RESUMO
The molecular complexity of tissues and the inaccessibility of most cells within a tissue limit the discovery of key targets for tissue-specific delivery of therapeutic and imaging agents in vivo. Here, we describe a hypothesis-driven, systems biology approach to identifying a small subset of proteins induced at the tissue-blood interface that are inherently accessible to antibodies injected intravenously. We use subcellular fractionation, subtractive proteomics and bioinformatics to identify endothelial cell surface proteins exhibiting restricted tissue distribution and apparent tissue modulation. Expression profiling and gamma-scintigraphic imaging with antibodies establishes two of these proteins, aminopeptidase-P and annexin A1, as selective in vivo targets for antibodies in lungs and solid tumours, respectively. Radio-immunotherapy to annexin A1 destroys tumours and increases animal survival. This analytical strategy can map tissue- and disease-specific expression of endothelial cell surface proteins to uncover novel accessible targets useful for imaging and therapy.
Assuntos
Endotélio Vascular/metabolismo , Perfilação da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Proteômica , Radioimunoterapia/métodos , Aminopeptidases/imunologia , Aminopeptidases/metabolismo , Animais , Anexina A1/imunologia , Anexina A1/metabolismo , Anticorpos/administração & dosagem , Anticorpos/imunologia , Anticorpos/uso terapêutico , Biologia Computacional , Sistemas de Liberação de Medicamentos/métodos , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/radioterapia , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Modelos Biológicos , Especificidade de Órgãos , Ratos , Taxa de SobrevidaRESUMO
Azetidine-2-carboxylic acid (Aze) 1 is a non-protein amino acid present in sugar beets and in table beets (Beta vulgaris). It is readily misincorporated into proteins in place of proline 2 in many species, including humans, and causes numerous toxic effects as well as congenital malformations. Its role in the pathogenesis of disease in humans has remained unexplored. Sugar beet agriculture, especially in the Northern Hemisphere, has become widespread during the past 150 years, and now accounts for nearly 30% of the world's supply of sucrose. Sugar beet byproducts are also used as a dietary supplement for livestock. Therefore, this study was undertaken as an initial survey to identify Aze-containing links in the food chain. Herein, we report the presence of Aze 1 in three sugar beet byproducts that are fed to farm animals: sugar beet molasses, shredded sugar beet pulp, and pelleted sugar beet pulp.
Assuntos
Ácido Azetidinocarboxílico/química , Beta vulgaris/química , Ração Animal , Alimentos , Tubérculos/químicaRESUMO
Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by restricted, stereotyped behaviors and impairments in social communication. Although the underlying biological mechanisms of ASD remain poorly understood, recent preclinical research has implicated the endogenous cannabinoid (or endocannabinoid), anandamide, as a significant neuromodulator in rodent models of ASD. Despite this promising preclinical evidence, no clinical studies to date have tested whether endocannabinoids are dysregulated in individuals with ASD. Here, we addressed this critical gap in knowledge by optimizing liquid chromatography-tandem mass spectrometry methodology to quantitatively analyze anandamide concentrations in banked blood samples collected from a cohort of children with and without ASD (N = 112). Findings: Anandamide concentrations significantly differentiated ASD cases (N = 59) from controls (N = 53), such that children with lower anandamide concentrations were more likely to have ASD (p = 0.041). In keeping with this notion, anandamide concentrations were also significantly lower in ASD compared to control children (p = 0.034). Conclusions: These findings are the first empirical human data to translate preclinical rodent findings to confirm a link between plasma anandamide concentrations in children with ASD. Although preliminary, these data suggest that impaired anandamide signaling may be involved in the pathophysiology of ASD.
Assuntos
Ácidos Araquidônicos/sangue , Transtorno Autístico/sangue , Agonistas de Receptores de Canabinoides/sangue , Endocanabinoides/sangue , Alcamidas Poli-Insaturadas/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , MasculinoRESUMO
Endothelial cells can function differently in vitro and in vivo; however, the degree of microenvironmental modulation in vivo remains unknown at the molecular level largely because of analytical limitations. We use multidimensional protein identification technology (MudPIT) to identify 450 proteins (with three or more spectra) in luminal endothelial cell plasma membranes isolated from rat lungs and from cultured rat lung microvascular endothelial cells. Forty-one percent of proteins expressed in vivo are not detected in vitro. Statistical analysis measuring reproducibility reveals that seven to ten MudPIT measurements are necessary to achieve > or =95% confidence of analytical completeness with current ion trap equipment. Large-scale mapping of the proteome of vascular endothelial cell surface in vivo, as demonstrated here, is advisable because distinct protein expression is apparently regulated by the tissue microenvironment that cannot yet be duplicated in standard cell culture.
Assuntos
Células Endoteliais/metabolismo , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Espectrometria de Massas/métodos , Proteínas de Membrana/metabolismo , Microcirculação/metabolismo , Proteoma/metabolismo , Animais , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Masculino , Proteômica/métodos , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Azetidine-2-carboxylic acid (L-Aze) is a toxic and teratogenic non-protein amino acid. In many species, including man, L-Aze is misincorporated into protein in place of proline, altering collagen, keratin, hemoglobin, and protein folding. In animal models of teratogenesis, it causes a wide range of malformations. The role of L-Aze in human disease has been unexplored, probably because the compound has not been associated with foods consumed by humans. Herein we report the presence of L-Aze in the garden or table beet (Beta vulgaris).