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W L Bigbee

Publications and source records attributed to W L Bigbee.

54 records · Page 3Linked to original sources
PubMed1989

Evidence for an elevated frequency of in vivo somatic cell mutations in ataxia telangiectasia.

Somatic cell mutation frequency in vivo was measured in individuals with high cancer risk who were from ataxia telangiectasia (A-T) families. The assay for somatic mutation measures the frequency of variant erythrocytes which are progeny of erythroid precursor cells with mutations that result in a loss of gene expression at the polymorphic glycophorin A (GPA) locus. Samples from 14 of 15 A-T homozygotes showed high frequencies of GPA gene expression-loss variant cells with normal expression of only one of the two alleles at the GPA locus (i.e., GPA hemizygous variant cells). The mean elevation of the frequency of hemizygous variant cells over those in normal controls and unaffected family members was 7-14-fold. A-T homozygotes also showed an increase in the frequency of cells in which one allele at the GPA locus had lost expression and in which the remaining allele was expressed at a homozygous level (i.e., GPA homozygous variant cells). Family members who are obligate A-T heterozygotes did not appear to have a significantly elevated frequency of GPA hemizygous or homozygous variant cells. These indications of elevated in vivo frequencies of variant erythrocytes in A-T homozygotes support a causal link between susceptibility to somatic mutation and susceptibility to cancer.

Adult↗
PubMed1988

A family study of multiple mutations of alpha and delta glycophorins (glycophorins A and B).

Glycophorins alpha and delta are the carriers of the antigens of the MNSs blood system; this report documents the presence of three glycophorin mutations in two individuals of a 16 member family. Erythrocytes were examined by serology, sodium dodecyl sulfate electrophoresis, and immunoblotting. The inheritance pattern and immunoblot profile revealed: (1) A variant Dantu glycophorin showed properties consistent with a delta-alpha glycophorin hybrid structure, previously noted in other individuals. The gene responsible for the Dantu glycophorin in this family is linked to a gene coding for an M-specific alpha glycophorin. (2) Another variant glycophorin, Mi-III glycophorin, was first revealed by immunoblotting and subsequently confirmed by erythrocyte antigen typing. This autosomal dominant trait is associated with N blood group activity and the inheritance pattern indicates that it could be a variant of delta glycophorin. (3) In the individuals with both Dantu and Mi-III glycophorins a delta glycophorin deficiency was observed suggesting that a deletion or alteration of delta gene may exist cis to the Dantu gene. Our findings that document clustering of multiple mutations in MNSs gene loci in the propositus family are very unusual as such variants are relatively rare.

Female↗
PubMed1987

Evidence for increased somatic cell mutations at the glycophorin A locus in atomic bomb survivors.

A recently developed assay for somatic cell mutations was used to study survivors of the atomic bomb at Hiroshima. This assay measures the frequency of variant erythrocytes produced by erythroid precursor cells with mutations that result in a loss of gene expression at the polymorphic glycophorin A (GPA) locus. Significant linear relations between variant frequency (VF) and radiation exposure were observed for three different variant cell phenotypes. The spontaneous and induced VFs agree with previous measurements of radiation-induced mutagenesis in other systems; this evidence supports a mutational origin for variant cells characterized by a loss of GPA expression and suggests that the GPA assay system may provide a cumulative dosimeter of past radiation exposures. VFs for some survivors differ dramatically from the calculated dose response, and these deviations appear to result primarily from statistical fluctuations in the number of mutations in the stem-cell pool. These fluctuations allow one to estimate the number of long-lived hemopoietic stem cells in humans.

Antibodies, Monoclonal↗
PubMed1987

Coordinate glycosylation and cell surface expression of glycophorin A during normal human erythropoiesis.

The expression of two epitopes on glycophorin A (GPA) during erythroid development was examined on normal human bone marrow using quantitative flow cytometry. The highly correlated binding of two monoclonal antibodies, one sensitive and the other insensitive to glycosylation, indicated that the two epitopes were coordinately expressed during erythroid development. Both antigens reached a maximum expression during the early normoblast stage and were maintained at a constant amount per cell throughout further maturation to erythrocytes. These data suggest that glycosylation of GPA, as detected by antibodies recognizing blood group (M) and (N) antigens, does not increase during erythroid maturation.

Antibodies, Monoclonal↗
PubMed1987

Structural polymorphism of glycophorins demonstrated by immunoblotting techniques.

We have explored the polymorphism of the glycophorin system in the human erythrocyte membrane using the immunoblotting techniques and examining 52 individuals selected without prior bias as to their serologic state and ten documented serologic variants of M, N, S, s blood group system. Polyclonal antisera to alpha glycophorin and to alpha glycophorin CNBr carboxyl terminal fragment C (residues 82-131) and M and N specific monoclonal antibodies (MoAbs) were used. The first two reagents detect specific regions of the alpha glycophorin molecule and all electrophoretically resolved species of glycophorins immunologically related to alpha and delta glycophorins (delta glycophorin, [alpha-delta] hybrids and other glycophorins with an alteration in the carboxyl terminal segment); the M and N MoAbs identified the glycophorin species containing or lacking the M or N determinant in the amino terminal octapeptide structures. We find that immunoblotting confirmed in all cases the serologically determined phenotype; we also find that polymorphic forms of the glycophorin system are relatively infrequent; immunoblotting, independent from serologic testing, was capable of detecting five mutants, two most likely S-s-U-phenotypes; a new glycophorin species was detected in normal red cells with both antiglycophorin and antipeptide C sera, which is not evident with MoAbs; immunoblots of known glycophorin variants (En(a-), U-, Mg, Mi I, II, III, V, and Sta) confirmed but also extended our knowledge of the abnormal glycophorins involved; and the He+ and Wrb(-) cells showed normal patterns.

Glycophorins↗
PubMed1986

Measurements of the frequency of human erythrocytes with gene expression loss phenotypes at the glycophorin A locus.

An assay method is described for determining the frequency of human erythrocytes having a gene expression loss phenotype at the glycophorin A locus presumably due to in vivo somatic mutational events in erythroid precursor cells. Monoclonal antibodies specific for the M and N glycophorin A alleles are used to identify variant cells that lack the expression of one allele in blood samples from MN heterozygotes. Flow cytometry and sorting are used to enumerate and purify variant cells. Using three different antibody combinations which are sensitive to the loss of either the M or the N allele, we find that variant cells occur at a frequency of 1 X 10(-5) in normal donors. We also detect variant cells with an apparent homozygous phenotype suggesting that events leading to homozygosity may occur at similar frequencies to gene loss events. Significant increases in variant cell frequency are observed in cancer patients after exposure to mutagenic chemotherapy drugs.

Adult↗
PubMed1985

Monoclonal antibodies specific for sickle cell hemoglobin.

Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility.

Amino Acid Sequence↗
PubMed1985

Flow cytometric characterization of normal and variant cells with monoclonal antibodies specific for glycophorin A.

Quantitative immunofluorescence measurements were performed on erythrocytes labeled with monoclonal antibodies to glycophorin A (GPA) to assess the level of binding of these antibodies to normal and variant cell types. The seven antibodies used in this study include two that bind preferentially to the M form of GPA, three that bind preferentially to the N form, and two that bind equally well to both. Flow cytometric analysis of mixtures of cells differing in M,N type showed binding specificities of greater than 100-fold for most of the antibodies, and showed that three antibodies bind cell-bound GPA with an affinity of approximately 10(9) M(-1). These data also showed that the level of expression of GPA varies by less than 10% from cell to cell and from individual to individual. Flow measurements were also done on human erythrocytes with the following variant forms of glycophorin: Mc, Mg, Mk, En(F), En(UK), Mi-I, Mi-II, Mi-III, S-s-U-, Tn+, and St(a+). Other cell types analyzed included erythrocytes from chimpanzee, rhesus, African green, and capuchin monkeys, and cells from the human erythroleukemia cell line, K562. Flow analysis with our seven antibodies showed these cell types have distinctive labeling patterns consistent with the known or inferred altered glycophorins presented on these cells. In most cases, variant alleles were expressed at normal levels. Our results support other observations that the variants En(UK) and St(a+) contain hybrid GPA-GPB proteins, and suggest that their level of expression is largely determined by the 3' end of the hybrid genes.

Animals↗
PubMed1984

Binding specificities of eight monoclonal antibodies to human glycophorin A--studies with McM, and MkEn(UK) variant human erythrocytes and M- and MNV-type chimpanzee erythrocytes.

Four newly derived mouse monoclonal antibodies to human glycophorin A are described. Three of these antibodies bind preferentially to the N form of glycophorin A; the fourth recognizes a shared determinant of the M and N forms. All four antibodies are directed toward the 39 amino acid, amino-terminal portion of the protein, and the N-specific antibodies require for binding the presence of N-acetyl-neuraminic acid on the glycosidically linked oligosaccharides. Cross-reaction of the N-specific antibodies to homozygous MM erythrocytes appears to result from binding to glycophorin B. In addition, these antibodies together with four previously reported glycophorin monoclonal antibodies, including two that specifically recognize the M form of glycophorin A, were tested for binding to McM and MkEn(UK) variant human erythrocytes and M- and MNV-type chimpanzee erythrocytes. Results obtained for five of the six M- or N-specific monoclonal antibodies point to the general immunodominance of the amino-terminal serine-leucine polymorphism and the requirement for sialic acid. One of the two M-specific monoclonal antibodies, 9A3, discriminates between the M, N, and Mc forms of glycophorin A solely on the basis of the amino-terminal serine-leucine polymorphism. The other M-specific antibody, 6A7, requires a more complex determinant involving the glycine-glutamic acid polymorphism at the fifth position in the sequence as well. The epitopes for all three N-specific monoclonal antibodies include the amino terminal leucine that occurs in the N form of glycophorin A and may also include the glutamic acid that occurs at position five. Our studies support the proposed Lepore-type glycophorin A-B hybrid gene rearrangement for the En(UK) allele found in the English En(a-) family. The data also confirm the expression of the M-like glycoprotein on chimpanzee erythrocytes and the presence of a human glycophorin B-like antigen on the MNV-type cells.

Amino Acid Sequence↗
PubMed1983

Monoclonal antibodies specific for the M- and N-forms of human glycophorin A.

Four mouse monoclonal antibodies directed against the red cell membrane protein glycophorin A have been isolated and characterized. They are produced by hybridomas derived from SP2/0 myeloma cells and spleen cells from Biozzi mice immunized with a mixture of human erythrocytes from homozygous blood group M and N individuals. These antibodies recognize and bind to purified glycophorin A and to glycophorin on the red cell surface. All are of the IgGl, kappa light chain subclass and bind to determinants presented on the 39 amino acid, trypsin-sensitive, N-terminal peptide of glycophorin A. Three display differential specificities for the two allelic forms of glycophorin A; two are exquisitely specific for the M-form and one preferentially binds the N-form. Treatment of red cells with neuraminidase, which removes N-acetylneuraminic acid from glycophorin A, abolishes the binding of these three antibodies. The binding of the N-specific antibody is also sensitive to modification of the amino-terminal residue of the antigen. The fourth antibody binds equally well to both the M- and N-forms as well as to neuraminidase-treated red cells; thus it recognizes a public, N-acetylneuraminic acid independent glycophorin A determinant.

Agglutination Tests↗
PubMed1981

Cell sorter immunofluorescence detection of human erythrocytes labelled in suspension with antibodies specific for hemoglobin S and C.

We have developed an immunochemical method for labeling human red blood cells in suspension with hemoglobin-specific antibodies. A membrane permeable cross-linking reagent, dimethyl suberimidate, is used to covalently bind, in situ, a fraction of the intracellular hemoglobin to integral membrane proteins. Hypotonic lysis and washing of the cells removes the unbound hemoglobin resulting in red blood cell ghosts which are permeable to macromolecules. Fluorescein-labeled antibodies for the hemoglobin variants S and C bind specifically to hemoglobin AS and AC ghosts, respectively, and not to normal hemoglobin AA ghosts. This technique can be used to prepare ghost suspensions for cell sorter analysis in which large numbers (10(9)--10(10)) of normal ghosts can be rapidly screened for the presence of rare anti-hemoglobin S and anti-hemoglobin C binding ghosts. In reconstruction experiments using mixtures of AS and AA cells and anti-hemoglobin S, AS ghosts as rare as 3 X 10(-5) were quantitatively recovered. Fluorescence artifacts prevented direct enumeration of AS ghosts at lower frequencies, but a two-step flow sorting-fluorescence microscope visual scanning procedure allows semiquantitative detection of anti-hemoglobin S-labeled ghosts as low as 10(--7). This method can be used for rapidly screening blood samples from individuals of normal hemoglobin A genotype for the presence of rare anti-hemoglobin S and anti-hemoglobin C binding ghosts.

Antibody Specificity↗
PubMed1978

Characterization of plasminogen activator in human cervical cells.

Plasminogen activator activity was detected in human gynecologic specimens using a synthetic fluorogenic peptide substrate assay and confirmed by an 125I-labeled fibrin plate assay. Epithelial cells in these samples contain enzymatic activity that biochemically resembles both the well-characterized plasminogen activator, urokinase, and the less-specific plasminogen activator, trypsin. Inhibition of the cervical cell activity by diisopropylfluorophosphate and p-nitrophenyl-p'-guanidinobenzoate demonstrates that, like urokinase and trypsin, this plasminogen activator is also a serine protease. Polyacrylamide gel electrophoresis of plasminogen that had been incubated with cervical cells indicated the same mechanism of plasminogen activation as exhibited by urokinase. We attempted to correlate plasminogen activator activity of each sample with cytomorphologic diagnosis. Three of the four dysplastic samples analyzed showed higher plasminogen activator activity than did the normal samples.

Cervix Uteri↗
PubMed1977

Magnetic resonance investigation of ionizable residues at the active site of thermolysin.

The details of the pH dependence of the thermodynamic and magnetic interactions of the active-site region of thermolysin in which manganese has replaced the active-site zinc atom and the inhibitor N-trifluoroacetyl-D-phenylalanine have been examined. These show a number of ionizable groups in the active-site region. A cooperative displacement of manganese at the catalytic site is observed as pH is lowered. This appears to be the result of the protonation of histidine-142 and -146 which act as metal ligands. The metal is 50% displaced at pH 6.0. At higher pH values, the environment of the bound manganese changes as a result of the ionization of at least two groups of approximate pKa = 8.5 and 9.5. These values are assigned to tyrosine-157 and to the water molecule which acts as a metal ligand at the active site. The binding behavior of the inhibitor strongly suggests that two molecules of inhibitor bind to the enzyme. The weaker site is competitive with the synthetic substrate FAGLA (furylacryloylglycyl-leucinamide), while the strong site has no effect on FAGLA hydrolysis. This second site is in the vicinity of the active site with a distance of 8 A or less between the trifluoromethyl group and manganese bound at the active site.

Binding Sites↗
PubMed1976

Role of Calcium in the thermal stability of thermolysin.

The effect of calcium ion on the thermal stability of thermolysin has been investigated. The native protein undergoes an irreversible structural change and autolysis at high temperature. Analysis of the calcium ion dependence of the apparent melting temperature observed spectroscopically gives an apparent deltaH of -x (130 kcal/mol) where x is either 1 or 2. Neither zinc ion, where bound at the active site, nor terbium ion, which binds very tightly to the double calcium binding site, shows a stabilizing effect. These sites are therefore presumably not coupled to the transition which leads to autolysis. Removal of calcium ion from the native enzyme at temperatures below 50 degrees C results in inactivation but not major autolysis. The addition of 1 equiv of terbium before calcium removal results in a protein species which is 40% active and is no longer subject to thermal stabilization by calcium. These results suggest a pathway for the thermal inactivation of the enzyme which involves an irreversible structural change at one or both of the single calcium ion sites. This change propagates to the active site and results in inactivation. The binding of calcium ion to either or both single sites completely inhibits this structural change. The structural change is apparently cooperative and may correspond to a localized denaturation of the native structure.

Apoproteins↗
PubMedDate not supplied

Plasminogen activator as a diagnostic marker for preneoplastic cells in human gynecologic specimens.

A modified fibrin-agar overlay assay was used to measure the fibrinolytic activity in human cervical epithelial cells. The fibrinolytic activity results from a cell-associated serine protease that acts as a plasminogen activator. An enzymatic and morphologic comparison of individual normal and abnormal cells reveals that about 50% of the dysplastic and carcinoma in situ cells contain high levels of the activator. Forty percent of morphologically normal metaplastic cells also exhibit high activities of plasminogen. Forty percent of morphologically normal metaplastic cells also exhibit high activities of plasminogen activator. Most normal intermediate and superficial epithelial cells and all erythrocytes and leukocytes are inactive. The frequency of plasminogen activator-containing cells was somewhat higher in cytomorphologically abnormal samples than in normal samples.

Carcinoma in Situ↗