Completed research The recurrent theme that has emerged from the structural study of proteins involved in signal transduction and transcriptional regulation is that multiple independently folded globular domains in these proteins often cooperate in macromolecular recognition. We have used the modularity of highly conserved arnino acid signature motifs as an approach to studying a protein's structure and function. The RING finger motif is a very abundant sequence motif in the protein database. This highly conserved module of approximately 50 amino acids is defined by eight cysteines and/or histidines that coordinate two zinc ions in a unique cross braced fashion. Upon cloning of BRCA1, the most recognizable signature motif in the amino acid sequence was a RING finger at the N-terminus. Moreover, genetic analysis of breast cancer kindreds identified naturally occuring germline mutations that lead to single amino acid substitutions of highly conserved residues in the RING finger and predispose individuals to disease. BAP-1, BRCA1 Associated Protein 1, is a novel, exclusively nuclear ubiquitin carboxy-terminal hydrolase that was identified by the Rauscher lab in a yeast two-hybrid interaction screen for proteins that would interact with the RING finger motif of BRCA1 (Jensen et al. Oncogene 16:1097-1112, 1998). Utilizing a putative coiled-coil structural motif located near the C-terminus of BAP-1, we performed a second, subsequent two-hybrid screen to identify additional proteins that may interact with BAP-1. Sequence from five positive clones, two independent fusions, identically matched nucleotide sequence which encodes the C-terminus of ß-catenin. Quantitative ß-gal assays of yeast extracts revealed the interaction was weak, indicating that either a larger portion of ß-catenin is necessary for optimal interaction or that the interaction might be very transient. To determine if a direct interaction occurred between BAP-1 and ß-catenin, 35 S-met labeled in vitro translates of BAP-1 and ß-catenin were tested for their ability to bind to GST fusions of ß-catenin or the C-terminus of BAP-1 in vitro, respectively, ß-catenin was observed to show some specific association with the C-terminus of BAP-1. In order to verify an in vivo interaction between BAP-1 and ß-catenin, co-immunoprecipitation experiments were employed. In order to verify an in vivo interaction between BAP-1 and ß-catenin, co-immunoprecipitation of 35 S-met labeled protein from transiently transfection of CMV driven expression plasmids containing the entire open reading frames of BAP-1 and ß-catenin into COS1 cells was employed. All attempts to co-immunoprecipitate BAP1 and ß-catenin failed under all experimental conditions (i.e. whole cell lysates vs. nuclear extracts, high salt vs. low salt, different detergents) and washing stringencies. Likewise, immunoprecipitation of endogenous BAP-1 followed by Western analysis with anti-sera against ß-catenin failed to detect in vivo interaction when performed under similar conditions. Identical David C. Schultz,...

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ObjectiveThe authors present their 35-year experience with intra-arterial chemotherapeutic regionai perfusion of 1 139 patients with melanomas, using an extracorporeal oxygenated circuft and heart-lung apparatus. Summary Background DataIntra-arterial chemotherapy produces improved responses in many tumors. By isolating and sustaining the area with extracorporeal oxygenated circulation, high doses can be delivered to the tumor area, limited only by local toxicity. Drug levels up to 10 times those achieved by systemic administration are obtained. MethodsTechniques for hyperthermic perfusion were developed for limbs, pelvis, head, neck, and skin of the breast. Melphalan (Burroughs Wellcome, Research Triangle Park, NC) was used in 753 patients. Combinations with melphalan or other drugs were used in remaining cases at temperature of 38 to 40 C for 30 to 60 minutes. ResultsChemotherapy perfusion followed by tumor excision or node dissection, was performed where indicated. The cumulative 1 0-year survival for patients with localized melanomas was 70%. For patients with local recurrences or satellites within 3 cm, survival was 61 %. For those with regionally confined intransit tumors, survival was 30%; for those with regional node involvement, 38%; for those with intransit and nodal metastases, 16%; for those with distant metastases and perfusion-mainly to save functional limbs-survival was 7%. Multiple perfusions were performed in 158 patients with recurrent disease on 366 occasions. Patients with indolent regionally confined melanomas were benefited by prolongation of useful life. ConclusionsSafe perfusion techniques are available for most anatomic regions. Increased chemotherapeutic doses are delivered to isolated areas limited only by local toxicity. Adjunct perfusion in poor prognosis stage cases is useful in reducing local recurrence, and intransit or lymph node metastases. Regional perfusion reduces the need for major amputation. Multiple perfusion can be useful in treating recurrent chronic melanoma. 520

Transcription factor IIIA (TFIIIA) and p43 zinc finger protein form distinct complexes with 5S ribosomal RNA in Xenopus oocytes. Additionally, TFIIIA binds the internal promoter of the 5S RNA gene and supports assembly of a transcription initiation complex. Both proteins have nine tandemly repeated zinc fingers with almost identical linker lengths between corresponding fingers, yet p43 has no detectable affinity for the 5S RNA gene. TFIIIA zinc fingers 1-3 are connected by highly conserved linkers, first identified in the Drosophila protein Krüppel, that are found in many DNA binding zinc finger proteins. To understand the role of these linkers in RNA and DNA binding we exchanged three TFIIIA linker amino acids with the equivalent amino acids from p43. The major effect of linker substitution is a 50-fold reduction in DNA specificity, concomitant with an 8-fold reduction in affinity. N-Terminal zinc fingers from either TFIIIA or p43 bind to multiple specific sites on 5S RNA that are resistant to competition by tRNA or poly(rA). This mode of RNA binding is unaffected by linker substitution. These data suggest that zinc finger linkers significantly facilitate the specificity of DNA binding.

Metastatic involvement of the gallbladder in melanoma is rare, but constitutes the most common metastatic lesion involving this organ. Two cases of metastatic melanoma to the gallbladder with radiographic evidence of gallbladder abnormality prior to surgery are presented. These cases are compared to the nine previously reported cases of metastatic melanoma to the gallbladder with abnormal cholecystograms. All eleven cases presented with signs and symptoms compatible with cholecystitis. Nine of the eleven patients had a previous melanoma primary and most had other extrabiliary metastases. Associated cholelithiasis appeared to be only incidental. In addition, nine reported cases of "primary" biliary melanoma were reviewed. Clinical and pathologic presentations in the latter cases were similar to the former cases with metastases. Seventy-eight percent had extrabiliary sites of metastasis at some time in the course of their disease, tending to refute the impression of "primary" biliary melanoma. Melanoma in the gallbladder is much more likely to have metastasized from a regressed skin primary than to have arisen de novo. The two reported cases and the 18 cases from the literature indicate that the physician must consider gallbladder metastasis in melanoma patients presenting with symptoms compatible with cholecystitis.

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Marjolin's ulcers have a grave prognosis, especially when regional nodes are involved. Recent studies suggest such cancers are in an immunologically privileges sites due to the dense scar tissue. The prognosis has been shown to be much worse for tumors not having a round cell infiltrate prior to surgery, as in Marjolin's ulcers. The use of topical 5-fluorouracil (5-FU) induces a round cell infiltrate. Three case reports of large Marjolin's ulcers are presented which were first treated with topical 5-FU. Radical ablative surgery was avoided in these patients with a successful outcome.

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