Structural analysis of a set of proteins resulting from a bacterial genomics project.

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.

Immunohistochemical staining for desmogleins 1 and 2 in keratinocytic neoplasms with squamous phenotype: actinic keratosis, keratoacanthoma and squamous cell carcinoma of the skin.

Desmosomes are intercellular junctions that have been shown to be down-regulated in certain types of carcinoma and that may play a role in suppression of invasion and metastasis. This paper describes an immunohistochemical study of three types of epidermal neoplasms with monoclonal antibody to desmoglein in order to determine how desmosomal staining correlates with the clinical, biological and histopathological features of these neoplasms. Actinic keratosis (AK) is the most common keratinocytic premalignant neoplasm that was reported to have a 10-20% rate of malignant transformation into squamous cell carcinoma (SCC). Keratoacanthoma (KA) is a benign neoplasm that involutes spontaneously after a few months of rapid growth. SCC is a malignant tumour capable of metastasis. Electron microscope studies of KA and SCC showed significantly reduced staining for desmosomes in SCC but not in KA. We have examined staining for desmoglein using the monoclonal antibody 33-3D, a mouse IgM monoclonal antibody, that recognizes the cytoplasmic domains of desmoglein (Dsg)1 and Dsg2 on frozen sections. Immunohistochemical staining of normal skin with this antibody revealed strong pericellular localization of the antigen, outlining the cell membranes of the keratinocytes. A series of 30 AKs, 12 KAs and 24 SCCs was stained immunohistochemically with 33-3D monoclonal antibody. All examined KAs showed extensive pericellular staining for Dsg. By contrast, juxtanuclear staining for Dsg was noted in 12 SCCs, and completely negative staining in seven SCCs. The five remaining SCCs showed focal pericellular staining for the Dsg marker. The most common finding in AK was focal pericellular staining for Dsg, with complete absence of staining in dysplastic areas (25 cases). In five cases negative pericellular staining in dysplastic areas was associated with juxtanuclear accumulation of the Dsg marker. A strong negative correlation between Dsg staining and degree of dysplasia was obtained. The Dsg pattern in KA is similar to normal epidermis and shows a clear difference between KA and SCC. AK has a limited loss of Dsg expression in a SCC-like pattern that is congruent with its premalignant nature. As the stain works on frozen tissue, it may be helpful for rapid differentiation in selected cases in cutaneous oncology and Mohs micrographic surgery. This antibody may also have great potential for the detection of the effects of chemopreventive agents in skin cancer.

The use of antidesmoglein stains in Mohs micrographic surgery. A potential aid for the differentiation of basal cell carcinoma from horizontal sections of the hair follicle and folliculocentric basaloid proliferation.

BACKGROUND: Histopathologic differentiation between benign and malignant tissue is of utmost importance for the Mohs surgeon. Folliculocentric basaloid proliferation (FBP) shares many histologic features with basal cell carcinoma (BCC). It is most commonly associated with tumors of areas with abundant hair follicles such as nasal and perinasal skin. Residual BCC incorrectly identified as a horizontally sectioned hair follicle undoubtedly increases the risk of tumor recurrence. Excision of additional layers of normal tissue to remove "funny looking follicles" may have profound impacts on tissue conservation, preservation of function, and cosmesis. Electron microscope studies of BCC revealed a significant reduction of desmosomes compared with normal basal cells and hair follicle keratinocytes. OBJECTIVE: This study has assessed the potential of rapid staining with monoclonal antidesmoglein antibody (33-3D) to discriminate between BCC, horizontally sectioned hair follicles, and FBP. METHODS: A rapid immunoperoxidase technique with 33-3D antidesmoglein antibody was performed on Mohs frozen sections. We selected 18 patients with BCC of nasal and perinasal locations where histologic discrimination between residual tumor and tumor-free margins with FBP or horizontally sectioned hair follicle was equivocal. RESULTS: Fourteen sections disclosed the preservation of desmoglein marker delineating the cell membranes ("perimembranous" pattern) consistent with normal hair follicles. The sections were identified as tumor-free and no additional stages were performed. The remaining four sections revealed absent perimembranous pattern but presence of diffuse cytoplasmic staining. These were diagnosed as positive for residual BCC requiring the excision of another layer of tissue to obtain tumor-free margins. A follow-up period ranging from 6 to 24 months revealed no instance of recurrent disease. CONCLUSION: Rapid detection of desmoglein with 33-3D antibody is a promising tool for discrimination between residual BCC and FBP or horizontally sectioned hair follicles. It may enhance the sensitivity of Mohs surgery by disclosing the hidden foci of BCC, thus preventing tumor recurrence and unnecessary excision of normal tissue.

Lymphoepithelioma-like carcinoma of the skin treated with Mohs micrographic surgery in combination with immune staining for cytokeratins.

Lymphoepithelioma-like carcinoma of the skin (LLCS) is a rare cutaneous neoplasm that histologically resembles nasopharyngeal lymphoepithelioma. Conventional surgical excision carries a considerable rate of recurrence (three of 11 reported cases with such treatment, with one patient dying of metastatic disease). We report the first case of lymphoepithelioma-like carcinoma of the skin treated with Mohs micrographic surgery. Because of its tendency to occur on the face and its potential for recurrence after incomplete removal, this tumor is a good candidate for treatment with Mohs micrographic surgery. Immunohistochemical staining of frozen sections for cytokeratins may help to detect neoplastic cells that may be obscured by the dense lymphoplasmacytic infiltrate associated with this tumor.

Immunohistochemical techniques in Mohs micrographic surgery: their potential use in the detection of neoplastic cells masked by inflammation.

BACKGROUND: Histopathologic evaluation of tissue obtained from Mohs micrographic surgery is the key step in obtaining complete tumor removal. Residual undetected tumor may result in recurrence. OBJECTIVE: In circumstances in which the histopathologic interpretation is difficult, we assessed the potential use of immunohistochemical techniques to detect tumor in Mohs micrographic surgical specimens. METHODS: A rapid immunoperoxidase technique with monoclonal anticytokeratin antibodies was performed on Mohs frozen sections. Cases selected included morpheaform basal cell carcinomas, perineural tumors, and sections with dense inflammation without apparent tumor. RESULTS: Four cases are described as examples that highlight the potential usefulness of immunostaining of Mohs tissue sections. Anticytokeratin antibodies helped to confirm free tumor margins, thus avoiding the unnecessary sacrifice of normal tissue, and to delineate tumor not identified in hematoxylin and eosin frozen sections. CONCLUSION: Immunohistochemical staining of Mohs micrographic surgical specimens with anticytokeratin antibodies is particularly useful when dense inflammatory infiltrate is present, because the latter may obscure any residual tumor. Application of this technique to difficult cases may prevent tumor recurrences or unnecessary excision of normal tissue.

Immunohistochemical margin control applied to Mohs micrographic surgical excision of dermatofibrosarcoma protuberans.

BACKGROUND: Surgical treatment of dermatofibrosarcoma protuberans has a high rate of recurrence presumably secondary to persistent residual tumor. Recently an antigenic marker, CD34, has demonstrated specificity for this tumor. OBJECTIVE: To improve the microscopic detection of dermatofibrosarcoma protuberans tumor elements in Mohs micrographic surgical sections by incorporating immunohistologic staining. METHODS: Standard Mohs micrographic surgical technique was used, coupled with standard immunohistochemical procedures using an antibody to the CD34 antigen. RESULTS: Immunohistochemical staining with anti-CD34 of Mohs micrographic sections clearly delineated the extent of the tumor elements. CONCLUSIONS: We anticipate that the application of this immunohistochemical-modified Mohs surgical technique will further enhance the detection of insidious portions of tumor thereby enhancing removal and reducing recurrence.