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F Arakawa

Publications and source records attributed to F Arakawa.

At least 37 records · Page 2Linked to original sources
PubMed1993

Signal intensity of brain metastases on T2-weighted images: specificity for metastases from colonic cancers.

In this report, we present and discuss the signal intensity of brain metastases from colon cancer on both T1- and T2-weighted images. In five of 6 cases, metastases were seen as markedly hypointense areas on T2-weighted images. This finding should alert one to the possibility of a primary cancer of the colon. Some haemorrhagic metastases from other malignancies also showed marked hypointensity. They usually exhibited hyperintensity on T1-weighted images. A case of colon metastasis was also haemorrhagic, and in this case a hyperintense area was observed on T1-weighted images. The marked hypointense area corresponded to peripheral necrosis and probably some viable tumour. Aetiologically, such hypointensity was not induced by severe fibrosis, calcification or excessive iron deposition.

Adolescent↗
PubMed1993

Construction and expression of two mouse-human chimeric antibodies with high specificity and affinity for carcinoembryonic antigen.

We have previously reported that a group of monoclonal antibodies (MAbs) to carcinoembryonic antigen (CEA), designated Group F MAbs, are able to discriminate CEA in tumor tissues from the CEA-related normal antigens and that CEA assay systems utilizing at least one Group F MAb show the improved cancer diagnosis. In this study, we cloned the genes coding for two Group F MAbs (F11-35 and F11-39) and deduced the amino acid sequences of the variable regions for their heavy and light chains. The variable region for the heavy chain of F11-35 contained a possible N-glycosylation site (Asn/Asp/Thr) at amino acid positions 89-91. Then, we constructed two mouse-human chimeric antibodies by using the F11-35 and F11-39 variable region genes of heavy and light chains (VH and V kappa) and human heavy and light chain constant region genes (gamma 1 and kappa) derived from a human plasma cell leukemia line (ARH77). The chimeric gene constructs were sequentially co-transfected into murine non-Ig-producing myeloma (P3-U1) or hybridoma (Sp2/0) cells by electroporation. The resulting chimeric heavy chain of F11-35 showed a slightly but significantly higher molecular weight than that of F11-39, but the molecular weights of their unglycosylated peptides synthesized in the presence of tunicamycin were similar, indicating the glycosylation at the possible N-glycosylation site in the variable region of the Ch F11-35 heavy chain. Both chimeric antibodies exhibited the same specificity and affinity for CEA as those of the parental murine hybridoma antibodies, respectively. Ascites production of Sp2/0 transfectomas is sufficiently high (600-900 micrograms/ml) for initial clinical studies with the chimeric antibodies.

Amino Acid Sequence↗
PubMed1992

Apolipoprotein E1 Lys-146----Glu with type III hyperlipoproteinemia.

During the screening of samples obtained from 5 individuals with type III hyperlipidemia, we identified a variant of apolipoprotein (apo) E which exhibited a discrepancy in apo E phenotype showing the E3/E1 isoform on isoelectric focusing (IEF) analysis and E3/E3 on gene analysis. Sequence analysis of the DNA of the proband that was amplified by PCR and subcloned, revealed a single substitution of one lysine (AAG) for one glutamic acid (GAG) at position 146, thereby adding two negatively charged units to apo E3. This defect had been described only for apo E1 to date (Mann et al. (1989) Clin. Res. 37, 520A (abstract)). In this case, PCR-mediated site-directed mutagenesis was used to identify the structural alterations forming the abnormal E1 genotype in the proband's family. Purified apo E1 Lys-146----Glu showed less than 10% of binding activity to apo B, E receptor on human skin fibroblasts compared with apo E3. This substitution demonstrates that Lys-146 is essential for the binding of apo E to the receptor.

Adult↗
PubMed1992

Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface.

The cDNA coding for the human dipeptidyl peptidase IV (DPPIV) has been isolated and sequenced. The nucleotide sequence (3465 bp) of the cDNA contains an open reading frame encoding a polypeptide comprising 766 amino acids, one residue less than those of rat DPPIV. The predicted amino acid sequence exhibits 84.9% identity to that of the rat enzyme, and contains nine potential N-linked glycosylation sites, one site more than those in the rat enzyme. A putative catalytic triad for serine proteinases, serine, aspartic acid and histidine, are found in a completely conserved COOH-terminal region (positions 625-752).

Amino Acid Sequence↗
PubMed1992

Reaction profiles of seven enzyme immunoassay kits for carcinoembryonic antigen (CEA) analyzed with purified preparations of CEA and related normal antigens.

Antigenic reactivities of 4 purified CEA preparations and 4 different CEA-related normal antigens (NCA from lungs, NCA-2 from meconium, and NFA-1 and NFA-2 from adult feces) were comparatively analyzed with seven commercially available EIA kits [Abbott CEA-EIA Monoclonal, D-ZYME CEA, Imzyne CEA, CEA MITSUI II, CEA Roche EIA, Immunoball-CEA (N), and Glaozyme CEA]. All kits employ a sandwich-type solid-phase method using polystyrene beads and monoclonal anti-CEA antibodies as either capture antibody and/or tracer antibody. In general, all CEAs reacted well with these assay kits. Reactivity differences in weight among the CEA preparations were, however, observed in all but one assay kit (D-ZYME CEA) in which all CEAs showed fairly homogeneous reactivity. The degree of reaction intensity among the preparations used varied depending on the assay kits used. NCA, which is well known to be partially cross-reactive with CEA, revealed a negligible reactivity in all kits. NFA-1 which is also partially cross-reactive with CEA but antigenically unrelated to NCA, showed very strong reactivity in 2 kits [CEA Roche EIA and Immunoball-CEA (N)] and weak reactivity in another kit (CEA MITSUI II). NCA-2 in meconium and NFA-2 in normal adult feces, which both have immunochemical and physicochemical properties very similar to those of CEA, reacted to a greater or lesser extent with all kits. However, one assay kit (D-ZYME CEA), whose reactivity to different CEAs was fairly homogeneous, could discriminate CEAs from NCA-2 and NFA-2. Although the reactivity of NFA-2 was less than that of any CEA in all kits, that of NCA-2 was higher than that of some CEAs in 3 kits (Abbott CEA-EIA Monoclonal, CEA MITSUI II, and CEA Roche EIA). These differences in reactivity and specificity of currently available EIA kits for CEA should be borne in mind when selecting an assay kit.

Antibodies, Monoclonal↗
PubMed1992

Biochemical characterization of 25 distinct carcinoembryonic antigen (CEA) epitopes recognized by 57 monoclonal antibodies and categorized into seven groups in terms of domain structure of the CEA molecule.

The chemical nature of 25 distinct carcinoembryonic antigen (CEA) epitopes, which were recognized by 57 different monoclonal antibodies and categorized into 7 groups (Groups A to G) in terms of domain structure of the CEA molecule, was analyzed and the findings obtained were compared with the results of our previous studies using recombinant CEA proteins. All 21 epitopes of Groups A to F defined by 48 MAbs were resistant to periodate oxidation and were to a greater or lesser extent retained after deglycosylation of CEA, indicating that they are all protein in nature. The 21 epitopes were detected in recombinant CEA proteins expressed in Chinese hamster ovary (CHO) cells. Seven of the 21 epitopes of protein nature were partially or completely sensitive to reduction and alkylation of CEA and not detected or only slightly revealed in the recombinant CEA proteins expressed in E. coli, indicating that those epitopes are dependent on the tertiary structure of the peptide chain, which is formed by disulfide bonds. All 4 epitopes of Group G defined by 9 MAbs were sensitive to mild periodate oxidation and deglycosylation, but resistant to reduction and alkylation and to digestion with pepsin or pronase, indicating that those 4 epitopes are carbohydrate in nature. Although none of the 4 epitopes of Group G were detected in the recombinant CEA proteins expressed in E. coli, two were detected in those expressed in CHO cells. The biochemical studies reported here thus provide information as to the nature of the epitopes on the CEA molecule and help form the basis for selecting the anti-CEA MAbs for use in biological study and potential clinical applications of CEA.

Animals↗
PubMed1992

Determination of epitope specificities of a large number of monoclonal antibodies by solid-phase mutual inhibition assays using biotinylated antigen.

A generally applicable method for the determination of the epitope specificities of a large number of monoclonal antibodies (MAbs) is presented. The method is based on the solid-phase mutual inhibition assay using 96-well plates coated with the respective MAbs, competitor MAbs, biotinylated antigen and avidin-peroxidase conjugate. Using carcinoembryonic antigen (CEA) as a model antigen the method was applied to the determination of epitope specificities of anti-CEA MAbs. A constant amount of biotinylated CEA was incubated with a given MAb immobilized on wells of 96-well plates in the presence of increasing amounts of soluble competitor MAbs. The biotinylated CEA bound to the immobilized antibody were then reacted with avidin-peroxidase conjugate and the activity of the bound peroxidase was determined by the use of o-phenylenediamine and hydrogen peroxidase. The method used here alleviates the laborious procedures of labeling all antibodies to be tested and the confusion caused by differential labeling among different MAbs, and is convenient for mapping analysis of many MAbs if the corresponding purified antigen is available.

Animals↗
PubMed1991

Molecular cloning of nonspecific cross-reacting antigens in human granulocytes.

To clarify the molecular structures of nonspecific cross-reacting antigens (NCAs), a family of glycoproteins antigenically related to carcinoembryonic antigen (CEA), in human granulocytes, we have screened a cDNA library of human leukocytes using a cDNA probe for the N-terminal domain (domain-N) of NCA-50, an NCA species in tumor cells. In 95 positive clones randomly selected, we identified six NCA or NCA-related cDNA clones including NCA-50, biliary glycoprotein protein a, and W272 (CGM6) which have previously been reported, and three new clones, W236, W264, and W282, encoding three novel NCA species. W236 and W264 consist of a domain-N, a putative transmembrane domain, and a possible cytoplasmic domain. The domain-N of W264 is 89% similar to that of NCA-50 at amino acid level, whereas the domain-N of W236 is only 49 and 43% similar to those of NCA-50 and pregnancy-specific beta 1-glycoprotein-11 (PSG11), respectively, indicating that W236 belongs to a new subfamily within the CEA family. The third clone W282 encodes a protein consisting of a domain-N virtually identical to that of W264 and a short hydrophilic C-terminal domain. W264 and W282 seem to be derived from a single gene by alternative splicing of RNA. These three new species are particularly unique in respect that they lack the repetitive immunoglobulin-related domains that have been universally found in the human CEA gene family members. The biochemical and immunochemical properties of the recombinant proteins of these cDNA clones, however, did not coincide with those of six NCA species previously identified in granulocytes at protein level, suggesting that, in granulocytes, there exist at least 12 NCA or NCA-related species whose expression is under complex control.

Amino Acid Sequence↗
PubMed1991

A specific heterotypic cell adhesion activity between members of carcinoembryonic antigen family, W272 and NCA, is mediated by N-domains.

The Ca(2+)-independent homotypic and heterotypic cell adhesion activities of a carcinoembryonic antigen (CEA) family member, W272 (CGM6), whose cDNA has recently been isolated from libraries of human peripheral leukocytes of apparently normal subjects (Arakawa, F., Kuroki, Mo., Misumi, Y., Oikawa, S., Nakazato, H., and Matsuoka, Y. (1990) Biochem. Biophys. Res. Commun. 166, 1063-1071) and spleen of chronic myelogenous leukemia patients (Berling, B., Kolbinger, F., Grunert, F., Thompson, J. A., Brombacher, F., Buchegger, F., von Kleist, S., and Zimmermann, W. (1990) Cancer Res. 50, 6534-6539) has been examined. Chinese hamster ovary cells transfected with the cDNA for W272, CEA, nonspecific cross-reacting antigen (NCA), and various antigens containing chimeric N-domain have been used. The W272 producers did not show homotypic binding at all but bound only to the cells expressing NCA and a chimeric CEA whose N-domain is substituted by that of NCA, indicating the major contribution of N-domain of NCA in the specific binding. The importance of the N-terminal region of NCA N-domain for the W272-NCA binding has been shown by detailed analysis using COS-1 cells producing various NCA whose N-domain are chimera of that of NCA and CEA. The strict heterotypic nature of the W272-NCA adhesion strongly suggests that the cell adhesion activities exhibited by CEA family members are not the fortuitous activity but the specific one which have some important physiological roles.

Amino Acid Sequence↗
PubMed1991

Three novel molecular forms of biliary glycoprotein deduced from cDNA clones from a human leukocyte library.

Three cDNA clones that encode three novel variants of biliary glycoprotein a (BGPa), a glycoprotein belonging to the CEA gene family, were identified in a human leukocyte cDNA library. The domain structures of the predicted proteins of the three clones W211, W233 and W239 are N-A1-B1-A2, N-A1-B1 and N-A1-B1-C, respectively; they lack the transmembrane and cytoplasmic domains that exist in the four BGP species (BGPa, BGPb, BGPc and BGPd) previously reported. Their sequences from N to B1 or to A2 are virtually identical to those of BGPa-d. Comparison with the genomic sequence for BGPa-d suggested that these three new BGP variants as well as BGPa-d are generated from the same single gene by alternative splicing of RNA.

Antigens, CD↗
PubMed1991

Immunological and biochemical characterization of the nonspecific cross-reacting antigen epitopes using twenty-three monoclonal antibodies.

Twenty-three monoclonal antibodies (MAbs) reactive with nonspecific cross-reacting antigen (NCA) were prepared and used for constructing a serological map of the NCA molecule. The MAbs were generated using purified NCA or carcinoembryonic antigen (CEA) as immunogen. The MAbs could be divided into two groups: Group X, 10 clones reactive with NCA and CEA; and Group Y, 13 clones specific for NCA. Cross-competition enzyme immunoassays between MAbs of the individual groups revealed that at least 8 different subgroups can be defined i.e., 5 and 3 subgroups in Groups X and Y, respectively. The chemical nature of the epitopes recognized by those MAbs was tested using chemically or enzymatically treated antigens; all MAbs reacted with periodate-treated NCA and deglycosylated NCA, indicating that all the epitopes identified appeared to be protein in nature. Reduction and alkylation, pepsin digestion or pronase treatment of NCA, however, gave some differential results with respect to MAb binding. The serologic mapping and biochemical studies reported here thus provide information as to the range and nature of the epitopes on the NCA molecule and help form the basis for selecting the anti-NCA MAbs for use in biological and immunological study of NCA.

Animals↗
PubMed1990

The nucleotide and deduced amino acid sequences of a cDNA encoding a new species of pregnancy-specific beta 1-glycoprotein (PS beta G).

We screened a cDNA library of a human placenta with cDNA for nonspecific cross-reacting antigen, a member of the carcinoembryonic antigen gene family. One of the positive clones, PS34, was found to encode a 426 amino acid protein belonging to pregnancy-specific beta 1-glycoprotein (PS beta G). The mature PS34 protein consisted of domains, N, A1, A2, B2 and C. The domain-N of PS34 showed sequence similarities of 79.8-83.5% to those of the PS beta G members so far reported, indicating PS34 is a new member of PS beta G and also of the carcinoembryonic antigen gene family.

Amino Acid Sequence↗
PubMed1990

Characterization of a cDNA clone encoding a new species of the nonspecific cross-reacting antigen (NCA), a member of the CEA gene family.

To clarify the molecular structures of the nonspecific cross-reacting antigens (NCAs) produced by human granulocytes, we cloned cDNAs from libraries of normal white blood cells. A clone, NCA-W272, was found to code a protein similar to NCA of tumor cells. The protein consisted of a signal peptide (34 aa), domain-N (108 aa), -A1 (92 aa), -B1 (86 aa) and -M (29 aa). Similarity of the amino acid sequence of each domain to that of the tumor NCA was 72, 92, 76 and 79%, respectively. COS-1 cells transfected with an expression vector carrying the cDNA synthesized a 70 kDa glycoprotein, which was reactive with anti-NCA antibody and released from cell surface by phosphatidylinositol-specific phospholipase C. Thus the clone NCA-W272 was indicated to encode a new species of NCA distinct from the tumor NCA.

Amino Acid Sequence↗
PubMed1988

Active production and membrane anchoring of carcinoembryonic antigen observed in normal colon mucosa.

Normal colon mucosa was found to produce carcinoembryonic antigen (CEA) quite actively as cancerous tissues do when maintained in an organ culture, although the fresh normal mucosae contained a very small quantity of CEA unlike cancerous tissues. This is consistent with an active expression of CEA mRNA in normal mucosa comparable to that in cancerous tissues actively producing CEA, and suggests that the normal cell product was rapidly released into the lumen of digestive tract and turned into normal fecal antigens (NFAs) previously found in feces. 3H-Labeled precursors of glycophospholipid such as ethanolamine and stearic acid were incorporated into CEA produced by both normal and cancerous tissues, suggesting that CEA in normal mucosa is anchored to the cell membrane through a glycophospholipid as in cancerous tissues.

Blotting, Northern↗
PubMed1987

Antigenic heterogeneity of carcinoembryonic antigen in the circulation defined by monoclonal antibodies against the carbohydrate moiety of carcinoembryonic antigen and closely related antigens.

Six mouse monoclonal antibodies reactive with carcinoembryonic antigen (CEA) were prepared and used for the analysis of the antigenic heterogeneity of CEA in patient sera. Their reaction specificity and the chemical nature of antigenic epitopes recognized by them were analyzed by radioimmunoassay on the basis of reactivities with different preparations of CEA, normal fecal antigen 2, and nonspecific cross-reacting antigen 2 before and after chemical and/or enzymatic treatment. Two antibodies, F3-30 and F4-82, raised with CEA were reactive with different peptide epitopes on the antigen molecules and revealed a quite universal reactivity with all CEA, normal fecal antigen 2, or nonspecific cross-reacting antigen 2 preparations tested. The serum CEA values obtained with these antibodies were highly correlated with those obtained with conventional radioimmunoassays for CEA. The other four antibodies (F4-11 and F33-37 raised with CEA, F8-52 with normal fecal antigen 2, and F48-60 with nonspecific cross-reacting antigen 2) were found to recognize carbohydrate epitopes with different specificities and revealed very heterogeneous reactivities. The serum CEA values estimated with these four antibodies were highly variable depending on the antibody used, suggesting that the expression of carbohydrate epitopes on the CEA molecules in patient sera was quite heterogeneous. The antigenic heterogeneity of the carbohydrate epitopes was detected even in a single patient serum by affinity chromatography. The causes that give rise to the difference in CEA values between the Roche and the Daiichi kits were analyzed on the basis of reactivities of three groups of patient sera, which showed extremely different ratios for the Roche and Daiichi kits, with monoclonal anti-carbohydrate antibodies. The results obtained suggest that, at least in part, the diversity of antigenic expression on carbohydrate chains on the CEA molecules in patient sera and the variation in specificity or quantity of anti-carbohydrate antibodies in the polyclonal antibody preparations used for the respective assay systems may result in the differences in the estimated CEA values.

Antibodies, Monoclonal↗
PubMed1987

Epitope mapping of the carcinoembryonic antigen by monoclonal antibodies and establishment of a new improved radioimmunoassay system.

A comprehensive mapping of epitopes on the carcinoembryonic antigen (CEA) molecule has been achieved by analyses of the specificities of 146 monoclonal antibodies (MAbs) from more than 300 hybridomas established recently. The reactivities of MAbs were analyzed by radioimmunoassays (RIA) with highly purified preparations of CEA and related antigens including normal fecal antigen-1 (NFA-1), NFA-2 in normal adult feces, nonspecific cross-reacting antigen (NCA) in lung and NCA-2 in meconium. The MAbs could be divided into five groups: group I, 23 clones directed to the NCA-common part of the CEA molecule; group II, 31 clones directed to the normal fecal cross-reacting antigen (NFCA)-common part; group III, 46 clones directed to the NFA-1-common part; group IV, 33 clones reactive with the heterogeneous carbohydrate part; and group V, 13 clones directed to the CEA-distinctive part which seemed to be highly specific for CEA. Mutual inhibitions of CEA binding between MAbs of the individual groups revealed that at least 25 different subgroups can be defined i.e., 4, 7, 8, 4, and 2 subgroups in groups I to V, respectively. The epitopes recognized by the group IV MAbs were found to be sensitive to oxidation with periodate, while the epitopes defined by MAbs of the other groups were resistant to this treatment. A solid-phase sandwich-type RIA system for CEA was established by using 2 MAbs from groups II and III as the CEA catcher and an MAb of group V as the tracer. This assay was shown to exhibit improved cancer-specificity and accuracy in the estimation of serum CEA levels.

Antibodies, Monoclonal↗