7 research outputs found
Controlling Mammalian Gene Expression by Allosteric Hepatitis Delta Virus Ribozymes
We engineered small molecule responsive
allosteric ribozymes based
on the genomic hepatitis delta virus (HDV) ribozyme by replacing the
P4-L4 stem-loop with an RNA aptamer through a connector stem. When
embedded in the 3′ untranslated region of a reporter gene mRNA,
these RNA devices enabled regulation of <i>cis</i>-gene
expression by theophylline and guanine by up to 29.5-fold in mammalian
cell culture. Furthermore, a NOR logic gate device was constructed
by placing two engineered ribozymes in tandem, demonstrating the modularity
of the RNA devices. The significant improvement in the regulatory
dynamic range (ON/OFF ratio) of the RNA devices based on the HDV ribozyme
should provide new opportunities for practical applications
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cAMP and cGMP Play an Essential Role in Galvanotaxis of Cell Fragments.
Cell fragments devoid of the nucleus and major organelles are found in physiology and pathology, for example platelets derived from megakaryocytes, and cell fragments from white blood cells and glioma cells. Platelets exhibit active chemotaxis. Fragments from white blood cells display chemotaxis, phagocytosis, and bactericidal functions. Signaling mechanisms underlying migration of cell fragments are poorly understood. Here we used fish keratocyte fragments and demonstrated striking differences in signal transduction in migration of cell fragments and parental cells in a weak electric field. cAMP or cGMP agonists completely abolished directional migration of fragments, but had no effect on parental cells. The inhibition effects were prevented by pre-incubating with cAMP and cGMP antagonists. Blocking cAMP and cGMP downstream signaling by inhibition of PKA and PKG also recovered fragment galvanotaxis. Both perturbations confirmed that the inhibitory effect was mediated by cAMP or cGMP signaling. Inhibition of cathode signaling with PI3K inhibitor LY294002 also prevented the effects of cAMP or cGMP agonists. Our results suggest that cAMP and cGMP are essential for galvanotaxis of cell fragments, in contrast to the signaling mechanisms in parental cells
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cAMP and cGMP Play an Essential Role in Galvanotaxis of Cell Fragments.
Cell fragments devoid of the nucleus and major organelles are found in physiology and pathology, for example platelets derived from megakaryocytes, and cell fragments from white blood cells and glioma cells. Platelets exhibit active chemotaxis. Fragments from white blood cells display chemotaxis, phagocytosis, and bactericidal functions. Signaling mechanisms underlying migration of cell fragments are poorly understood. Here we used fish keratocyte fragments and demonstrated striking differences in signal transduction in migration of cell fragments and parental cells in a weak electric field. cAMP or cGMP agonists completely abolished directional migration of fragments, but had no effect on parental cells. The inhibition effects were prevented by pre-incubating with cAMP and cGMP antagonists. Blocking cAMP and cGMP downstream signaling by inhibition of PKA and PKG also recovered fragment galvanotaxis. Both perturbations confirmed that the inhibitory effect was mediated by cAMP or cGMP signaling. Inhibition of cathode signaling with PI3K inhibitor LY294002 also prevented the effects of cAMP or cGMP agonists. Our results suggest that cAMP and cGMP are essential for galvanotaxis of cell fragments, in contrast to the signaling mechanisms in parental cells
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Identification of DES1 as a vitamin A isomerase in Müller glial cells of the retina.
Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternative visual cycle for regenerating opsins in daylight. Here we identified dihydroceramide desaturase-1 (DES1) as a retinol isomerase and an excellent candidate for isomerase-2 in this alternative pathway. DES1 is expressed in retinal Müller cells, where it coimmunoprecipitates with cellular retinaldehyde binding protein (CRALBP). Adenoviral gene therapy with DES1 partially rescued the biochemical and physiological phenotypes in Rpe65(-/-) mice lacking isomerohydrolase (isomerase-1). Knockdown of DES1 expression by RNA interference concordantly reduced isomerase-2 activity in cultured Müller cells. Purified DES1 had very high isomerase-2 activity in the presence of appropriate cofactors, suggesting that DES1 by itself is sufficient for isomerase activity
Identification of DES1 as a vitamin A isomerase in Müller glial cells of the retina
Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternate visual cycle for regenerating opsins in daylight. Here, we identified dihydroceramide desaturase-1 (DES1) as a retinol isomerase and an excellent candidate for isomerase-2 in this alternate pathway. DES1 is expressed in retinal Müller cells where it co-immunoprecipitates with cellular retinaldehyde binding protein (CRALBP). Adenoviral gene therapy with DES1 partially rescued the biochemical and physiological phenotypes in rpe65 (−/−) mice lacking isomerohydrolase (isomerase-1). Knockdown of DES1 expression by RNA-interference concordantly reduced isomerase-2 activity in cultured Müller cells. Purified DES1 possessed very high isomerase-2 activity in the presence of appropriate cofactors, suggesting that DES1 by itself is sufficient for isomerase activity