RESUMO
A major hurdle in the structural analysis of membrane proteins is the expression of a functional and homogeneous form of the protein. Except for rhodopsin, most G protein-coupled receptors (GPCRs) are endogenously expressed at very low levels. Heterologous expression of GPCRs in bacteria, yeast, insect cells or mammalian cell lines often yields proteins with large amounts of misfolded proteins and heterogeneous posttranslational modifications. Here, we report a novel mammalian "in vivo" system for the expression of the chemokine receptor CXCR1. This receptor was expressed in liver of mice infected with adenovirus encoding CXCR1. Liver plasma membranes from infected mice displayed high-levels of (125)I-labeled human interleukin-8 (IL-8) binding. The pharmacological profile of the recombinant CXCR1 expressed "in vivo" was similar to those expressed in neutrophils. We found that the incorporation of the detergent solubilized CXCR1 into phospholipid vesicles in the presence of Gi/Go proteins is required for the reconstitution of (125)I-IL-8 binding. On the basis of the presence of the several endogenous His residues and glycosylation moieties in CXCR1 we fractionated the detergent-solubilized plasma membranes by employing Ni- and Concanavalin A-based chromatography. Fractions enriched with CXCR1 were monitored by (125)I-IL-8-bound to the receptor and Western blots with anti-CXCR1 antibodies. This robust expression system could be readily applied for the expression of GPCRs and other eukaryotic membrane proteins.
Assuntos
Adenoviridae/genética , Receptores de Interleucina-8A/metabolismo , Adenoviridae/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Células Cultivadas , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Dados de Sequência Molecular , Coelhos , Receptores de Interleucina-8A/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Although DNA ploidy correlates with prognosis in certain childhood cancers, e.g., neuroblastoma, its significance in rhabdomyosarcoma (RMS) is unclear and controversial. METHODS: Ploidy by flow cytometry (FCM) and image analysis (IA) in 26 of 27 children with RMS (17 embryonal, 3 mixed embryonal/alveolar, 5 alveolar, 1 anaplastic, 1 ectomesenchymoma) and 4 adults with pleomorphic RMS were evaluated. Statistical comparisons were analyzed between DNA content and gender, age, localization, Intergroup Rhabdomyosarcoma Study (IRS) group, and histopathologic subtype. Survival analyses were performed by the Kaplan-Meier test using the approximate chi-square statistic for the log rank test. RESULTS: The concordance rate between FCM and IA was 26 of 30 (87%); FCM was not performed in one tumor. Image analysis was more sensitive than FCM in detecting aneuploidy. Furthermore, DNA content was associated significantly with histologic subtype (P = 0.031); embryonal histology commonly was hyperdiploid (mean, 1.44; median, 1.27), whereas alveolar histology usually was near-tetraploid (mean, 1.83; median, 1.95). All four adult patients with pleomorphic RMS were aneuploid, with one showing multiple DNA peaks. No correlation between DNA content and survival was observed in the children with RMS. However, IRS group (P = 0.011) and patient age (P = 0.036) were independent prognostic indicators significantly related to survival. All adult patients died of their disease. CONCLUSIONS: Although ploidy correlates with histologic subtype, DNA content is not significantly predictive of prognosis in patients with RMS. Age at diagnosis and IRS group are independent predictors of clinical outcome in children with RMS.
Assuntos
Ploidias , Rabdomiossarcoma/genética , Neoplasias de Tecidos Moles/genética , Adolescente , Adulto , Distribuição de Qui-Quadrado , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Humanos , Processamento de Imagem Assistida por Computador , Lactente , Masculino , Prognóstico , Estudos Retrospectivos , Rabdomiossarcoma/patologia , Neoplasias de Tecidos Moles/patologia , Coloração e Rotulagem , Análise de SobrevidaRESUMO
Round atelectasis is an unusual pulmonary pseudotumor often associated with primary pleural pathologic conditions or pleural effusion. We report the case of a 20-year-old woman who was found to have a large, circular pulmonary lesion demonstrated on both plain chest x-ray film and computed chest tomography. The patient underwent exploratory thoracotomy, at which time this uncommon entity was identified.
Assuntos
Atelectasia Pulmonar/diagnóstico por imagem , Adulto , Feminino , Humanos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Complicações Pós-Operatórias , Atelectasia Pulmonar/patologia , Tomografia Computadorizada por Raios X , TonsilectomiaRESUMO
Topically applied O-butyryl timolol, O-pivaloyl timolol and levobunolol (0.25 micrograms) antagonized isoproterenol-induced ocular hypotension for 8 hrs whereas timolol (0.25 micrograms) was shorter acting (4 hrs). Timolol (25 micrograms) produced greater antagonism of isoproterenol-induced tachycardia than did O-butyryl and O-pivaloyl timolol (25 micrograms). These results suggest that, at similar doses, O-butyryl and O-pivaloyl timolol produce high concentrations of timolol in ocular tissues and undergo redistribution more slowly into the systemic circulation than does topical timolol. Under certain circumstances, prodrugs may provide a mechanism for increasing selectivity and extending the duration of action in the target organ as well as decreasing systemic effects.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Olho/efeitos dos fármacos , Coração/efeitos dos fármacos , Levobunolol/farmacologia , Pró-Fármacos/farmacologia , Timolol/farmacologia , Animais , Frequência Cardíaca/efeitos dos fármacos , Pressão Intraocular/efeitos dos fármacos , Isoproterenol/antagonistas & inibidores , Isoproterenol/farmacologia , Masculino , CoelhosRESUMO
Dopamine (DA1 and DA2) receptors have been demonstrated functionally in the anterior segment of the eye. Previous results have indicated that bromocriptine, a relatively selective DA2 agonist, can lower intraocular pressure (IOP) in laboratory animals as well as in normal and glaucomatous humans. Other ergoline derivatives (pergolide, lergotrile, LY141865) have also been demonstrated to lower IOP in laboratory animals. Cianergoline, a new ergoline derivative, was tested for: 1) ocular hypotensive activity in normal and sympathectomized (SX) rabbits and in normal capuchin monkeys, 2) inhibition of induced ocular hypertension by waterloading in rabbits and 3) suppression of contractions of the cat nictitating membrane (CNM) elicited by electrical stimulation of sympathetic nerves and by intraarterial (i.a.) injection of norepinephrine (NE). Topically administered cianergoline (0.022-0.22 mg) produced dose-related, unilateral ocular hypotension in normal but not in SX rabbits. In addition, cianergoline (0.5 mg) produced a slight reduction of IOP in capuchin monkeys. There were no significant effects on iris function in rabbits, however, miosis did occur in the monkeys. Cianergoline (0.22mg) suppressed ocular hypertension induced by waterloading in rabbits, and this effect was antagonized by metoclopramide, a relatively selective DA2 antagonist. Cianergoline (1-333 micrograms, i.a.) also produced dose-related inhibition of neuronally mediated contractions of the CNM. Cianergoline inhibited low frequency (2 & 4 Hz) contractile responses of the CNM more than high frequency (6 & 8 Hz) responses. Contractions of the CNM caused by i.a. NE were also inhibited by higher concentrations of cianergoline. These data demonstrate that cianergoline, like bromocriptine, can lower IOP and that the predominant mechanism involves inhibition of sympathetic neuronal function at prejunctional (DA2) and postjunctional (alpha 1) adrenoceptors.