The glycerophosphodiester phosphodiesterase enzyme family involved in the hydrolysis of glycerophosphodiesters has been characterized in bacteria and recently identified in mammals. Here, we have characterized the activity and function of GDE3, one of the seven mammalian enzymes. GDE3 is up-regulated during osteoblast differentiation and can affect cell morphology. We show that GDE3 is a glycerophosphoinositol (GroPIns) phosphodiesterase that hydrolyzes GroPIns, producing inositol 1-phosphate and glycerol, and thus suggesting specific roles for this enzyme in GroPIns metabolism. Substrate specificity analyses show that wild-type GDE3 selectively hydrolyzes GroPIns over glycerophosphocholine, glycerophosphoethanolamine, and glycerophosphoserine. A single point mutation in the catalytic domain of GDE3 (GDE3R231A) leads to loss of GroPIns enzymatic hydrolysis, identifying an arginine residue crucial for GDE3 activity. After heterologous GDE3 expression in HEK293T cells, phosphodiesterase activity is detected in the extracellular medium, with no effect on the intracellular GroPIns pool. Together with the millimolar concentrations of calcium required for GDE3 activity, this predicts an enzyme topology with an extracellular catalytic domain. Interestingly, GDE3 ectocellular activity is detected in a stable clone from a murine osteoblast cell line, further confirming the activity of GDE3 in a more physiological context. Finally, overexpression of wild-type GDE3 in osteoblasts promotes disassembly of actin stress fibers, decrease in growth rate, and increase in alkaline phosphatase activity and calcium content, indicating a role for GDE3 in induction of differentiation. Thus, we have identified the GDE3 substrate GroPIns as a candidate mediator for osteoblast proliferation, in line with the GroPIns activity observed previously in epithelial cells.
Background:The known mammalian glycerophosphodiester phosphodiesterases hydrolyze glycerophosphodiesters. Results: New members of the glycerophosphodiester phosphodiesterase family, GDE4 and GDE7, cannot hydrolyze glycerophosphodiesters but show lysophospholipase D activity. Conclusion: GDE4 and GDE7 can hydrolyze 1-acyl-lyso-PC and lyso-PAF to produce 1-acyl-lysophosphatidic acid (LPA) and alkyl-LPA, respectively. Significance: The mammalian glycerophosphodiester phosphodiesterase family may have a new function in LPA signaling.
T-DNA binary vectors are often used in plant transformation experiments. Because they are usually very large and have few restriction sites suitable for DNA ligation reactions, cloning DNA fragments into these vectors is difficult. We provide herein an alternative to cloning DNA fragments into very large vectors. Our yeast-based recombineering method enables DNA fragments to be cloned into certain types of T-DNA binary vectors by one-step transformation without the requirement of specific recombination sites or precisely positioned restriction ends, thus making the cloning process more flexible. Moreover, this method is inexpensive and is applicable to multifragment cloning.
Summary
Background: The effect of activin on differentiated cells is known to be different from that on undifferentiated cells. Cultured gastric epithelial cells in complete serum‐free conditions grew into matured mucous cells after treatment with epidermal growth factor (EGF).
Aim: To elucidate the effect of activin on the growth of differentiated and undifferentiated gastric mucosal cells.
Methods: Cultured guinea pig gastric epithelial cells were prepared using the method of Ogihara et al.
1Synthesis of activin was analysed by Western blot using monoclonal anti‐activin A antibody. Cell proliferation was assessed by counting the number of cells. Mucin production was assessed by histochemical study using periodic acid‐Schiff (PAS) reaction.
Results: Western blot analysis indicated that activin was synthesized in cultured guinea pig gastric mucosal cells. One hundred nanomolar EGF induced a 3‐fold increase in cell count and the appearance of PAS‐positive granules. Five nanograms activin per millilitre without EGF stimulated proliferation of the cells that showed almost negative PAS staining. When activin was added after treatment with 100 n
m EGF for 24 h, cell proliferation induced by EGF was inhibited by activin at concentrations higher than 5 ng/mL.
Conclusion: These results suggest that activin stimulates proliferation of undifferentiated cells and inhibits growth of differentiated cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.