An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc.

The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation.     

E.s.r. of spin-trapped radicals in aqueous solutions of peptides. Reactions of the hydroxyl radical.

The reactions of hydroxyl radicals with 30 dipeptides and several larger peptides were studied in aqueous solutions. The OH radicals were generated by U.V. photolysis of H2O2. The short-lived peptide radicals were spin-trapped using t-nitrosobutane and identified by e.s.r. For dipeptides containing the amino terminal residues glycine, alanine and phenylalanine, abstraction of the hydrogen from the carbon adjacent to the peptide nitrogen was the major process leading to the spin-adducts. Such radicals will be referred to as backbone radicals. Dipeptides with a carbonyl terminal serine residue and also glycylglutamic acid form both backbone and side-chain radicals, with the latter being formed in larger quantities. For dipeptides, side-chain radicals were detected on either the carboxyl or amino terminal residues of both. The effect of pD on the e.s.r. sectrum of the spin-adducts of glycylglycine was studied and the pK of the carboxyl group of this radical was determined to be 2.5. For (Ala)3 and (Ala)n, with an average value of n = 1800, backbone and minor side-chain radicals were observed. For ribonucleases-S-peptide, containing 20 amino acid residues, both backbone and side-chain radicals were detected.     

Free radicals in U.V.-irradiated aqueous solutions of substituted amides: an e.s.r. and spin-trapping study.

The radicals produced by reactions of hydroxyl radicals with alkyl substituted ureas and amides in aqueous solutions have been investigated. Hydroxyl radicals were produced by U.V. photolysis of H2O2 and the short-lived amide and urea radicals were spin-trapped by t-nitrosobutane and identified by e.s.r. For all N-alkyl derivatives of urea and acetamide, and for N,N-dimethyl propionamide and N,N-diethyl formamide, only radicals centred on N-alkyl groups were detected. Radicals situated only on alkyl groups attached to the carbonyl carbon were observed for dimethyl acetamide, trimethyl acetamide and butyramide. However, for N,N-dimethyl butyramide, N, N-diethyl butyramide, N-methyl propionamide and N, N-diethyl propionamide, free radicals were formed which were localized on the alkyl group attached to the amide carbon as well as those attached to nitrogen. The hydrogen atom bound to the carbonyl carbon was abstracted in N-ethyl formamide. Acyl radicals formed by C-N scission due to direct U.V. photolysis of N, N-dimethyl butyramide and N,N-dimethyl propionamide were also detected.     

Mutant KRAS in the initiation of pancreatic cancer

Pancreatic ductal adenocarcinoma is the most common pancreatic neoplasm. There are approximately 33,000 new cases of pancreatic ductal adenocarcinoma annually in the United States with approximately the same number of deaths. Surgery represents the only opportunity for cure, but this is restricted to early stage pancreatic cancer. Pancreatic ductal adenocarcinoma evolves from a progressive cascade of cellular, morphological and architectural changes from normal ductal epithelium through preneoplastic lesions termed pancreatic intraepithelial neoplasia (PanIN). These PanIN lesions are in turn associated with somatic alterations in canonical oncogenes and tumor suppressor genes. Most notably, early PanIN lesions and almost all pancreatic ductal adenocarcinomas involve mutations in the K-ras oncogene. Thus, it is believed that activating K-ras mutations are critical for initiation of pancreatic ductal carcinogenesis. This has been proven through elegant genetically engineered mouse models in which a Cre-activated K-Ras(G12D) allele is knocked into the endogenous K-Ras locus and crossed with mice expressing Cre recombinase in pancreatic tissue. As a result, mechanistic insights are now possible into how K-Ras contributes to pancreatic ductal carcinogenesis, what cooperating events are required, and armed with this knowledge, new therapeutic approaches can be pursued and tested.     

Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture

This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.     

L<Subscript>2</Subscript>-norm multiple kernel learning and its application to biomedical data fusion

Background  

This paper introduces the notion of optimizing different norms in the dual problem of support vector machines with multiple kernels. The selection of norms yields different extensions of multiple kernel learning (MKL) such as L _∞, L ₁, and L ₂ MKL. In particular, L ₂ MKL is a novel method that leads to non-sparse optimal kernel coefficients, which is different from the sparse kernel coefficients optimized by the existing L _∞ MKL method. In real biomedical applications, L ₂ MKL may have more advantages over sparse integration method for thoroughly combining complementary information in heterogeneous data sources.