Two-dimensional differential in-gel electrophoresis (DIGE) of leaf and roots of Lycopersicon esculentum.

In this report we present a detailed protocol for the analysis of differential protein expression between two plant tissue samples. The protocol involves harvesting of leaves and roots from mature tomato plants, preparing protein extracts from the harvested tissues, fluorescent labeling of each sample prior to differential in-gel electrophoresis (DIGE), first- and second-dimension electrophoretic separations, and image analysis to visualize and quantify differential protein expression. This protocol is adaptable for use with a wide variety of plant materials and can be used to measure protein expression changes occurring in response to abiotic stress, biotic stress, genetic manipulation, selective breeding, and many other conditions. In addition to the detailed protocol, we also present the results of a representative experiment analyzing subtle changes in protein expression in the roots of tomato plants grown under control and salt-stress conditions.

The wildcat toolbox: a set of perl script utilities for use in peptide mass spectral database searching and proteomics experiments.

We describe in this communication a set of functional perl script utilities for use in peptide mass spectral database searching and proteomics experiments, known as the Wildcat Toolbox. These are all freely available for download from our laboratory Web site (http://proteomics.arizona.edu/toolbox.html) as a combined zip file, and can also be accessed via the Proteome Commons Web site (www.proteomecommons.org) in the tools section. We make them available to other potential users in the spirit of open source software development; we do not have the resources to provide any significant technical support for them, but we hope users will share both bugs and improvements with the community at large.

Retinal capillary pericyte proliferation and c-Fos mRNA induction by prostaglandin D2 through the cAMP response element.

PURPOSE: Cycloxygenase inhibitors have been shown to prevent angiogenesis in some circumstances, suggesting that growth of capillary pericytes or endothelial cells may be regulated by prostaglandins (PGs). The present study tests the effects of PGs on the growth of human retinal capillary pericytes. METHODS: Cell growth was assayed by formazan formation and 5-bromo-2'-deoxyuridine (BrdU) incorporation. The expression of mRNAs corresponding to c-fos, PG receptors, and VEGF was examined by RT-PCR. Signal transduction was evaluated by immunoblot analysis using phosphospecific antibodies against mitogen-activated protein kinases (MAPKs) and cAMP response element-binding protein (CREB). Synthesis of cAMP was inhibited with the adenyl cyclase inhibitor SQ22536. A reporter gene (luciferase) assay was conducted using the expression vector pSVOADelta5' containing the 379-bp c-fos promoter with and without a mutation in cAMP response element (CRE). RESULTS. PGD2 treatment induced c-fos mRNA, stimulated pericyte growth, and increased expression of VEGF mRNA. PGE2 and -F(2alpha) had similar effects on c-fos induction and pericyte growth, whereas PGI2 was ineffective. RT-PCR confirmed that mRNAs corresponding to the receptors for PGD2, -E2, -F(2alpha), and -I(2) were expressed in human retinal pericytes. Stimulation by PGD2 led to phosphorylation of CREB, but had negligible effect on phosphorylation of p44/42 MAPK. The adenylyl cyclase inhibitor inhibited CREB activation and c-fos induction by PGD2. In a reporter gene assay, c-fos induction occurred only with wild-type c-fos promoter. Mutation in CRE eliminated the response to PGD2. CONCLUSIONS: PGD2 promotes the growth of retinal capillary pericytes by signaling through cAMP and CREB. The findings underscore the importance of PGs in the growth of human retinal capillary pericytes and raise the possibility that PGs may play a role in proliferative retinopathies.