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M J Krantz

Publications and source records attributed to M J Krantz.

At least 19 recordsLinked to original sources
PubMed1992

Ciliated respiratory epithelial surface changes after formaldehyde exposure.

The investigation sought to identify alterations of specific ciliated epithelial surface components after exposure to formaldehyde (HCHO) levels that decrease respiratory ciliary function. Bovine tracheae were reacted with an analog of N-hydroxysuccinimidobiotin to label epithelial surface-accessible components before exposure to HCHO. The tracheae were then exposed to 0, 16, 33, and 66 micrograms HCHO/cm2 epithelial surface for 30 min. Cilia were isolated from the epithelium, separated into membrane and internal axonemal portions, analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and either stained to detect proteins or transblotted to detect biotin-labeled components. Densitometric analysis of axoneme proteins showed a decrease in the total amount extracted with increased HCHO concentration, including axoneme-specific proteins, dynein, and tubulin. However, biotinylated proteins in the axoneme fractions proportionately increased. Membrane fractions showed little change in protein with increasing HCHO concentration. The majority of these is not biotin-labeled and thus not surface-accessible components. Biotinylated material in the membrane fractions showed a significant decrease with increased HCHO concentration, particularly of bands at 92, 98, and 105 kD. These data suggest that increasing HCHO exposure reduces both extractable ciliary axonemes and detergent-soluble surface components, possibly by stabilizing respiratory epithelial membranes. This process apparently strengthens association of certain surface components with the internal axoneme, thereby reducing subsequent solubilization in detergent.

Adenosine Triphosphatases↗
PubMed1990

Identification of surface components of mammalian respiratory tract cilia.

Cilia isolation methods were modified to retain respiratory tract ciliary membranes and to identify accessible surface components. Prior to isolation of cilia, halves of cow tracheae were treated with the extended spacer arm analog of N-hydroxysuccinimido-biotin (NHS-LC-biotin) to label accessible membrane constituents. Mechanical disruption of the epithelium and substitution of CHAPS for Triton X-100 provided a good yield of cilia with membranes and with minimal contamination. Subsequent extraction of these cilia with Triton X-100 solubilized the membranes and released soluble matrix proteins. Proteins of membrane + matrix and axoneme fractions were analyzed after electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The major biotin-labeled components in the membrane + matrix fraction were 105, 98, and 92 kd, were glycosylated, and remained with reconstituted, pelleted membrane vesicles along with the major non-biotinylated protein at 51 kd. Other membrane + matrix proteins at 126 and 76 kd bound streptavidin even from nonlabeled trachea, but remained soluble. Several biotin-labeled proteins distinct from those in the membrane fraction remained with Triton X-100-extracted axonemes. Streptavidin-colloidal-gold (SAG) particles appeared to bind randomly along the length of cilia. The peripheral join between A and B microtubules was a predominant nonspecific location of SAG on axonemes. Axonemes with biotin label also bound significant numbers of SAG to outer dynein arms, confirming the streptavidin reaction with separated proteins on transfers. These results suggest close association of the membrane with the axoneme in respiratory tract cilia and a membrane composition somewhat different from protozoan cilia.

Animals↗
PubMed1986

Epitopes of carcinoembryonic antigen defined by monoclonal antibodies prepared from mice immunized with purified carcinoembryonic antigen or HCT-8R cells.

A library of 18 monoclonal antibodies (MAbs) reactive with purified carcinoembryonic antigen (CEA) has been prepared. The specificity of these MAbs was tested and they have been separated into nine subgroups, each recognizing a different region of the CEA molecule. Seven MAbs from four of the groups also react with the nonspecific cross-reacting antigen. Some of the MAbs are directed against conformational determinants: three of the MAb groups bind poorly to sodium dodecyl sulfate-treated CEA, while five of the groups are not reactive with reduced and alkylated CEA. Three of the groups react with purified CEA but not with the cell surface CEA of HCT-8R cells, while the other groups react with both forms. The MAbs were tested for binding to fragments of CEA obtained by chemical cleavage and the groups of MAbs were found to react with different subsets of such fragments.

Antibodies, Monoclonal↗
PubMed1986

Characterization of monoclonal antibodies to carcinoembryonic antigen with increased tumor specificity.

Nine monoclonal antibodies reacting with carcinoembryonic antigen (CEA) were produced after immunization of mice with either purified CEA or a CEA-producing human cell line. Their specificities were assessed by immunohistochemistry on tissue sections of neoplastic and nonneoplastic lesions. These monoclonal antibodies have different patterns of tissue reactivity. Two of them, D14 and B18, were found to have a high degree of specificity for colonic carcinoma and did not react with formalin-fixed paraffin-embedded sections of normal colon with standardized staining conditions. Most cases of noncolonic adenocarcinomas and normal epithelial structures were not stained by these two monoclonal antibodies. The specificity of the monoclonal antibodies was further investigated immunochemically using intact, reduced, and alkylated or chemically fragmented CEA. Liquid phase radioimmunoassays and antibody competition immunoenzymatic assays confirmed that the antibodies recognize different epitopes of CEA. These data support the concept of CEA heterogeneity and the reactivity of the D14 and B18 monoclonal antibodies with colonic adenocarcinomas indicates that they are useful immunohistochemical probes.

Animals↗
PubMed1985

Carbohydrate determinants associated with carcinoembryonic antigen (CEA).

The reactivities of eight purified preparations of carcinoembryonic antigen with monoclonal antibodies directed to tumor-associated carbohydrate determinants have been studied. All eight preparations showed strong reactivities with AH6, which defines Y structure (Fuc alpha 1----2Gal beta 1----4[Fuc alpha 1----3] GlcNAc beta 1----R), whereas only a few preparations showed reactivity with FH4-defining dimeric X determinants, (Gal beta 1----4 [Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4 [Fuc alpha 1----3]GlcNA beta 1----3Gal beta 1----R). No other antibodies tested showed any reactivity with these preparations. These carbohydrate markers associated with carcinoembryonic antigen will be useful to enhance the diagnostic value of the antigen.

Carbohydrate Sequence↗
PubMed1985

Monoclonal antibody recognizing human melanoma-carcinoma cross-reacting oncofetal antigen epitopically associated with carcinoembryonic antigen.

By fusion of mouse NS1 myeloma cells with splenocytes from a BALB/c mouse immunized with human melanoma cells, an IgG1 monoclonal antibody, designated as 140.72, was produced. By the mixed hemadsorption antibody binding assay, 140.72 was shown to react with 17 of 20 melanoma cell lines and with 5 of 14 carcinoma cell lines. This antibody also reacted with 3 of 3 normal melanocyte cultures in much lower titers. It did not react with any of 35 other normal and malignant lines, including neuroblastoma, glioblastoma, sarcoma, teratoma, fibroblast, and lymphoid cell lines. Absorption with fresh melanoma and carcinoma homogenates confirmed the results of direct tests. Fetal reactivity of antibody 140.72 was determined by positive absorption with 10 of 11 tissue homogenates derived from different fetuses of 10-16 weeks' gestation. The reactivity of this antibody was completely removed by absorption with a highly purified preparation of carcinoembryonic antigen (CEA) derived from a colon carcinoma. The antigenic activity was detected in the culture medium of reactive cell lines. Immunoprecipitation analyses of melanoma and carcinoma cells indicated that the antigenic determinant recognized by antibody 140.72 is on a glycoprotein with an apparent molecular weight of 95,000-150,000 common to both serologically reactive cell types. Additionally, a 200,000-molecular-weight glycoprotein corresponding to the CEA molecule was detected only on the reactive carcinoma cells. These data confirmed previous findings obtained with polyclonal anti-CEA antisera for the existence of shared CEA-related antigenic determinants on human carcinomas and melanomas and provided additional molecular characterization of these glycoproteins. Further characterization of the molecules bearing the antigenic determinant recognized by antibody 140.72 should be performed with a view to exploring its potential in the immunodiagnosis and immunotherapy of patients with melanoma.

Animals↗
PubMed1983

Preparation of fragments of carcinoembryonic antigen and identification of a major subset of antigenic determinants.

The detailed immunochemistry of a limited molecular domain of carcinoembryonic antigen (CEA) was examined, by using new methods to prepare and purify immunoreactive fragments. Fragments of CEA were prepared by digestion of the reduced and alkylated glycoprotein with protease V8, chymotrypsin or trypsin, or by chemical cleavage at cysteine residues with 2-nitro-5-thiocyanobenzoic acid. The products were fractionated by high performance liquid chromatography (HPLC) on a reversed phase support, and the most immunoreactive fragment was recovered from each of the four mixtures. The Mr of these purified fragments ranged from 30,000-35,000, the amino acid compositions were similar, and all contained carbohydrate. The four independently derived fragments gave similar inhibitions of CEA binding to anti-CEA antisera; maximum values were 15-55% depending on the antiserum employed. Competitive assays with pairs of fragments in different combinations showed that the four contained essentially the same subset of antigenic determinants. The fragments were quite resistant to further enzymatic digestion, but comparison of the HPLC profiles of mild acid hydrolysates of two of the fragments showed substantial similarity. These results suggest that the CEA molecule includes a region of about 30,000 mol. wt that is contained between consecutive cysteine residues, is relatively resistant to a number of residue-specific proteases, and constitutes a significant subset of antigenic determinants. Assays to identify cleavage products with different antigenic specificities were negative, suggesting that other determinants were destroyed or that the corresponding antibody populations were too small to detect.

Amino Acids↗
PubMed1983

Purification and characterization of a 50,000 mol. wt glycoprotein antigen, and localization of determinants involved in cross-reactivity with carcinoembryonic antigen.

A 50,000 mol. wt glycoprotein antigen (50 k) was purified from metastatic colon tumor. The initial purification was monitored by cross-reactivity of the antigen with an unabsorbed antiserum against carcinoembryonic antigen (CEA, Mr 180,000). Six mg of antigen was obtained from 250 g of tissue, with a recovery of 14% and a relative purification of 143-fold. The amino acid composition of 50 k was similar to that of CEA. The carbohydrate content, primarily glucosamine and mannose, totaled about 25%. Immunodiffusion showed that 50 k lacked some of the CEA epitopes, and that normal spleen contained an antigen identical to 50 k. Radioimmunoassays showed that the high avidity anti-CEA antibodies in the unabsorbed antiserum were mainly cross-reactive with 50 k, probably via a similar but not identical antigenic site. The immunochemical properties of 50 k thus correspond to those of the non-specific cross-reacting antigen (NCA). The molecular size and carbohydrate composition reported for various NCA-like antigens have differed, so identity of 50 k an NCA cannot be established on this basis. Immunoreactive fragments of 50 k were prepared by digestion with each of three different proteolytic enzymes, and were purified by high performance liquid chromatography (HPLC). A polypeptide of 20,000-30,000 mol. wt containing about half of the 50 k determinants, was recovered in each case. Experiments with mixtures showed that the three purified fragments all contained the same subset of antigenic determinants. The fragment-localized subset represented most, if not all of the 50 k determinants involved in CEA cross-reactivity. Similarly, a CEA fragment was shown to contain essentially all of the CEA determinants involved in 50 k cross-reactivity. The fragments of 50 k and CEA were quite resistant to further proteolytic digestion, and comparison of a CEA fragment with a 50 k fragment by partial acid hydrolysis and HPLC peptide mapping failed to reveal structural homology to account for the cross-reactivity.

Amino Acids↗
PubMed1977

Biochemical and immunological characterization of the major envelope glycoprotein gp69/71 and degradation fragments from Rauscher leukemia virus.

Analysis of the proteins of Rauscher murine oncornavirus by immunoprecipitation showed that antiserum to the purified envelope glycoprotein of approximately 69,000 and 71,000 daltons (gp69/71) reacted as well with a number of other components of several murine oncornaviruses of approximately 45,000, 32,000, and 15,000 daltons. Polypeptides of similar size were also produced by limited proteolysis of purified gp69/71; these degradation fragments were shown to contain carbohydrate by the incorporation of (3)H from sodium boro[(3)H]hydride after neuraminidase and galactose oxidase treatment. Each of these glycoproteins was isolated by preparative polyacrylamide gel electrophoresis and was analyzed by tryptic peptide mapping. The major virion components of 69,000 and 71,000 daltons were nearly identical, as were the primary degradation fragments. Analysis of the immunological properties of the glycoproteins showed that the 71,000-, 69,000-, and 32,000-dalton glycoproteins behaved similarly with respect to type and group-specific antigenic determinants. In contrast, the 45,000-dalton glycoprotein lacked detectable interspecies and some of the group-specific reactivity. Components of about 45,000 and 32,000 daltons isolated directly from virions were also identified as constituents of the major envelope glycoprotein by immune precipitation and tryptic peptide mapping. These results indicate that all of the examined virion glycoproteins of approximately 71,000, 69,000, 45,000, and 32,000 daltons are derived from the same viral gene and that these lower-molecular-weight glycoproteins can readily be produced from the major envelope glycoprotein.

Carbohydrates↗
PubMed1976

2-Imino-2-methoxyethyl 1-thioglycosides: new reagents for attaching sugars to proteins.

Cyanomethyl 1-thioglycosides ofD-galactose, D-glucose, 2-acetamido-2-deoxy-D-glucose, and D-mannose were prepared from the respective pseudothiourea derivatives and chloroacetonitrile. The nitrile group in these cyanomethyl thioglycosides can be converted to a methyl imidate group by treatment with sodium methoxide or HC1 in dry methanol to yield 2-imino-2-methoxyethyl 1-thioglycosides (IME-thioglycosides). The factors influencing the yield of IME-thioglycosides were investigated. The most convenient method of preparing IME-thioglycosides was treating 0.1 M cyanomethyl thioglycoside peracetate in dry methanol with 0.01 M sodium methoxide at room temperature for 24-48 h (50-60% yield). These IME-thioglycosides reacted readily with simple amines, amino acids, and proteins in mildly alkaline buffer solutions. Alpha-amylase and lysozyme modified with these reagents under appropriate conditions retained full activities. Thus the IME-thioglycosides constitute a new group of reagents for attaching sugars to proteins.

Acetylglucosamine↗
PubMed1976

Attachment of thioglycosides to proteins: enhancement of liver membrane binding.

Thioglycosides of D-galactose, D-glucose, N-acetyl-D-glucosamine, and D-mannose were covalently attached to Aspergillus oryzae alpha-amylase, hen's eggs lysozyme, and bovine serum albumin by amidination, diazo coupling, and amide formation. The binding of the newly formed glycoproteins (neoglycoproteins) to rabbit liver membranes was measured, using asialoorosomucoid as a reference. Attachment of D-galactosides by any of the three methods enhanced binding by several orders of magnitude. Coupling of a comparable number of D-mannosides or N-acetyl-D-glucosaminides had little or no effect. Attachment of D-glucosides also enhanced binding but to a variable extent depending on the method of attachment. Thus, the behavior of neoglycoproteins toward rabbit liver membranes closely paralleled that of serum glycoproteins (Ashwell and Morell, 1974) with respect to sugar specificity.

Acetylglucosamine↗
PubMed1973

Dopamine-beta-hydroxylase: a tetrameric glycoprotein.

Dopamine-beta-hydroxylase (EC 1.14.2.1) has been isolated as a pure protein from bovine-adrenal glands. Although the molecular weight of the native protein is well established (290,000), sodium dodecyl sulfate-gel electrophoresis under dissociating conditions gave a single band with a molecular weight of about 75,000. The enzyme contains about 4% carbohydrate, which consists of residues of mannose, glucosamine, galactose, glucose, fucose, and sialic acid. The pure enzyme also contains about 4 atoms of copper per molecule. It is concluded that dopamine-beta-hydroxylase is a tetrameric glycoprotein.

Adrenal Glands↗