Endothelial cells use alpha 2 beta 1 integrin as a laminin receptor

Human umbilical vein endothelial cells attach and spread on laminin-coated substrates. Affinity chromatography was used to identify the attachment receptor. Fractionation of extracts from surface-iodinated endothelial cells on human laminin-Sepharose yielded a heterodimeric complex, the subunits of which migrated with molecular sizes corresponding to 160/120 kD and 160/140 kD under nonreducing and reducing conditions, respectively. The purified receptor bound to laminin and slightly less to fibronectin and type IV collagen in a radioreceptor assay. This endothelial cell laminin receptor was classified as an alpha 2 beta 1 integrin because monoclonal and polyclonal antibodies directed against the alpha 2 and bet 1 subunits immunoprecipitated the receptor. Cytofluorometric analysis and immunoprecipitation showed that the alpha 2 subunit is an abundant integrin alpha subunit in the endothelial cells and that the alpha subunits associated with laminin binding in other types of cells are expressed in these cells only at low levels. The alpha 2 beta 1 integrin appears to be a major receptor for laminin in the endothelial cells, because an anti-alpha 2 monoclonal antibody inhibited the attachment of the endothelial cells to human laminin. These results define a new role for the alpha 2 subunit in laminin binding and suggest that the ligand specificity of the alpha 2 beta 1 integrin, which is known as a collagen receptor in other types of cells, can be modulated by cell type-specific factors to include laminin binding.     

Characterization of the class III collagen receptor, a phosphorylated, transmembrane glycoprotein expressed in nucleated human cells

We previously identified a 90-kDa cell surface glycoprotein, termed the class III collagen receptor (CRIII), that bound to collagen in affinity chromatography experiments (Wayner, E. A., and Carter, W. G. (1987) J. Cell Biol. 105, 1873-1884). Here, we utilize monoclonal antibodies to define three domains of the CRIII, hydrophobic transmembrane, phosphorylated cytoplasmic, and glycosylated extracellular. The domain designations are based on the following characteristics. (i) Differential extraction, phase partitioning with Triton X-114, and incorporation into liposomes all indicate that the CRIII is an intrinsic membrane receptor with a hydrophobic domain. After incorporation into liposomes the CRIII binds collagen. (ii) Immunofluorescence microscopy reveals that most nucleated cells express the CRIII and that after extraction with Triton X-100, the Triton-insoluble CRIII distributes in a fibrillar pattern at the cell periphery and in closed loops that partially co-distributed with vimentin. The CRIII contains phosphoserine residues which are located on a cytoplasmic domain that may interact with the cytoskeleton. (iii) The CRIII contains 25% carbohydrate in 8-10 asparagine-linked carbohydrate chains of 2800 daltons each bound to a 65-kDa core peptide in the extracellular domain. Peptide mapping with trypsin defined a glycosylated 27-kDa extracellular fragment and a phosphorylated and glycosylated 35-kDa transmembrane fragment. These data suggest a model for the CRIII that links the cytoskeleton with the extracellular matrix.     

Affinity chromatographic isolation of the melanoma adhesion receptor for the IIICS region of fibronectin and its identification as the integrin alpha 4 beta 1

Eukaryotic cells adhere to at least two different regions of the fibronectin molecule: a central domain present in all fibronectin isoforms, and the type III connecting segment domain (IIICS), the expression of which is controlled by complex alternative splicing of precursor mRNA. Using affinity chromatography on a matrix containing a synthetic peptide ligand (CS1) representing the strongest active site within the IIICS, we have isolated the human melanoma cell receptor recognizing this region of fibronectin. The receptor is a complex of two polypeptides with subunit molecular masses of 145 and 125 kDa. This heterodimeric structure resembles that of receptors for other extracellular matrix proteins. Immunological analysis with specific antibodies identified these polypeptides as the integrin subunits alpha 4 and beta 1. In addition, antifunctional monoclonal antibodies directed against either alpha 4 or beta 1, but not against other integrin subunits, were potent inhibitors of CS1-mediated melanoma cell spreading. Furthermore, when the function of the central cell-binding domain was blocked, anti-alpha 4 and anti-beta 1 antibodies abolished spreading of A375-M cells on fibronectin, indicating that alpha 4 beta 1 is an authentic fibronectin receptor. Taken together, these results identify the human fibronectin IIICS receptor as the integrin heterodimer alpha 4 beta 1.     

Andrenocorticotropin and β-lipotropin in the hypothalamus

Adrenocorticotropin and β-lipotropin (β-LPH) have been localized by immunoperoxidase methods in nerve cells and fibers of the hypothalamus and brain stem of the ewe. 6-μm sections were immunostained first for either ACTH or β-LPH. The reaction products and the antibody complexes were then eluted completely from the tissue, and the same section was immunostained for the second peptide. Absorption of the primary antisera with a variety of peptide fragments of ACTH and β-LPH demonstrated, immunocytochemically as well as by radioimmunoassay, that the ACTH and β-LPH antisera were directed to the COOH- and NH(2)-termini of the peptides, respectively. Neither antiserum recognized any portion of the heterologous peptide. In the sequential staining procedure on the same tissue section, preincubation of the antisera with the homologous peptide abolished the staining, whereas preincubation with the heterologous peptide did not affect it, regardless of the order followed. Every nerve cell in the arcuate nucleus that contained ACTH also contained β-LPH, but β-LPH cells appeared, probably falsely, to be twice as numerous as ACTH cells. β-LPH-positive fibers in and beyond the hypothalamus were also more numerous and stained more intensively than ACTH fibers. The salient exception was fibers in the infundibular zona externa, where the opposite was true. Our observations establish that ACTH and β-LPH are contained in the same nerve cells They stongly favor biosynthesis in brain, probably from a common precursor molecule, as has been demonstrated in the pituitary gland. The complexity of the cytologic distribution pattern described suggests that the two peptides are not processed in the same manner by the nerve cell.     

Structural origins of fibrin clot rheology

The origins of clot rheological behavior associated with network morphology and factor XIIIa-induced cross-linking were studied in fibrin clots. Network morphology was manipulated by varying the concentrations of fibrinogen, thrombin, and calcium ion, and cross-linking was controlled by a synthetic, active-center inhibitor of FXIIIa. Quantitative measurements of network features (fiber lengths, fiber diameters, and fiber and branching densities) were made by analyzing computerized three-dimensional models constructed from stereo pairs of scanning electron micrographs. Large fiber diameters and lengths were established only when branching was minimal, and increases in fiber length were generally associated with increases in fiber diameter. Junctions at which three fibers joined were the dominant branchpoint type. Viscoelastic properties of the clots were measured with a rheometer and were correlated with structural features of the networks. At constant fibrinogen but varying thrombin and calcium concentrations, maximal rigidities were established in samples (both cross-linked and noncross-linked) which displayed a balance between large fiber sizes and great branching. Clot rigidity was also enhanced by increasing fiber and branchpoint densities at greater fibrinogen concentrations. Network morphology is only minimally altered by the FXIIIa-catalyzed cross-linking reaction, which seems to augment clot rigidity most likely by the stiffening of existing fibers.     

Identification and characterization of a prevacuolar compartment in stigmas of nicotiana alata

The stigmas of the ornamental tobacco plant Nicotiana alata accumulate large quantities of a series of 6-kD proteinase inhibitors (PIs) in the central vacuole that are derived from a 40-kD precursor protein, Na-PI. The sorting information that directs Na-PI to the vacuole is likely to reside in a C-terminal propeptide domain of 25 amino acids that forms an amphipathic alpha helix. Using cell fractionation techniques, we have examined transit of Na-PI through the endomembrane system and have identified a prevacuolar compartment that contains Na-PI with an intact targeting signal. In contrast, the targeting signal is not present on the predominant form of Na-PI in the vacuole. The prevacuolar compartment is marked by the presence of homologs of both the t-SNARE, PEP12p, and the putative vacuolar sorting receptor BP-80. Cross-linking and affinity precipitation studies revealed that Na-PI associates with BP-80 within this compartment, providing in vivo evidence for the function of BP-80 as a sorting receptor for a protein with a C-terminal vacuolar targeting signal.     

Use of a Single-Chain Antibody Library for Ovarian Cancer Biomarker Discovery

The discovery of novel early detection biomarkers of disease could offer one of the best approaches to decrease the morbidity and mortality of ovarian and other cancers. We report on the use of a single-chain variable fragment antibody library for screening ovarian serum to find novel biomarkers for the detection of cancer. We alternately panned the library with ovarian cancer and disease-free control sera to make a sublibrary of antibodies that bind proteins differentially expressed in cancer. This sublibrary was printed on antibody microarrays that were incubated with labeled serum from multiple sets of cancer patients and controls. The antibodies that performed best at discriminating disease status were selected, and their cognate antigens were identified using a functional protein microarray. Overexpression of some of these antigens was observed in cancer serum, tumor proximal fluid, and cancer tissue via dot blot and immunohistochemical staining. Thus, our use of recombinant antibody microarrays for unbiased discovery found targets for ovarian cancer detection in multiple sample sets, supporting their further study for disease diagnosis.Despite many advances in the treatment of cancer, early detection and tumor removal remains the best prospect for overcoming disease. Ovarian cancer is an excellent example of the potential prognostic value of early detection because diagnosis at a localized stage has a 5-year survival rate of 93%. However, only 19% of cases are diagnosed at this stage, and by the time the disease has evolved to an advanced stage, the 5-year survival rate drops to 31% (1).Much effort has been expended to find early detection markers of ovarian cancer, and some success has been achieved. Most notable is CA125, the only approved marker for the detection of recurrence of ovarian cancer (2). Other leading targets are mesothelin and HE4, which have been examined by several groups for their efficacy as early detection markers (3–8). Nevertheless, several conditions necessitate the discovery of more specific and sensitive ovarian cancer markers: the heterogeneity of this disease, the ambiguity of its symptoms, its low incidence in the general population, and the low sensitivity and specificity of currently available markers.One of the difficulties in finding markers in blood is the complexity of the plasma/serum proteome, estimated in the tens to hundreds of thousands of proteins, as well as its large range in constituent protein concentrations, which can span 12 orders of magnitude (9). However, along with its easy accessibility, the fact that blood is in contact with virtually every tissue and contains tissue- and tumor-derived proteins makes it a preferred source for disease biomarker discovery.Our previous results (10, 11) and those of others (12–14) using high density, full-length IgG antibody microarrays to validate and discover cancer serum biomarkers demonstrated that this platform is valuable for simultaneously comparing the levels of hundreds of proteins on dozens of serum samples from cancer patients and healthy controls. We confirmed overexpression of CA125, mesothelin, and HE4 in ovarian cancer samples using this high density microarray platform, validating our array methodology for measurement of cancer serum biomarkers and yielding new putative biomarkers for this disease (10, 11).Previously reported approaches are typically limited to a few hundred antibodies. The methodology reported here allows us to exploit the specific advantages of antibodies as high affinity capture reagents to detect differential expression of thousands of tumor biomarkers using a diverse (2 × 10⁸ binding agents) single-chain variable fragment antibody (scFv)¹ library for detection of ovarian cancer markers in serum, tumor cyst fluid, and ascites fluid. Our results build on previous reports of phage display library microarrays to discover autoantibody (15–18) and other protein (12, 19, 20) cancer biomarkers. Our scFv are high affinity capture reagents consisting of the variable regions of human antibody heavy and light chains joined by a flexible linker peptide. These recombinant antibodies are able to recognize a wide variety of antigens, including many previously thought difficult, such as self-antigens and proteins that are not normally immunogenic in animals (21–24). Using a highly diverse recombinant antibody library, one has the ability to overcome the complexity of the serum proteome. It has been calculated that for an immune repertoire to be complete (at least one antibody in the repertoire has reasonable affinity for every epitope possible in nature) it requires a diversity of at least 10⁶ antibodies (25). The reported diversity of our scFv library exceeds this value by 100-fold (21).To enrich for antibodies that differentiate disease status, we performed a selection or panning of the naïve library for proteins that are differentially expressed in cyst fluid, ascites fluid, or serum of cancer patients with respect to healthy serum. We printed this sublibrary on activated hydrogel slides that were queried with three different sets of labeled case and control sera to further select those that discriminate cancer status in a statistically significant manner. Next, we identified some of the targets that bind to the individual scFv using high density nucleic acid programmable protein arrays (NAPPAs) expressing a total of over 7000 proteins. Finally, we validated the effectiveness of the selection process by confirming overexpression of these targets in cancer serum, cyst fluid, and ascites fluid as well as in tumor sections.     

Requirement of the integrin beta 3 subunit for carcinoma cell spreading or migration on vitronectin and fibrinogen

FG human pancreatic carcinoma cells use integrin alpha v beta 5 as their primary vitronectin receptor since they fail to express integrin alpha v beta 3. These cells are unable to form focal contacts, spread, or migrate on vitronectin but readily do so on collagen in a beta 1 integrin-dependent manner. Transfection of FG cells with a cDNA encoding the integrin beta 3 subunit results in the surface expression of a functional integrin alpha v beta 3 heterodimer providing these cells with novel adhesive and biological properties. Specifically, FG cells expressing beta 3 acquire the capacity to attach and spread on vitronectin as well as fibrinogen with beta 3 localization to focal contacts. Moreover, these cells gain the capacity to migrate through a porous membrane in response to either vitronectin or fibrinogen. These results demonstrate that the beta 3 and beta 5 integrin subunits when associated with alpha v, promote distinct cellular responses to a vitronectin extracellular environment.     

Quantitative measurement of the total, peroxyl radical-trapping antioxidant capability of human blood plasma by controlled peroxidation. The important contribution made by plasma proteins

Plasma obtained from fasted humans has been analysed for total radical-trapping antioxidant content by subjecting it to controlled peroxidation using the thermal decomposition of water-soluble azobis (2-amidinopropane hydrochloride) at 37 degrees C to produce peroxyl radicals at a known, steady rate. It is found that the total radical-trapping antioxidant content is rather similar for the 7 subjects that have been tested and, furthermore, it is 10-20-times larger than the effect attributable to vitamin E alone. Although it is shown that urate and ascorbate augment the contribution from vitamin E, their contributions (21-34 and 0-2%, respectively) still leave 57-73% of the total antioxidant content unaccounted for. Evidence is presented to show that this previously unrecognized large reserve of antioxidant capacity is attributable to the plasma proteins.