Identification of novel tumor antigens with patient-derived immune-selected antibodies.

The identification of tumor antigens capable of eliciting an immune response in vivo may be an effective method to identify therapeutic cancer targets. We have developed a method to identify such antigens using frozen tumor-draining lymph node samples from breast cancer patients. Immune responses in tumor-draining lymph nodes were identified by immunostaining lymph node sections for B-cell markers (CD20&CD23) and Ki67 which revealed cell proliferation in germinal center zones.

Expression of the bovine papillomavirus type 1 E5B gene reveals a protein-protein interaction of the E5A and E5B gene products.

The bovine papillomavirus type 1 (BPV-1) genome has been shown to contain a small open-reading frame designated E5B (nucleotides 4013-4167) which is predicted to encode a hydrophobic, 52 amino acid protein. In order to detect and characterize the E5B protein, an 18 nucleotide sequence encoding a 6 amino acid epitope was added to the 3' end of the E5B open-reading frame which was then expressed in COS-1 cells using a SV40 vector. Immunoprecipitation, immunofluorescence, and cell fractionation studies identified the E5B protein as a 4-kDa protein and localized it primarily to membranes of the endoplasmic reticulum and nucleus.

Rolling circle amplification: a new approach to increase sensitivity for immunohistochemistry and flow cytometry.

Immunohistochemistry is a method that can provide complementary diagnostic and prognostic information to morphological observations and soluble assays. Sensitivity, specificity, or requirements for arduous sample preparation or signal amplification procedures often limit the application of this approach to routine clinical specimens. Rolling circle amplification (RCA) generates a localized signal via an isothermal amplification of an oligonucleotide circle.

GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming.

The GreA and GreB proteins of Escherichia coli show a multitude of effects on transcription elongation in vitro, yet their physiological functions are poorly understood. Here, we investigated whether and how these factors influence lateral oscillations of RNA polymerase (RNAP) in vivo, observed at a protein readblock. When RNAP is stalled within an (ATC/TAG)(n) sequence, it appears to oscillate between an upstream and a downstream position on the template, 3 bp apart, with concomitant trimming of the transcript 3' terminus and its re-synthesis.

E5 oncoprotein mutants activate phosphoinositide 3-kinase independently of platelet-derived growth factor receptor activation.

The E5 oncoprotein of bovine papillomavirus type 1 is a Golgi-resident, 44-amino acid polypeptide that can transform fibroblast cell lines by activating endogenous platelet-derived growth factor receptor beta (PDGF-R). However, the recent discovery of E5 mutants that exhibit strong transforming activity but minimal PDGF-R tyrosine phosphorylation indicates that E5 can potentially use additional signal transduction pathway(s) to transform cells. We now show that two classes of E5 mutants, despite poorly activating the PDGF-R, induce tyrosine phosphorylation and activation of phosphoinositide 3-kinase (PI 3-K) and that this activation is resistant to a selective inhibitor of PDGF-R kinase activity, tyrphostin AG1296.