUMEN
1. The functional profile of the long form of the human cloned 5-HT7 receptor (designated h5-HT7(a)) was investigated using a number of 5-HT receptor agonists and antagonists and compared with its binding profile. Receptor function was measured using adenylyl cyclase activity in washed membranes from HEK293 cells stably expressing the recombinant h5-HT7(a) receptor. 2. The receptor binding profile, determined by competition with [3H]-5-CT, was consistent with that previously reported for the h5-HT7(a) receptor. The selective 5-HT7 receptor antagonist SB-258719 ((R)-3,N-Dimethyl-N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]ben zene sulfonamide) displayed high affinity (pKi 7.5) for the receptor. 3. In the adenylyl cyclase functional assay, 5-CT and 8-OH-DPAT were both full agonists compared to 5-HT and the rank order of potency for agonists (5-CT > 5-HT > 8-OH-DPAT) was the same in functional and binding studies. 4. Risperidone, methiothepin, mesulergine, clozapine, olanzapine, ketanserin and SB-258719 antagonised surmountably 5-CT-stimulated adenylyl cyclase activity. Schild analysis of the antagonism by SB-258719 gave a pA2 of 7.2+/-0.2 and slope not significantly different from 1, consistent with competitive antagonism. 5. The same antagonists also inhibited basal adenylyl cyclase activity with a rank order of potency in agreement with those for antagonist potency and binding affinity. Both SB-258719 and mesulergine displayed apparent partial inverse agonist profiles compared to the other antagonists tested. These inhibitory effects of antagonists appear to be 5-HT7 receptor-mediated and to reflect inverse agonism. 6. It is concluded that in this expression system, the h5-HT7(a) receptor shows the expected binding and functional profile and displays constitutive activity, revealing inverse agonist activity for a range of antagonists.
Asunto(s)
Piperidinas/farmacología , Receptores de Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , Sulfonamidas/farmacología , Adenilil Ciclasas/metabolismo , Línea Celular , Clonación Molecular , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Unión Proteica , Receptores de Serotonina/genética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
One open reading frame of a 13 kb genomic clone of the human beta 1-adrenergic receptor, which lacks introns, encodes the previously isolated cDNA. Transcript(s) between 4.7 and 5.1 kb are detected in total RNA, whereas a approximately 3 kb transcript is detected only in polyadenylated RNA. The poly (A+) transcript is most highly expressed in the pancreas, liver, heart, kidney, thalamus, adrenal, and salivary glands. Primer extension and ribonuclease protection analyses suggest that the major transcriptional start site is located at -263. Transient expression of luciferase reporter gene constructs indicates that the region from -444 to -360 possesses the primary promoter, consistent with the transcriptional start site at -263. Negative transcriptional regulatory elements are located from -3118 to -2730 and -2730 to -2241, while a positive element is located between -2241 and -1790. The present study suggests that, despite similarities, the expression and transcriptional regulation of the human gene are distinct from those of the genes of other species.
Asunto(s)
Receptores Adrenérgicos beta 1/genética , Animales , Secuencia de Bases , Corteza Cerebral/metabolismo , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Datos de Secuencia Molecular , Miocardio/metabolismo , Placenta/metabolismo , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mapeo Restrictivo , Distribución Tisular , Transcripción GenéticaRESUMEN
Four experiments are reported which attempt to externalize subjects' mental representation of conditional sentences, using novel research methods. In Experiment 1, subjects were shown arrays of coloured shapes and asked to rate the degree to which they appeared to be true of conditional statements such as "If the figure is green then it is a triangle". The arrays contained different distributions of the four logically possible cases in which the antecedent or consequent is true or false: TT, TF, FT, and FF. For example, a blue triangle would be FT for the conditional quoted above. In Experiments 2 to 4, subjects were able to construct their own arrays to make conditional either true or false with any distribution of the four cases they wished to choose. The presence and absence of negative components was varied, as was the form of the conditional, being either "if then" as above or "only if": "The figure is green only if it is a triangle". The first findings was that subjects represent conditional in fuzzy way: conditional that include some counter-example TF cases (Experiment 1) may be rated as true, and such cases are often included when subjects construct an array to make the rule true (Experiments 2 to 4). Other findings included a strong tendency to include psychologically irrelevant FT and FF cases in constructed arrays, presumably to show that conditional statements only apply some of the time. A tendency to construct cases in line with the "matching bias" reported on analogous tasks in the literature was found, but only in Experiment 4, where the number of symbols available to construct each case was controlled. The findings are discussed in relation to the major contemporary theories of conditional reasoning based upon inference rules and mental models, neither of which can account for all the results.
Asunto(s)
Aprendizaje por Asociación , Percepción de Color , Reconocimiento Visual de Modelos , Solución de Problemas , Semántica , Adulto , Femenino , Lógica Difusa , Humanos , MasculinoRESUMEN
A seroepidemiologic hepatitis B survey of students and staff at schools for the mentally retarded in the City of Toronto found 2.5% of students to be carriers, 5% to be immune without being immunized, 11.3% to have immunization underway or complete, and 81.2% to be susceptible. The first two groups were older and more likely to have been born in countries with intermediate or high hepatitis B marker prevalence. Among staff tested, 4.1% were immune without immunization while all others were susceptible. Because hepatitis B control recommendations could not be made on the basis of these results alone, local Medical Officers of Health also considered other studies and practical experience to recommend the following: (1) Use Ontario Ministry of Health Guidelines for mentally retarded hepatitis B carriers in regular schools; (2) Offer hepatitis B vaccine to all susceptible students and staff in schools specifically for the mentally retarded.