Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.

Previously, we identified the expression of a prostate-specific form of T cell receptor ␥ chain (TCR␥) mRNA in the human prostate and demonstrated that it originates from epithelial cells and not from infiltrating T lymphocytes. Here, we show that this prostatespecific transcript is also expressed in three breast cancer cell lines and breast cancer tissues. Analysis of the cDNA sequence predicts that this transcript can encode two protein products of 7 and 13 kDa, and in vitro translation experiments showed that both proteins were made. The longer ORF encodes a 13-kDa truncated version of TCR␥, whereas the shorter alternative reading frame encodes a 7-kDa protein with five leucine residues in heptad repeats followed by a basic region. Studies with specific antibodies against each protein product revealed that both prostate and breast cancer cells contain only the 7-kDa protein, which is located in the nucleus. We have named this protein TCR␥ alternate reading frame protein (TARP). These results demonstrate that an alternative protein product is encoded by the TCR␥ locus in cells other than T lymphocytes.expressed sequence tag ͉ leucine zipper ͉ nucleus

We report here syntenic loci in humans and mice incorporating gene clusters coding for secreted proteins each comprising 10 cysteine residues. These conform to three-fingered protein/Ly-6/urokinase-type plasminogen activator receptor (uPAR) domains that shape three-fingered proteins (TFPs). The founding gene is PATE, expressed primarily in prostate and less in testis. We have identified additional human PATElike genes (PATE-M, PATE-DJ, and PATE-B) that co-localize with the PATE locus, code for novel secreted PATE-like proteins, and show selective expression in prostate and/or testis. Anti-PATE-B-specific antibodies demonstrated the presence of PATE-B in the region

Chondrichthyan fishes are a diverse class of gnathostomes that provide a valuable perspective on fundamental characteristics shared by all jawed and limbed vertebrates. Studies of phylogeny, species diversity, population structure, conservation, and physiology are accelerated by genomic, transcriptomic and protein sequence data. These data are widely available for many sarcopterygii (coelacanth, lungfish and tetrapods) and actinoptergii (ray-finned fish including teleosts) taxa, but limited for chondrichthyan fishes.  In this study, we summarize available data for chondrichthyes and describe resources for one of the largest projects to characterize one of these fish, Leucoraja erinacea, the little skate.  SkateBase ( http://skatebase.org) serves as the skate genome project portal linking data, research tools, and teaching resources.

To identify unknown membrane proteins that could be used as targets for breast and prostate cancer immunotherapies and secreted proteins to be used as diagnostic markers, a cDNA library was generated from membrane-associated polyribosomal RNA derived from four breast cancer cell lines, one normal breast cell line, and a prostate cancer cell line. The membrane-associated polyribosomal cDNA library was subtracted with RNA from normal brain, liver, lung, kidney, and muscle. Of the 15,581 clones sequenced from the subtracted cDNA library, sequences from 10,506 clones map to known genes, but 5,075 sequences, representing 3,181 unique transcripts, are not associated with known genes. As one example, we experimentally investigated expression of a previously uncharacterized breast cancer gene that encodes a secreted protein designated BASE (breast cancer and salivary gland expression). BASE is expressed in many breast cancers but not in essential normal tissues including the five organs used for subtraction. Further analysis of this library should yield additional gene products of use in the diagnosis or treatment of breast or prostate cancer.

This is a report of the assessment of the predictions made for the CASP6 protein structure prediction experiment conducted in 2004 in the New Fold (NF) category. There were nine protein domains that were judged to have new folds (NF) and 16 for which a similar structure was known but the sequence similarity was judged to be too low for them to be easily recognized (FR/A). We selected all NF targets and eight of the 16 FR/A targets judged to be at the borderline between NF and FR/A for evaluation in the NF category. A total of 165 prediction groups submitted over 7400 structural models for these targets. The quality of these models was evaluated using the GDT_TS scores of the structural similarity detection program LGA and by visual inspection of the top-scoring models. The best models submitted bore an overall similarity to the target structure for three or four of the nine NF targets and for all but one of the FR/A targets. High-scoring models for the NF targets were submitted by several different groups. When both the NF and FR/A targets were considered, Baker group dominated by submitting best models for seven of the 17 targets, but 14 other groups also managed to submit best models for one or more targets.

We present an analysis of the domain boundary prediction, a new category, in the sixth community-wide experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP6). There were 1011 predictions submitted for 63 targets. Each prediction was compared to the set of domains defined manually by visual inspection of the experimental structure. The comparison was scored using a new domain prediction scoring scheme. As the definition of a domain is subjective, many targets were assigned alternate definitions. For such targets, each prediction was compared with all different definitions and the best score was chosen. The predictors found it difficult to accurately predict domain boundaries when the target protein contained many domains or domains made of multiple sequence segments. The CBRC-DR (P0536) and Sternberg (P0237) groups were the most successful among human experts, while Baker-Rossettadom (P0353) and Baker-Robetta-Ginzu (P0421) did well among servers.

Recent advances in high-throughput DNA sequencing technologies have equipped biologists with a powerful new set of tools for advancing research goals. The resulting flood of sequence data has made it critically important to train the next generation of scientists to handle the inherent bioinformatic challenges. The North East Bioinformatics Collaborative (NEBC) is undertaking the genome sequencing and annotation of the little skate (Leucoraja erinacea) to promote advancement of bioinformatics infrastructure in our region, with an emphasis on practical education to create a critical mass of informatically savvy life scientists. In support of the Little Skate Genome Project, the NEBC members have developed several annotation workshops and jamborees to provide training in genome sequencing, annotation and analysis. Acting as a nexus for both curation activities and dissemination of project data, a project web portal, SkateBase (http://skatebase.org) has been developed. As a case study to illustrate effective coupling of community annotation with workforce development, we report the results of the Mitochondrial Genome Annotation Jamborees organized to annotate the first completely assembled element of the Little Skate Genome Project, as a culminating experience for participants from our three prior annotation workshops. We are applying the physical/virtual infrastructure and lessons learned from these activities to enhance and streamline the genome annotation workflow, as we look toward our continuing efforts for larger-scale functional and structural community annotation of the L. erinacea genome.