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Introduction Background What are Phosphoinositide 3-kinases? The Experiment Monitoring Regulation of PI3K Why is this Important Hook Me Up Bibliography Resources	The Experiment
	Twenty-five years ago, scientists discovered that phosphoinositide metabolism might experience an inhibitory affect from exposure to methylxanthines. However there was no research done to follow up this initial discovery. Recently, Peter Shepherd and his colleagues have begun to study the relationship and have unveiled some interesting results. Beginning their research with the hypothesis that methylxanthines would inhibit PI3-K, they carefully planned out several assays to determine if their hypothesis was sound or not
	The first experimental procedure concerned Protein Expression, Isolation, and Analysis. Baculoviral expression, a technique used to produce proteins on a large scale by using a recombinant vector, was applied to Sf9 insect cells which come from a species of parasitic insect know as the fall armyworm or Spodoptera frugiperda. By infecting these cells with recombinant vectors, three Class I recombinant heterodimers were prepared: p85a/p110a; p85a/p110b; and p85a/p110g as well as a Class II p110d monomer. Using ATP and phosphatidylionsitol as the substrates, the team conducted in vitro kinase assays with each of these recombinant isoforms using a incrementally increasing concentrations of caffeine and theophylline. They discovered that each of these PI3-K isoforms were sensitive to different concentrations of the methylxanthines top	Figure 12 Tables reflecting the inhibitory affects of Caffeine and Theophylline on the four respective recombinant PI3-K isoforms.
	Figure 13 These graphs demonstrate the PI3-K percentage of activity in the CHO-IR cells after treatment with Caffeine and Theophylline.	The next step to take was to determine whether or not the methylxanthines would have the same effect endogenically. In order to do this, CHO-IR cells were used. CHO-IR cells are Chinese Hamster Ovary cells which express human insulin receptors. Immunoprecipitate from these cells was collected. The same isoforms of Class I and II were tested for their Phosophoinositol 3-Kinase activity in the presence of caffeine and theophylline. The caffeine and theophylline were found to inhibit the activity of these lipid kinases as potently as in the in vitro experiment. top
	In order to determine what aspect of the methylxanthines inhibited the PI3-K isoformes, four synthetic derivatives of the core methylxanthine structure were tested to determine their inhibitory potency. Of the four, 3-isobutyl-1-methylxanthine; alloxazine; PD116948; and 3-propylxanthine, none was found to be as effective an inhibitor of the naturally occurring caffeine and theophylline. top
	Cambel Berk cberk@email.arizona.edu University of Arizona