RESUMEN
The amyloid b-protein (Ab) deposited in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid b-protein precursor (APP). Generation of Ab from the APP requires two sequential proteolytic events: an initial b-secretase cleavage at the amino terminus of the Ab sequence followed by g-secretase cleavage at the carboxyl terminus of Ab. We describe the development of a robust in vitro assay for g-secretase cleavage by showing de novo Ab production in vitro and establish that this assay monitors authentic gamma-secretase activity by documenting the production of a cognate g-CTF, confirming the size of the Ab produced by mass spectrometry, and inhibiting cleavage in this system with multiple inhibitors that alter g-secretase activity in living cells. Using this assay, we demonstrate that the g-secretase activity 1) is tightly associated with the membrane, 2) can be solubilized, 3) has a pH optimum of 6.8 but is active from pH 6.0 to pH >8.4, and 4) ascertain that activities of the g-40 and g-42 are indeed pharmacologically distinct. These studies should facilitate the purification of the protease or proteases that are responsible for this unusual activity, which is a major therapeutic target for the treatment of AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidasas/análisis , Proteínas de la Membrana/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Animales , Células CHO , Sistema Libre de Células , Cricetinae , Concentración de Iones de Hidrógeno , Modelos Biológicos , Oligopéptidos/farmacología , Inhibidores de Proteasas/farmacología , SolubilidadRESUMEN
The monoamine oxidase-B inhibitor L-deprenyl (Selegiline) is effective in treating Parkinson's disease and possibly Alzheimer's disease. The neuroprotective property of L-deprenyl may be unrelated to the inhibition of monoamine oxidase-B. Since nitric oxide (NO) modulates activities including cerebral blood flow and memory, we examined the effect of L-deprenyl on NO. L-Deprenyl induced rapid increases in NO production in brain tissue and cerebral vessels. Vasodilation was produced by endothelial NO-dependent as well as NO-independent mechanisms in cerebral vessels. The drug also protected the vascular endothelium from the toxic effects of amyloid-beta peptide. These novel actions of selegiline may protect neurons from ischemic or oxidative damage and suggest new therapeutic applications for L-deprenyl in vascular and neurodegenerative diseases.
Asunto(s)
Circulación Cerebrovascular/efectos de los fármacos , Inhibidores de la Monoaminooxidasa/farmacología , Óxido Nítrico/biosíntesis , Selegilina/farmacología , Vasodilatación/efectos de los fármacos , Péptidos beta-Amiloides/toxicidad , Animales , Bovinos , Arterias Cerebrales/efectos de los fármacos , Arterias Cerebrales/patología , Arterias Cerebrales/fisiología , Interacciones Farmacológicas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Técnicas In Vitro , Músculo Liso Vascular/patología , Estimulación QuímicaRESUMEN
Cerebrovascular effects of beta-amyloid were investigated using bovine mid-cerebral arteries. beta-amyloid-induced endothelial damage was evidenced by increased vasoconstriction, diminished vasodilation and was evident on electron microscopy. The endothelial dysfunction was mediated by reactive oxygen radicals. Vascular damage by beta-amyloid may be an early event in the development of the pathology of Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Endotelio Vascular/efectos de los fármacos , Animales , Bovinos , Arterias Cerebrales/efectos de los fármacos , Técnicas In Vitro , Serotonina/farmacología , Vasoconstricción/efectos de los fármacosRESUMEN
beta-Amyloid (A beta) toxicity has a critical role in the pathology of Alzheimer's disease (AD) but its function in the neurodegenerative process is not clearly established. Recently, we demonstrated a novel action of beta-amyloid on peripheral blood vessels: endothelial dysfunction through reactive oxygen species. Here we report the direct effect of A beta on cerebrovascular endothelium. Following treatment with A beta 1-40, bovine cerebral arteries showed characteristic features of endothelial dysfunction such as increased contraction to vasoconstrictor and diminished relaxation to endothelium-dependent vasodilators. Electron microscopy revealed significant damage to the endothelium by A beta. Pretreatment with the antioxidant superoxide dismutase (SOD) and PBN (n-tert-butyl-alpha-phenylnitrone) antagonized the effects of A beta. Endothelial damage induced by A beta could produce ischemic and inflammatory changes contributing to the pathology of AD.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Arterias Cerebrales/fisiología , Endotelio Vascular/fisiología , Fragmentos de Péptidos/farmacología , Vasoconstrictores/farmacología , Vasodilatadores/farmacología , Péptidos beta-Amiloides/antagonistas & inhibidores , Análisis de Varianza , Animales , Antioxidantes/farmacología , Bradiquinina/farmacología , Bovinos , Óxidos N-Cíclicos , Modelos Logísticos , Óxidos de Nitrógeno/farmacología , Fragmentos de Péptidos/antagonistas & inhibidores , Superóxido Dismutasa/farmacologíaRESUMEN
Deposits of beta-amyloid are apparent in ageing and Alzheimer's disease, but the role of this peptide in neurodegeneration is unclear. The free-radical theory of ageing may also account for Alzheimer-type degeneration and consequently links between free-radical generation and beta-amyloid have been sought. We demonstrate here that beta-amyloid interacts with endothelial cells on blood vessels to produce and excess of superoxide radicals, with attendant alterations in endothelial structure and function. The superoxide radical can scavenge endothelium-derived relaxing factor and produce potent oxidizing agents, which can cause lipid peroxidation and other degenerative changes. The alterations in vascular tone and endothelial damage are prevented by the oxygen-radical-scavenging enzyme superoxide dismutase. These observations suggest a normal vasoactive role for beta-amyloid as well as a mechanism by which beta-amyloid may play a role in vascular abnormalities and neurodegeneration mediated by free radicals.
Asunto(s)
Péptidos beta-Amiloides/fisiología , Endotelio Vascular/metabolismo , Superóxidos/metabolismo , Acetilcolina/farmacología , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Aorta , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/ultraestructura , Técnicas In Vitro , Degeneración Nerviosa , Fenilefrina/farmacología , Ratas , Vasoconstricción , Vasoconstrictores/farmacología , Vasodilatadores/farmacologíaRESUMEN
Degloving injuries of the upper extremity may require extensive soft tissue coverage. Free tissue transfer is often useful in the management of these injuries. We describe the use of an extended rectus abdominis myofasciocutaneous vascularized free flap in the management of a massive longitudinal upper extremity traumatic soft tissue defect.
Asunto(s)
Traumatismos del Brazo/cirugía , Colgajos Quirúrgicos/métodos , Músculos Abdominales , Adulto , Humanos , MasculinoRESUMEN
The diagnosis of squamous cell carcinoma of the head and neck is not usually a problem for the otolaryngologist/head and neck surgeon. However, we describe a patient whose ultimate diagnosis of squamous cell carcinoma required 16 months and eluded several clinicians, despite an aggressive diagnostic regimen. The diagnostic difficulty was due to the fact that the small, centrally located tumor was surrounded by an intense inflammatory reaction that histologically mimicked a recently described rare entity of the head and neck, focal myositis. Our purpose is to make the otolaryngologist/head and neck surgeon aware of this previously unreported presentation of squamous cell carcinoma of the tongue; to review the pertinent findings associated with focal myositis; and to reinforce the need to maintain a constant vigil for the diagnosis of cancer, in spite of negative pathologic confirmation, when dictated by the clinical situation.