Search PubMed⌕ Search

Biomedical subjects

H G Wada

Publications and source records attributed to H G Wada.

32 records · Page 2Linked to original sources
PubMed1989

Detection of cell-affecting agents with a silicon biosensor.

Cellular metabolism is affected by many factors in a cell's environment. Given a sufficiently sensitive method for measuring cellular metabolic rates, it should be possible to detect a wide variety of chemical and physical stimuli. A biosensor has been constructed in which living cells are confined to a flow chamber in which a potentiometric sensor continually measures the rate of production of acidic metabolites. Exploratory studies demonstrate several applications of the device in basic science and technology.

Biosensing Techniques↗
PubMed1989

Detection of Neisseria meningitidis and Yersinia pestis with a novel silicon-based sensor.

A light-addressable potentiometric (silicon) sensor was used in an immunofiltration procedure for the detection of pathogenic bacteria. Yersinia pestis was detected by filtering the cells onto nitrocellulose membranes and then filtering anti-Y. pestis mouse monoclonal antibody and anti-mouse immunoglobulin G-horseradish peroxidase conjugate. For Neisseria meningitidis detection, mouse monoclonal antibody to the major outer membrane protein of this bacterium was coupled directly to horseradish peroxidase. N. meningitidis cell suspensions were filtered onto polycarbonate membranes, and the enzyme conjugate was allowed to react with the filtered bacteria. The presence of both enzyme conjugates was determined potentiometrically with the silicon sensor. The sensitivity of this technique relative to that of an enzyme-linked immunosorbent assay for N. meningitidis was determined. Fewer than 1,000 bacterial cells could be detected with the silicon sensor in a 20-min assay, whereas a 2.5-h enzyme-linked immunosorbent assay with the same antigen and antibody preparations was significantly less sensitive.

Antigens, Bacterial↗
PubMed1982

Enzyme immunoassay of the glycoprotein tropic hormones--choriogonadotropin, lutropin, thyrotropin--with solid-phase monoclonal antibody for the alpha-subunit and enzyme-coupled monoclonal antibody specific for the beta-subunit.

Monoclonal antibody technology has made it possible to produce homogeneous populations of antibodies to discrete determinants on an antigen surface. We have produced monoclonal antibodies to the alpha-subunit and beta-subunits of the glycoprotein hormones choriogonadotropin, thyrotropin, and lutropin, and developed two-site simultaneous enzyme-linked immunospecific assays for these hormones. The anti-alpha-subunit monoclonal antibody was used as the solid-phase (coated tube) capture antibody for all three hormones; the anti-beta-subunit monoclonal antibodies were coupled to horseradish peroxidase (EC 1.11.1.7). Cross reactions between the closely related choriogonadotropin and lutropin were apparently greater in this method than in RIA, with use of the same antibodies. Ka of the antibodies did not appear to be as critical to sensitivity of the sandwich assay as it was for RIA. The lower limit of detection was 0.2 microgram/L after a 2-h incubation with serum sample at room temperature and a 30-min incubation with enzyme substrate at room temperature after washing away excess enzyme conjugate. Within-assay precision (CV) was very good, less than 6%.

Antibodies, Monoclonal↗
PubMed1980

Distribution and characterization of alpha hCG in the serum and tumor cytosol of patients with breast cancer.

Serum and tumor tissue from breast cancer patients were evaluated for the presence of alpha subunit of hCG, by means of the specific radioimmunoassay. Elevated serum levels occurred in 0/50 (0%) of normal female controls, 1/17 (6%) of patients with benign breast disease, 3/53 (6%) of patients with primary breast cancer, and 12/84 (14%) of patients with metastatic breast cancer. Cytosols from 10/56 (18%) primary tumors and 10/37 (28%) metastatic tumors were found to contain the alpha subunit. Comparison of serum and tumor cytosol levels in nine patients with metastatic disease showed a correspondence between elevated cytosol levels and elevated serum levels. No similar correspondence in 25 patients with primary disease was observed. Alpha subunit was isolated from one tumor cytosol and was characterized with respect to its immunochemical cross-reactivity and molecular weight as compared with native alpha subunit from hCG. For both parameters, the native and ectopic alpha subunit were found to be identical.

Breast Neoplasms↗
PubMed1980

Expression of human placental cell surface antigens on peripheral blood lymphocytes and lymphoblastoid cell lines.

The expression of human placental cell surface antigens was examined in cells of lymphoid origin, including peripheral blood lymphocytes and cultured lymphoblastoid cells of bone marrow or thymus derivation. A select group of this defined set of surface antigens was detected on all three cell preparations. The most remarkable observation was the conspicuous absence of three subunits previously demonstrated to be present on all human cell surfaces examined to date. Antiserum directed against several placental components prevents adhesion and spreading of cells which grow attached to surfaces. These results suggest a role for these three glycoproteins in mediating cellular adhesion.

Antigens, Surface↗
PubMed1979

Demonstration of placental alkaline phosphatase in human breast cancer.

The screening of a series of 11 metastatic breast tumors for the presence of the placental isoenzyme of alkaline phosphatase (EC3.1.3.1) by RIA revealed one strong producer. The alkaline phosphatase of this tumor was characterized with respect to its immunochemical cross-reactivity, inhibition by L-phenylalanine and levamisole, subunit molecular weight (Mr) and isoelectric point (pl) in two-dimensional electrophoresis, and one-dimensional peptide map. In all parameters of the characterization, the tumor alkaline phosphatase, except for subunit molecular weight which was slightly lower (60,000 versus 64,000 for the placental isoenzyme). No strong placental alkaline phosphatase producers were found among 16 primary tumors examined by RIA. The screening of patients' sera for the placental alkaline phosphatase using RIA indicated elevated levels over post-menopausal controls in 20% of the metastatic patients. Only 3% of the primary patients had elevated serum levels. These results suggest that the placental isoenzyme of alkaline phosphatase may be a useful tumor marker for recurrent breast cancer.

Alkaline Phosphatase↗
PubMed1979

Characterization of antigenic sialoglycoprotein subunits of the placental brush border membranes: comparison with liver and kidney membrane subunits by two-dimensional electrophoresis.

The sialoglycoprotein subunits of human placental brush border membranes were labeled by sequential treatment with periodate and (3H)-sodium borohydride, which trititates sialic acid, and by lactoperoxidase-catalyzed (125I) iodination of tyrosine residues. The labeled subunits were characterized with respect to their affinity for antisera raised against Triton X-100 extracts of placental brush border membranes. The immunochemically reactive components were analyzed by two-dimensional electrophoresis according to a modification of the O'Farrell technique [20] enabling the assignment of estimated Mr and pI. Of the 33 3H-labeled brush border subunits present in Triton X-100-solubilized membrane preparations, 18 subunits reacted with antiplacental brush border antisera insolubilized on CNBr-activated Sepharose or in immunoprecipitates. Fourteen of these tritiated subunits were also labeled with 125I, confirming that these are glycoproteins. The plasma membranes of normal human liver and microsomes from kidney were examined for the placental brush border glycoprotein subunits by reaction with insolubilized antiplacental brush border antisera and two-dimensional electrophoresis of the reacting tritium-labeled subunits. Comparison of the two-dimensional electrophoretic maps of the immunochemically reacting glycoproteins from liver, kidney, and placenta resulted in the identification of seven placental subunits in common with liver and kidney on the basis of antigenic cross-reactivity, Mr, and pI. Four placental glycoproteins were not found in the other tissues and are potentially specific to the placenta. Three of the placental subunits were only seen in placenta and kidney. Three of the subunits ran at the dye front and could not be assigned molecular weights. One of the subunits was poorly labeled by tritiation of sialic acid and was not considered.

Antigens, Surface↗
PubMed1979

Human placental cell surface antigens:expression by cultured cells of diverse phenotypic origin.

The present work examined the expression of cell surface glycoprotein antigens in cultured human cell lines. The set of glycoproteins studied was defined by their immunoreactivity with antiserum developed to Triton-solubilized extracts of placental brush border membranes. Studies were performed using cell lines of trophoblastic (BeWo, JEG-3) and nontrophoblastic (Chang liver cells) origin, as well as diploid fibroblast cell lines (WI-38, GM-38). Antiplacental brush border antiserum reacts with at least 19 distinct antigens present in placental membrane preparations, each of which can be resolved and identified in two-dimensional electrophoresis. The subunit molecular weight and isoelectric point for all components were defined by their positions in the two-dimensional matrix. Thirteen of these could be detected among the five cell lines examined by lactoperoxidase-catalyzed cell surface iodination. One of these 13 antigens has been identified as the placental isoenzyme of alkaline phosphatase (PAP). The expression of this component is limited to choriocarcinoma cells and Chang liver cells and it is not present in diploid fibroblasts. Under normal circumstances expression of PAP is unique to the differentiated placenta but has been frequently demonstrated in both trophoblastic and nontrophoblastic neoplasms. Two other antigens are variably expressed among the different cell types examined in the present study and their presence or absence was independent of the trophoblastic, epithelial nontrophoblastic, or fibroblastic origin of the cells. Ten surface antigens were expressed in all five cell lines. Six of these had previously been found common to membranes from three adult differentiated tissues, including liver and kidney, as well as placenta (Wada et al, J Supramol Struc 10(3): 287-305, 1979). The presence of this set of antigens in cultured cells as well extends the possibility that these are ubiquitously expressed on human cell surfaces. Two other antigens observed in all cultured cells had been found in both placental and either kidney or liver membranes and may represent common functions shared by many tissues which are also necessary for growth in vitro. The two remaining placental antigens seen in all cultured cells have previously been shown to be absent in adult tissues. Their presence in cultured cells but not in the membranes of resting differentiated tissues may signify the expression of glycoproteins characteristic of trophoblasts in all cells adapted to growth in culture.

Alkaline Phosphatase↗
PubMed1979

Identification of transferrin receptors on the surface of human cultured cells.

We have examined the binding of human transferrin to cultured human choriocarcinoma cell lines and to detergent extracts of such cells. The results indicate the presence of a high-affinity saturable binding site (Ka = 4.25 x 10(8) M-1) that is specific for transferrin. This receptor has also been detected on three other human cell lines of different phenotypic origin, including Wil-2 (splenic lymphocytes of B-cell origin), RPMI-2650 (a quasi-diploid nasopharyngeal carcinoma), and WI-38 (embryonic lung fibroblasts). By using anti-human transferrin antiserum to immunoprecipitate the receptor-transferrin complex from detergent extracts of cells containing saturating levels of transferrin followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, a single polypeptide of 90,000 daltons has been identified as a subunit of the putative transferrin receptor. The protein shows immunochemical identity and coelectrophoreses in sodium dodecyl sulfate gels with a cell surface glycoprotein subunit, previously identified in placental brush border membrane preparations, on all human cultured cell lines examined. These results suggest that the recent demonstration of transferrin dependence of maximal cell growth in culture [e.g., Hutchings, S.E. & Sato, G.H. (1978) Proc. Natl. Acad. Sci. USA 75, 901-904] is mediated through expression of this glycoprotein receptor.

Cell Line↗
PubMed1979

Alkaline phosphatase of mouse teratoma stem cells: immunochemical and structural evidence for its identity as a somatic gene product.

The immunochemical and structural characteristics of the alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] from mouse teratoma stem cells derived from the OTT-6050 teratoma (ascitic and solid tumors and the F9 and PCC4 cell lines) have been compared to those of the alkaline phosphatases expressed in normal mouse placenta and several adult organs. Crossreactivity of the stem cell alkaline phosphatase with antisera reacting with placental, kidney, liver, and brain alkaline phosphatases indicated that the stem cell enzyme had common antigenic determinants. Structural studies utilizing two-dimensional electrophoresis of the (32)P-labeled alkaline phosphatase subunits showed that the stem cell, placental, and kidney alkaline phosphatases differed only in their sialic acid content and comigrated after removal of terminal sialic acid by neuraminidase digestion. Furthermore, one-dimensional peptide mapping of partial proteolysis fragments from (32)P-labeled enzymes demonstrated identical fragmentation patterns for the stem cell and somatic enzymes. These immunochemical and structural data indicate that the stem cell alkaline phosphatase is the same core enzyme as that produced in the mouse placenta and kidney, with different amounts of terminal sialic acid. The one mouse alkaline phosphatase examined that differed from the other enzymes was the intestinal alkaline phosphatase. This isoenzyme was not immunochemically crossreactive with the other alkaline phosphatases, did not comigrate in two-dimensional electrophoresis after neuraminidase digestion, and did not give identical peptide maps after partial proteolysis.

Alkaline Phosphatase↗
PubMed1977

The sialoglycoprotein subunits of human placental brush border membranes characterized by two-two-dimensional electrophoresis.

A brush border membrane enriched fraction was isolated from human full-term placenta. This membrane fraction exhibited large membrane fragments with microvilli projecting from the basal membrane in electron micrographs and was enriched tenfold in alkaline phosphatase, a brush border enzyme marker. The sialoglycoproteins associated with this membrane fraction were tritiated by mild periodate oxidation of sialic acid and reduction with tritiated NaBH4. The membranes were solubilized in 8 M urea, 2 percent Triton X-100, and the tritiated glycoprotein subunits were reduced with beta-mercaptoethanol and characterized by 2-dimensional poly-acrylamide gel electrophoresis using a method similar to that described by O'Farrell and Bhakdi, Knüferman, and Wallach. The tritiated subunits were detected in the gels by autofluorography. The 2-dimensional subunit "maps" resolved at least 17 major sialoglycoprotein subunits whereas only 10 major periodate-Schiff reagent staining components were resolved by 1-dimensional SDS polyacrylamide gel electrophoresis. Placental alkaline phosphatase (PAP) was identified on the subunit maps by inclusion of 32P-labeled PAP in the tritiated membrane sample. The 32P-labeled PAP corresponded to a major tritiated sialoglycoprotein subunit, which was heterogeneous with respect to charge as demonstrated by 3 closely running spots of the same molecular weight.

Cell Fractionation↗
PubMed1976

The plasma membrane of human placenta. Isolation of microvillus membrane and characterization of protein and glycoprotein subunits.

A microvillus plasma membrane-enriched fraction of human placenta was obtained by a combination of differential, isopycnic, and rate-zonal centrifugation techniques. Assays for enzyme markers from mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, and plasma membrane indicated a relative enrichment of plasma membrane between 10- and 20-fold over the most prominent contaminating enzyme markers. Electron microscopy verified the microvillus ultrastructure of the isolated placental membrane and the lack of significant contamination by identifiable organelles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the microvillus membrane fraction revealed a protein and glycoprotein subunit composition. There were 16 major protein subunits and 10 major glycoprotein subunits, and apparent molecular weights are assigned to these subunits. 32P-labeling of the microvillus membrane-associated alkaline phosphatase indicated that this enzyme is one of the major glycoproteins of the human placental microvillus membrane.

Cell Fractionation↗
PubMed1976

Characterization of two different alkaline phosphatases in mouse teratoma: partial purification, electrophoretic, and histochemical studies.

Alkaline phosphatase (E.C.3.1.3.1.) has been used as a marker for embryonal carcinoma cells which constitute the multipotential stem cells of the mouse teratoma. Studies by other investigators based on kinetics of thermal inactivation and L-phenylalanine inhibition have shown that the alkaline phosphatase of the teratoma differs from the mouse intestinal and liver isozymes, but resembles the isozymes of kidney and placenta. Since functional characterization of nonpurified enzymes is not the most accurate means for distinguishing different molecular forms of an enzyme, we have partially purified the enzymes from the ascitic (embryoid body) and solid tumor forms of the OTT-6050 teratoma line, and utilized the technique of electrophoresis in polyacrylamide gels to compare the teratoma enzyme with isozymes from kidney and placenta. Covalent 32PO4-labeling of the alkaline phosphatases and polyacrylamide gel electrophoresis in sodium dodecylsulfate was also used to compare the subunit molecular weights of the enzymes. The results indicate that the mouse teratoma enzyme is distinct from the kidney and placental isozymes. Since histochemical studies have localized the enzyme to the stem cell population of the teratoma, the results imply that stem cell alkaline phosphatase is a distinct isozyme. The embryoid bodies contain a second alkaline phosphatase which may correspond to the placental isozyme. This enzyme may be attributed to the outer cell layer of embryoid bodies of the ascitic tumor, since this cell type histochemically demonstrates alkaline phosphatase activity.

Alkaline Phosphatase↗