Complete amino acid sequence of a novel integrin beta subunit (beta 6) identified in epithelial cells using the polymerase chain reaction

The integrin family of adhesion receptors consists of several heterodimeric glycoproteins, each composed of one alpha and one beta subunit. Three different mammalian beta subunits, beta 1, beta 2, and beta 3, have been sequenced, but recent evidence suggests the existence of several others. Amplification of guinea pig airway epithelial cell cDNA with oligonucleotide primers designed to recognize consensus integrin beta subunit sequences led to the identification of a novel partial cDNA sequence. Clones containing portions of this sequence were used to screen cDNA libraries constructed from the human pancreatic carcinoma cell line FG-2 and identified a series of overlapping clones encoding the full-length sequence of the human homologue of this protein. This sequence of 788 amino acids is 43, 38, and 47% identical to the sequences of beta 1, beta 2, and beta 3, respectively. Features shared between this novel protein and the previously sequenced beta subunits include the positions of all 56 cysteine residues in the extracellular domain, the single putative transmembrane domain, and the short putative cytoplasmic domain. However, a unique 11-amino acid extension at the carboxyl terminus, not present in any of the other beta subunits, is suggestive of distinctive interactions with cytoplasmic components. Comparison of the human and guinea pig sequences reveals a high degree (94%) of cross-species conservation. Because this protein is clearly distinct from the two other recently described integrins beta 4 and beta 5, we propose to designate it beta 6.     

A novel vitronectin receptor integrin (alpha v beta x) is responsible for distinct adhesive properties of carcinoma cells

Carcinoma cells express a novel integrin involved in cell adhesion to vitronectin, but not to fibrinogen or von Willebrand factor, whereas melanoma and endothelial cells express a vitronectin receptor (alpha v beta 3) that promotes cell attachment to all of these matrix components. The integrin responsible for this adhesive phenotype of carcinoma cells is composed of an alpha subunit that is indistinguishable from the alpha v of the vitronectin receptor and a beta subunit (beta x) that is distinct from any known integrin beta subunit. Accordingly, Northern blot analysis identifies an mRNA for alpha v, but not for beta 3 in carcinoma cells. This receptor appears to mediate cell adhesion to vitronectin as well as fibronectin since an antibody directed to its alpha subunit blocked carcinoma cell adhesion to both of these matrix proteins. These results suggest that homologous integrins with identical alpha subunits and structurally distinct beta subunits can account for the functional recognition of different matrixes by two cell types.     

Transgenic HLA-DR alpha faithfully reconstitutes IE-controlled immune functions and induces cross-tolerance to E alpha in E alpha 0 mutant mice

We have constructed transgenic mice that express the human class II MHC molecule HLA-DR alpha on a genetic background in which the equivalent endogenous gene, H-2 IE alpha, is not expressed. In these mice, DR alpha complemented the E beta chain such that tissue-specific expression of an interspecies hybrid DR alpha-E beta heterodimer was obtained. Despite 25% amino acid differences between DR alpha and E alpha, immune responsiveness to IE-controlled antigens, clonal deletion of IE-reactive T cells, and alloantigenicity were quantitatively and qualitatively indistinguishable in IE-positive mice and in mice that had integrated at least four copies of the transgene. These results demonstrate a remarkable degree of structural, regulatory, and functional conservation. They also suggest that tolerance induction involves only discrete portions of MHC molecules.     

Chromosomal damage induced by maleic hydrazide in mammalian cells in vitro and in vivo

The induction of sister-chromatid exchanges (SCE) and chromosomal aberrations (Ch.Ab.) by the herbicide maleic hydrazide (MH) has been investigated in Chinese hamster ovary (CHO) cells grown in vitro and in bone marrow cells of mice treated in vivo. MH induces SCE and Ch.Ab. in CHO cells without metabolic activation; however, no induction of SCE was found in the in vivo experiments.     

Sulfated N-linked oligosaccharides in mammalian cells. III. Characterization of a pancreatic carcinoma cell surface glycoprotein with N- and O-sulfate esters on asparagine-linked glycans

In the preceding two papers, we described two new classes of sulfated N-linked oligosaccharides isolated from total cellular 35SO4-labeled macromolecules of different mammalian cell lines. The first class carries various combinations of sialic acids and 6-O-sulfate esters on typical complex-type chains, while the second carries heparin and heparan-like sequences. In this study, we have characterized a sulfophosphoglycoprotein of 140 kDa from FG-Met-2 pancreatic cancer cells whose oligosaccharides share some properties of both these classes. The molecule was localized to the cell surface by electron microscopy using a monoclonal antibody (S3-53) and by cell surface 125I-labeling. Metabolic labeling of the cells with radioactive glucosamine, methionine, inorganic sulfate, or phosphate all demonstrated a single 140-kDa molecule. Pulse-chase analysis and tunicamycin treatment indicated the glycosylation of a putative primary translation product of 110 kDa via an intermediate (120 kDa) to the mature form (140 kDa). Digestion with peptide:N-glycosidase F (PNGaseF) indicated a minimum of four N-linked glycosylation sites. PNGaseF released more than 90% of the [6-3H]GlcNH2 label and 40-70% of 35SO4 label from the immunoprecipitated 140-kDa molecule. The isolated oligosaccharides were characterized as described in the preceding two papers. The majority of [6-3H]GlcNH2-labeled molecules were susceptible to neuraminidase. More than 50% of the 35SO4 label was associated with only 5-10% of the 3H-labeled chains. Some of the sulfated chains were partly sialylated molecules with four to five negative charges. Treatment with nitrous acid released about 25% of the 35SO4 label as free sulfate, together with 6% of the [6-3H]GlcNH2 label, indicating the presence of N-sulfated glucosamine residues. Some of these oligosaccharides were degraded by heparinase and heparitinase. Therefore, while they are not as highly charged as typical heparin or heparan chains, they must share structural features that permit recognition by the enzymes. Thus, this 140-kDa glycoprotein contains at least four asparagine-linked chains substituted with a heterogeneous mixture of sulfated sequences. The heterogeneity of these molecules is as extensive as that described for whole-cell sulfated N-linked oligosaccharides in the preceding two papers.     

The monoclonal xenoantibody Q6/64 recognizes a determinant expressed by certain gene products of the A and B loci of the HLA region

A combination of serological (cytotoxicity, binding assay, lysostrip) and immunochemical (indirect immunoprecipitation, sequential immunoprecipitation, two-dimensional gel electrophoresis) assays have shown that the monoclonal antibody Q6/64 recognizes an antigenic determinant which is expressed by certain gene products of the A and B loci of the HLA region. The determinant identified by Q6/64 is spatially close to those which define the serological polymorphism of the HLA-A, B, C antigenic system. The results presented in this study in conjunction which those recently published by other investigators indicate that sharing of determinants among HLA-A and -B allospecificities is more frequent than originally assumed on the basis of the cross-reactivity pattern obtained with alloantisera. This conclusion is in agreement with the high degree of homology in the primary amino-acid sequence of A and B allospecificities.     

Cross-reactivity between human and murine lymphocyte antigens

The anti-H-2 alloantiserum D-32 [(BlO.A(2R) × C3H.SW) anti-C3H] is cytolytic to human lymphocytes. Fab₂ blocking assays, indirect immunoprecipitation and sequential immunoprecipitation experiments showed that the anti-H-2 alloantiserum D-32 recognizes antigenic determinants which are expressed on the heavy chain of subpopulations of HLA-A, B antigens. These determinants are different from those defining the serological polymorphism of the HLA-A, B, C system, are the same as or spatially close to those recognized by the anti-HLA-A, B monoclonal antibody Q6/64 and are expressed on rabbit, rat or guinea pig lymphocytes.     

The p53 tumor suppressor gene

p53 was originally considered to be a nuclear oncogene, but several convergent lines of research have indicated that the wild-type gene functions as a tumor suppressor gene negatively regulating the cell cycle. Mutations in the p53 gene have been detected in many tumor types and seem to be the most common genetic alterations in human cancer. In this preliminary study, sera of 92 patients (pts) with breast disease were analyzed for the presence of the mutant p53 protein (mp53) with a selective immunoenzyme assay employing a monoclonal antibody (PAb 240) specific for the majority of mammalian m p53 but not for the wild-type protein. Of the 10 patients with benign breast disease, only two (20%) showed detectable m p53 levels in the serum. In the breast cancer group, sera from 7 of the 30 pts (23%) without lymph node involvement were positive for m p53, as were 7 out of the 45 pts (15%) with metastatic lymph nodes and 1 out of the 7 pts (14%) with disseminated disease. The specifity of m p53 assay evaluated in 20 healthy controls was 100%. These preliminary results showed that serum positivity for m p53 is not related to breast disease extension. Further studies to assess the utility of m p53 as a possible prognosis factor in breast cancer are currently in progress.     

Monoclonal antibodies to HLA-A,B, and Ia-like antigens inhibit colony formation by human myeloid progenitor cells

Hybridomas derived from the fusion of murine myeloma cells with splenocytes from mice immunized with human cultured lymphoid cells secreted monoclonal antibodies to human cell surface antigens. Serologic and immunochemical assays showed that 4 monoclonal antibodies (Ab Q2/47, Q2/61, Q2/70, Q2/80) recognize framework determinants of Ia-like antigens and 1 monoclonal antibody (Ab Q1/28) reacts with determinants expressed on the heavy chain of HLA-A,B antigens. Both anti-HLA-A,B and anti-Ia-like antigen monoclonal antibodies caused complement-dependent inhibition of granulocyte-macrophage colony formation by human bone marrow grown in soft agar. Mixing experiments excluded the possibility of an indirect effect on progenitor cells by lysis of auxiliary cells. These results indicate that human myeloid progenitor cells express HLA-A,B and Ia-like antigens.