Genetic analysis of a possible case of canine X-linked ectodermal dysplasia.

In the present report, we describe targeted next-generation sequencing of the EDA gene of a male poodle with a clinical and histopathological diagnosis of X-linked hypohidrotic ectodermal dysplasia. The result was compared with the reference sequence and with the result of the sequencing of a normal dog's EDA gene. No point variant, small deletion or insertion were found in the exons and splice sites, but a transition and a transversion were found in the intron 6' and 3' UTR, respectively. The cause of the dysplasia of the affected dog in this study is neither a point variant nor a small deletion or insertion in the exons and splice sites of the EDA gene. Therefore, patients with phenotype of XLHED may have other types of variants in the EDA gene or variants in other genes of the EDA signalling pathway.

On the ratio distribution of energy windowing algorithms for radiation portal monitors.

This paper introduces a windowing algorithm for radiation portal monitors based on a transformation of random variables to calculate the ratio between the radiation intensity in two energy windows. Procedures to evaluate critical limits and establish detection limits are described. Simulations and experimental data illustrate a good agreement between derived and observed ratio distributions. The algorithm was compared with commonly-used algorithms. The results illustrate proper evaluation of critical and detection limits even in the case of shadow shielding by vehicles entering a radiation portal monitor.

Regulation of PTEN transcription by p53.

PTEN tumor suppressor is frequently mutated in human cancers and is a negative regulator of PI3'K/PKB/Akt-dependent cellular survival. Investigation of the human genomic PTEN locus revealed a p53 binding element directly upstream of the PTEN gene. Deletion and mutation analyses showed that this element is necessary for inducible transactivation of PTEN by p53. A p53-independent element controlling constitutive expression of PTEN was also identified. In contrast to p53 mutant cell lines, induction of p53 in primary and tumor cell lines with wild-type p53 increased PTEN mRNA levels. PTEN was required for p53-mediated apoptosis in immortalized mouse embryonic fibroblasts. Our results reveal a unique role for p53 in regulation of cellular survival and an interesting connection in tumor suppressor signaling.

An automated aequorin luminescence-based functional calcium assay for G-protein-coupled receptors.

We describe in detail an automated and highly sensitive functional assay for calcium-coupled receptors (those receptors whose activation results in an increase in intracellular calcium levels) utilizing coelenterazine-charged aequorin as a probe for intracellular calcium levels ([Ca(2+)](i)). The assay was originally established to investigate Galpha(q)-coupled prostanoid receptors, which are members of the G-protein-coupled receptor (GPCR) superfamily, signaling through elevation of [Ca(2+)](i), initially focusing on the human EP(1) prostanoid receptor (hEP(1)). The parental human embryonic kidney cell line 293-AEQ17, developed by Button and Brownstein (Cell Calcium 14, 663-671, 1993), constitutively expresses apoaequorin and was used to develop a clonal cell line which stably coexpresses hEP(1). This cell line was used to optimize assay parameters in order to maximize accuracy and throughput in an automated 96-well format with the result that each 96-well plate can be completed in 70 min. Use of this flexible system will greatly simplify the functional analysis of GPCRs and other receptors which when activated result in increases in [Ca(2+)](i).

Molecular characterization of a common fragile site (FRA7H) on human chromosome 7 by the cloning of a simian virus 40 integration site.

Common fragile sites are chromosomal loci prone to breakage and rearrangement, hypothesized to provide targets for foreign DNA integration. We cloned a simian virus 40 integration site and showed by fluorescent in situ hybridization analysis that the integration event had occurred within a common aphidicolin-induced fragile site on human chromosome 7, FRA7H. A region of 161 kb spanning FRA7H was defined and sequenced. Several regions with a potential unusual DNA structure, including high-flexibility, low-stability, and non-B-DNA-forming sequences were identified in this region. We performed a similar analysis on the published FRA3B sequence and the putative partial FRA7G, which also revealed an impressive cluster of regions with high flexibility and low stability. Thus, these unusual DNA characteristics are possibly intrinsic properties of common fragile sites that may affect their replication and condensation as well as organization, and may lead to fragility.

MP26, a protein of intercellular junctions in the bovine lens: electrophoretic and chromatographic characterization.

We have characterized the membrane protein of apparent molecular weight 26 kD from bovine lenses (MP26 or MIP) with respect to six different electrophoretic and chromatographic procedures. These include one- and two-dimensional gel electrophoretic procedures, as well as SDS-hydroxylapatite chromatography. The two-dimensional gels include isoelectric focusing with both conventional ampholytes and buffer focusing methods. With buffer focusing, the membranes are solubilized without the use of SDS and the isoelectric focusing is performed in the absence of SDS. As specific probes for MP26, a monoclonal antibody and an anti-MP26 rabbit serum were used, the latter prepared against electrophoretically purified MP26. These separation techniques were adapted to MP26 in order to permit a more detailed characterization of this protein and to search for any heterogeneity in this size range, specifically other junctional proteins or protein fragments. We have found evidence for charge heterogeneity in MP26, but no evidence for multiple membrane proteins of Mr 26,000 in urea-treated membranes. The charge heterogeneity appears to be related to a phosphorylation of MP26. The results reported here aid the interpretation of a variety of data, especially findings on the reconstitution of MP26 in artificial membranes and results from work with polyclonal MP26 antibodies. These investigations are all designed to evaluate the proposed role of MP26 as a protein of cell-to-cell channels in the lens fiber cell.

Enteric neuronal autoantibodies in pseudoobstruction with small-cell lung carcinoma.

Severe gastrointestinal dysmotility is a newly recognized paraneoplastic syndrome that occurs with small-cell lung carcinoma. Thirty-four patients with small-cell carcinoma, of whom 5 had chronic intestinal pseudoobstruction and 29 had no digestive symptoms, were studied serologically. Four of the 5 patients with gut dysmotility had immunoglobulin G antibodies reactive with neurons of the myenteric and submucosal plexuses of jejunum and stomach in an indirect immunofluorescence assay. Antibodies of this type were not found in any of the 29 patients who had no gut dysmotility, nor were they found in patients with chronic idiopathic intestinal pseudoobstruction (n = 8), ovarian cancer (n = 20), or epilepsy (n = 4) or in normal subjects (n = 9). In 4 of the patients with paraneoplastic pseudoobstruction, antibodies in highly diluted serum (1:4000-1:8000) bound selectively to nuclei and cytoplasm of neuronal elements in the gut. This novel autoantibody activity suggests that intestinal pseudoobstruction occurring in patients with small-cell carcinoma may have an autoimmune basis. From a clinical standpoint, serological testing offers a simple means for determining which patients with gut dysmotility syndromes may have associated small-cell carcinoma, thereby enabling earlier diagnosis and treatment of the tumor.

Culture of human neoplastic gastrointestinal smooth muscle cells.

Due to limited growth potential of primary cultures and the absence of continuous lines of healthy enteric smooth muscle, we have studied the culture behavior of neoplastic gastrointestinal smooth muscle cells. Forty-six human enteric smooth muscle neoplasms (leiomyomas and leiomyosarcomas) were studied while fresh and/or after culture in vitro and growth in vivo in athymic nude mice, with assessments made of morphology, growth characteristics, and biochemical markers of differentiation. The state of differentiation of the tumors varied, with well-differentiated tumors tending to express binding sites for the gastrointestinal hormone cholecystokinin, whereas less well-differentiated tumors did not. Poorly differentiated tumors were the easiest to establish in culture in vitro and to grow in vivo in nude mice. When the cells placed directly into culture proliferated to confluent density, they underwent morphologic differentiation from a spread, fibroblastlike shape to a slender spindle morphology, with these cells possessing fewer biosynthetic organelles and arranging themselves in characteristic "hill and valley" arrays. However, the highly differentiated characteristics of expression of desmin or cholecystokinin-binding sites were not observed in cultured cells. In contrast, cells that had been passaged in nude mice before culture displayed a proliferative phenotype and failed to undergo morphologic differentiation on reaching confluent density. Four human enteric smooth muscle cell lines (documented by chromosomal analysis) originating in stomach, jejunum, ileum, and rectum were established using this strategy.

Culture behavior of healthy bovine gallbladder muscularis smooth muscle cells.

In contrast to the extensive experience culturing vascular smooth muscle cells, little is known about the culture behavior of gastrointestinal smooth muscle cells. In this work, we have studied smooth muscle cells from bovine gallbladder muscularis in culture. Properties reflecting their state of differentiation, including cellular morphology, multicellular arrangements, intermediate filament protein expression, and content of contractile proteins, were studied after dispersed cell preparations were placed into short- and long-term culture on a variety of substrates. Immunocytochemical analysis of intact healthy gallbladder wall demonstrated that the muscularis smooth muscle cells express actin and the intermediate filament protein desmin, while being vimentin-negative. In this tissue they are, however, surrounded by sheets of vimentin-positive fibroblasts. Optimal microdissection of mucosa and serosa from the muscularis therefore still produced a combination of smooth muscle cells and fibroblasts; however, multiple strategies for enriching the yield and purity of muscularis smooth muscle cells for culture were partially successful. Like vascular smooth muscle cells in culture, these visceral smooth muscle cells rapidly underwent morphological dedifferentiation, losing their contactile phenotype. By 5 days in culture, the desmin-positive muscle cells took on a spread, fibroblast-like morphology, likely representing modulation to a proliferative, dedifferentiated state. After long-term culture, the muscle cells were observed to regain some markers of differentiation, but they were never observed to attain complete morphological and functional redifferentiation.

Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression.

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.

Clearing and release of basement membrane proteins from substrates by metastatic tumor cell variants.

A variety of normal cells and tumor cell lines of differing metastatic potential were evaluated for their effect on various substrates consisting of purified preparations of the basement membrane-associated proteins fibronectin, laminin, and type IV collagen, which were labeled with tritium and/or rhodamine isothiocyanate. Different degrees of clearing or release of material from the substrate were observed, depending on the cell-protein combination. Normal fibroblasts as well as transformed cells and cells of low metastatic potential showed extensive clearing of surfaces coated with fibronectin, laminin, and type IV collagen. This clearing of protein began at sites of initial cell adhesion and was restricted to areas beneath and/or along the apparent paths of cell migration and beneath cellular processes. Covalent attachment of adhesion proteins to glass coverslips nearly eliminated clearing and release. Studies showed that a substantial amount of the fibronectin released from the substrate by HT-1080 fibrosarcoma cells is due to proteolysis. Release and clearing of substratum-attached protein is reduced by approximately 50% by antibodies specific for the protein on the substrate. Tumor cells with low metastatic potential were found to produce higher levels of clearing and release of protein adsorbed to the substrate than tumor cells with high metastatic potential. This was true for variants of the murine K-1735 melanoma and the UV-2237 fibrosarcoma with high and low metastatic capability on all three basement membrane-associated protein substrates. The differences in clearing and release between high and low metastatic cells were not due to differences in initial cell adhesion to the substrates but may be associated with differences in the affinity, type of cell-substrate interactions, proteases, or other variables.

Lens junctional protein: analyzing MP26 with monoclonal antibodies.

Specific antibodies are versatile tools for analyzing cell surface proteins. This study involves the characterization of monoclonal antibodies which are specific for the junctional protein found in the lens fiber cell. This protein can be expected to include regions on the external membrane surface for junction formation, others on the cytoplasmic surface for regulation of junctional properties and, if cell-cell channels are indeed involved, transmembrane domains forming the hydrophilic connection between adjacent cytoplasms. Antibodies to these various regions would provide for an experimental analysis of the junctional protein, e.g., the identification of "active sites" for junction formation. Three monoclonal antibodies specific for the lens junctional protein in the chicken are described here. The first, termed B2, also recognizes the bovine junctional protein, MP26 (5). We have characterized the submolecular specificity of B2 and have found that it binds approximately ten amino acid residues from the C-terminus of MP26. In isolated lens junction preparations, B2 binds to the cytoplasmic surfaces of the lens junctions (both 12 nm and 16 nm thick forms). Thus, we consider MP26 a component of the lens junction. Monoclonal A4, the second antibody considered in detail here, was produced by immunization with lens membranes after treatment with low pH. We have found that lens junctional membranes are separated, or "split," by treatment at pH 2.5-3.0. It appears that A4 binds to the external surface of the junctional membrane; EM studies to confirm this are in progress. In order to map the A4 binding site within the chicken junctional protein and to explore the arrangement of this protein within the membrane, a number of procedures were used to generate fragments of MP26. These included reactions with N-chlorosuccinimide and proteases after acid treatment. Antibody binding to fragments was evaluated with immunotransfer ("Western") procedures. These studies mapped the A4 binding site to the center of the molecule and suggested that MP26 projected externally from the membrane at two different points. These results are consistent with a recent model, based on sequence data (6), for the arrangement of MP26 within the bovine lens membrane.

Junctions between lens fiber cells are labeled with a monoclonal antibody shown to be specific for MP26.

A monoclonal antibody (mcAb) that recognizes an intracellular domain of the major lens membrane protein in both chicken and bovine lenses is described. Mice were immunized with chicken lens fiber cell membranes that had been washed with 7 M urea. Hybridomas were screened by means of enzyme-linked immunosorbent assays and the molecular specificities of the mcAbs were determined using electrophoretic transfer procedures, "Westerns." One of these mcAbs, an IgG designated B2, reacted with a single band of 28,000 Mr from the chicken embryo lens (MP28) and the analogous 26,000 Mr protein in the bovine lens (MP26). Monoclonal B2 was shown to be specific for these proteins, since (a) heating in SDS caused MP26 to aggregate and reduced B2 binding to the protein band at an Mr of 26,000 in Western transfer analysis; (b) apparent dimers were bound by B2 in Western transfers; (c) soluble protein fractions from the lens contained no detectable B2 antigens; and (d) a cyanogen bromide fragment of MP26 was bound by B2. Studies with several proteases indicated that the antigenic site for B2 resides on a 2-kd, protease-sensitive region at the C-terminal end of MP26 and MP28. Evidence for B2 binding on the cytoplasmic side of the membrane comes from labeling studies done at the ultrastructural level. These studies, utilizing indirect methods with peroxidase and colloidal gold markers, clearly demonstrated that B2 labels two types of junctional profiles. In our calf lens membrane preparations after tannic acid staining, the predominant type (80%) measured 16-18 nn thick, with the second type measuring only 12-14 nm. Chick embryo lens cells that had differentiated in vitro and formed groups of lens fiber-like cells (termed lentoids), fluoresced brightly only when they had been permeabilized before labeling with B2 and a fluorochrome-conjugated antibody. This binding was concentrated at the plasma membranes of cells within the lentoids, even outside areas of cell-cell contact. Surrounding epithelioid cells were not stained. Solubilized lens cultures, examined by Westerns, displayed a single immunoreactive band, which co-migrated with MP28.

The role of cell adhesion proteins--laminin and fibronectin--in the movement of malignant and metastatic cells.

Metastasizing tumor cells must traverse diverse extracellular matrices during dissemination. Extracellular matrices consist of two basic types, interstitial stroma and basement membranes. Extracellular matrices are chemically complex structures that interact with cell surfaces by a number of mechanisms. There has been a great deal of effort in recent years to understand the molecular nature of extracellular matrices, especially as it relates to the adhesion of normal and malignant cell types. Adhesive noncollagenous glycoproteins, such as laminin and fibronectin, serve pivotal roles in basement membrane and stromal matrices, respectively. These proteins participate in establishing the architecture of extracellular matrices as well as in attaching to the surface of cells and affecting cellular phenotype. This phenotypic effect ranges from adhesion and motility to growth and differentiation. Changes in adhesive characteristics and motility of cells have long been suspected to play a role in mediating the spread of malignant neoplasms. This article is designed to review extracellular matrix constituents that are currently known that can mediate the adhesion and motility of malignant neoplasms. The adhesion of normal and malignant cells to matrices is a complex process mediated by several distinct mechanisms which are initially manifested by changes in cytoskeletal architecture. The topic of normal and malignant cell adhesion to matrices will also be discussed in this regard, since any explanation of tumor cell migration must account for the complex dynamic interactions of the cell surface with the substratum as well as with the cytoskeleton. Finally, current efforts designed to understand the molecular nature of tumor cell:matrix interactions that contribute to metastatic behavior will also be discussed. The rationale behind these studies is that selective inhibition of specific tumor:extracellular matrix interactions can provide an avenue for therapeutic intervention of metastatic cancer.

Arrangement of MP26 in lens junctional membranes: analysis with proteases and antibodies.

The major membrane protein of the bovine lens fiber cell is a 26-kilodalton (kD) protein (MP26), which appears to be a component of the extensive junctional specializations found in these cells. To examine the arrangement of MP26 within the junctional membranes, various proteases were incubated with fiber cell membranes that had been isolated with or without urea and/or detergents. These membranes were analyzed with electron microscopy and SDS-PAGE to determine whether the junctional specializations or the proteins were altered by proteolysis. Microscopy revealed no obvious structural changes. Electrophoresis showed that chymotrypsin, papain, and trypsin degraded MP26 to 21-22 kD species. A variety of protease treatments, including overnight digestions, failed to generate additional proteolysis. Regions on MP26 which were sensitive to these three proteases overlapped. Smaller peptides were cleaved from MP26 with V8 protease and carboxypeptidases A and B. Protein domains cleaved by these proteases also overlapped with regions sensitive to chymotrypsin, papain, and trypsin. Specific inhibition of the carboxypeptidases suggested that cleavage obtained with these preparations was not likely due to contaminating endoproteases. Since antibodies are not thought to readily penetrate the 2-3 nm extracellular gap in the fiber cell junctions, antibodies to MP26 were used to analyze the location of the protease-sensitive domains. Antisera were applied to control (26 kD) and proteolyzed (22 kD) membranes, with binding being evaluated by means of ELISA reactions on intact membranes. Antibody labeling was also done following SDS-PAGE and transfer to derivatized paper. Both assays showed a significant decrease in binding following proteolysis, with the 22 kD product showing no reaction with the anti-MP26 sera. These investigations suggest that MP26 is arranged with approximately four-fifths of the primary sequence "protected" by the lipid bilayer and the narrow extracellular gap. One-fifth of the molecule, including the C-terminus, appears to be exposed on the cytoplasmic side of the membrane.