The bovine a and murine P subunits of rod-photoreceptor cGMP-phosphodiesterase (PDEL and PDEp) were expressed in adenovirus-transformed 293 human embryonic kidney cells. RNA blots from transfected cells showed transcripts of 3.0 and 2.8 kb corresponding to PDEL and PDEp, respectively. Protein expression was analyzed by using affnity-purified antibodies against cGMP-PDE on immunoblots and by immunoprecipitation. PDEa and PDEp exhibited the expected mobility (and thus apparent molecular size) and had cGMP hydrolytic activity. Reconstitution of the PDE a13 heterodimer with the expressed proteins increased by =6-fold the activity of the individual a and (8 subunits. Addition of expressed (8 subunit to retinal extracts from 9-to 10-day-old rd/rd mice (which have only normal a and Y subunits of rod cGMP-PDE and thus minimal activity) increased enzyme activity by -3-fold. Our results therefore demonstrate that photoreceptor-specific cGMP-PDE can be synthesized in human kidney cells with consequent expression of enzymatic activity.Rod-photoreceptor cGMP-phosphodiesterase (cGMP-PDE) is one of the key enzymes of the visual phototransduction cascade in the vertebrate retina (1). The holoenzyme is a heterotetrameric complex, consisting of two large catalytic subunits, a (88 kDa; PDEa) and P (84 kDa; PDE9), and two identical inhibitory subunits y (11 kDa) (2, 3). cGMP-PDE activity is stimulated in vivo by removal of the inhibitory y subunits by activated transducin (4). cGMP-PDE can be stimulated in vitro by incubation with histones (5) or by limited proteolytic digestion with trypsin (6).The cDNAs encoding the three subunits of cGMP-PDE from different animal species have been cloned, and their nucleotide and amino acid sequences have been determined (7)(8)(9)(10). Sequence homology between the a and 8 subunits from bovine (9) and murine (11) rod photoreceptors is -72% and 74%, respectively. Each of these subunits contains two putative noncatalytic cGMP-binding sites, two y subunitbinding sites, a putative catalytic domain, and the CAAX motif for isoprenylation (9,(12)(13)(14)(15). In rod photoreceptor cells the a and ,B subunits are farnesylated and geranylgeranylated, respectively, which facilitates their association with the disc membranes. The domain organization of these proteins suggests that they can have independent cGMP-hydrolytic activity.To carry out studies on the structure and function of cGMP-PDE in normal retinas and in those affected with genetic diseases, each of the biologically active subunits of the enzyme is necessary. Although the separation of the catalytic subunits is possible under denaturing conditions, no reports have been published to date on the isolation of active PDEa or PDE,9 using classical preparative biochemical procedures. Difficulties inherent to these methods can be overcome with the use of recombinant DNA technology, which has been applied in many instances to express large amounts of active proteins in bacterial or mammalian systems. However, previous attempts to express the ...
Acute Myocardial Infarction (AMI) remains a leading cause of mortality in the United States. Finding accurate and cost effective solutions for AMI diagnosis in Emergency Departments (ED) is vital. Consecutive, or serial, ECGs, taken minutes apart, have the potential to improve detection of AMI in patients presented to ED with symptoms of chest pain. By transforming the ECG into 3 dimensions (3D), computing 3D ECG markers, and processing marker variations, as extracted from serial ECG, more information can be gleaned about cardiac electrical activity. We aimed at improving AMI diagnostic accuracy relative to that of expert cardiologists. We utilized support vector machines in a multilayer network, optimized via a genetic algorithm search. We report a mean sensitivity of 86.82%±4.23% and specificity of 91.05%±2.10% on randomized subsets from a master set of 201 patients. Serial ECG processing using the proposed algorithm shows promise in improving AMI diagnosis in Emergency Department settings.
The current diagnostic criteria for sensitization and chronic beryllium disease rely on the beryllium lymphocyte proliferation test. By understanding the novel immunologic mechanisms and genetic factors associated with sensitization and chronic beryllium disease, we may improve our ability to detect beryllium health effects with new diagnostics, and hopefully refine therapies for disease.
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