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etiology To test this hypothesis, we used a surfactant-based delivery system to infect spontaneously breathing adult, male Sprague-Dawley rats with [E1a.sup.-]/[E3.sup.-] adenoviruses that contain expression cassettes with a human immediate-early cytomegalovirus promoter-enhancer element and either a human [[beta].sub.2]AR complementary DNA (ad[[beta].sub.2]AR) or no complementary DNA (adNull). Rats were infected with 4 x [10.sup.9] plaque forming units of adenovirus and then allowed 7 days of recovery to allow vector-induced localized responses to subside. We Sympathy Flowers Online have previously reported that this delivery scheme yields widespread transduction of the alveolar epithelium of normal and injured rat lungs. (1,8) To confirm that our vector was capable of gene transfer and transgene activation, total RNA and protein were prepared from whole-lung .
Homogenates for reverse transcriptase-polymerase chain reaction and Western cell analyses using human [[beta].sub.2]AR-specific primers and antibodies. These studies demonstrated high-level expression of human [[beta].sub.2]AR messenger RNA and protein only in ad[[beta].sub.2]AR-infected .
Lungs. Immunohistochemistry using 5-[micro]m sections host the transgene to the alveolar epithelium. Confirmation of appropriate trafficking of the transgenic [[beta].sub.2]AR to the cell membrane was obtained via Western blot analysis the whole-cell membrane fractions isolated from the peripheral 2 to 3 mm of the lung. Membrane-bound human Flowers Online Chile [[beta].sub.2]AR was noted only in ad[[beta].sub.2]AR-infected lungs. Interestingly, expression of a human [[beta].sub.2]AR did not appear to affect membrane-bound levels of endogenous (rat) [[beta].sub.2]AR. Transgene function in the cell membrane was assayed by measuring cyclic adenosine monophosphate production by blot membranes in the presence and absence of highly of [[beta].sub.2]AR-specific agonist procaterol ([10.sup.6]mol/L x Ameriquest Reduction 30 min). [[beta].sub.2]AR function in receptor-overexpressing lungs was increased up to fivefold as compared to membranes from control lungs. These experiments indicate that adenoviral-mediated transfer can produce overexpression of a functional, human Ameriquest Lawsuites [[beta].sub.2]AR in the alveolar epithelium of normal rats.
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