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Biomedical subjects

R Kannagi

Publications and source records attributed to R Kannagi.

At least 127 records · Page 7Linked to original sources
PubMed1989

Parietal cell autoantigens involved in neonatal thymectomy-induced murine autoimmune gastritis. Studies using monoclonal autoantibodies.

Autoimmune gastritis accompanied by autoantibodies to parietal cells was induced in BALB/c nu/+ mice by neonatal thymectomy 2-4 days after birth. Three monoclonal autoantibodies, designated as 2B6 (IgG1), 2G10 (IgG2b), and 1H9 (IgG1), were obtained from one of these mice. All three reacted specifically with parietal cells, 2G10 recognizing species-specific antigenic determinants and 2B6 and 1H9 recognizing interspecies-specific antigenic determinants. All three recognized antigens on the membrane of intracellular secretory canaliculi and the cytoplasmic tubulovesicular system of parietal cells. At least two different molecular groups were recognized by these antibodies; 2B6 recognizing a 65,000-79,000-mol wt group and 1H9 recognizing a 92,000-120,000-mol wt group. Sera of most mice with autoimmune gastritis reacted with either or both groups. Both groups were consistently coprecipitated by any of the three antibodies when solubilized in NP-40. Sera, from patients with pernicious anemia, containing anti-parietal cell antibodies could also precipitate these two groups of antigens. Competition assay and physicochemical studies showed that the epitopes recognized by the three monoclonal antibodies are different.

Anemia, Pernicious↗
PubMed1989

Degradation of neurofilament protein in cerebral ischemia.

Degradation of neurofilament (NF) triplet proteins: NF200 (molecular weight (MW) 200,000), NF150 (MW 150,000), and NF68 (MW 68,000) as well as of other cytoskeletal proteins in the rat brain during ischemia was investigated. Sodium dodecyl sulfate-gel electrophoresis and immunoblot methods with anti-NF200 antibody were used for the study. Selective degradation of NF200 and NF150 was observed during the initial 10 to 15 minutes of ischemia. The degradation was demonstrated both in permanent ischemia caused by decapitation and in transient ischemia induced by four-vessel occlusion followed by reperfusion after 30 minutes of occlusion (modified Pulsinelli method). The degradation suggests that the activation of a protease occurs in the first 15 minutes of cerebral ischemia, which is the earliest irreversible neuronal change ever to be reported.

Animals↗
PubMed1989

Multiple forms of calpastatin in pig brain.

Pig brain was found to contain two calpain-specific, heat-stable inhibitory fractions which could be separated by DEAE-cellulose chromatography. CS-0.1, which was eluted from the column at 0.1 M NaCl, was identified as an ordinary, well-known calpastatin. CS-0.2, eluted at 0.2 M NaCl, was different from CS-0.1 in that it inhibited calpain 1 more strongly than calpain II and that it did not cross-react with anti-calpastatin antibodies. Partial purification indicated that CS-0.2 contained inhibitor proteins smaller than ordinary calpastatin, but whether they are the products derived from CS-0.1 or entirely different genetic products has not yet been determined.

Animals↗
PubMed1989

[Analysis of VH genes which encode the variable region of monoclonal antibodies directed to cancer-associated carbohydrate antigens].

Immune responses against cancer-associated, carbohydrate antigens are investigated by studying idiotypic determinants of specific antibodies with monoclonal anti-idiotypic antibodies, and by analyzing the structure of VH genes which encode the V region of the anti-carbohydrate antibodies. Four syngenic anti-idiotypic antibodies towards monoclonal antibodies which are specific to the sialyl Lewis A antigen and two kinds of SSEA-1 related antigens (sialyl SSEA-1 and fucosul SSEA-1) were obtained. Antibodies directed to carbohydrate antigens were mostly of IgM isotype, indicating these antigens are T-independent antigens, while anti-idiotypic monoclonal antibodies directed to those antibodies were mostly of IgG isotype, suggesting that T cells participate actively in the anti-idiotypic response. The Northern blotting analysis of VH genes of monoclonal antibodies directed to negatively-charged carbohydrate antigens such as gangliosides or sulfated glycolipids expressed the VH gene family J558 (group 1), followed by J606 (group 6) and Q52 (group 2) families. On the other hand, monoclonal antibodies directed to SSEA-1 related neutral carbohydrate antigens expressed VH genes of a minor family such as X24 (group 4), V31 (group 9), or 7183 (group 5). The same VH family as expressed in anti-SSEA-1 antibody (x24) was also expressed in the antibodies such as anti-i and anti-I antibodies, which are directed to the synthetic precursors of the SSEA-1 antigen. In either case, the antibodies directed to one particular carbohydrate antigen tended to express the VH gene of one particular family exclusively. This suggests idiotypic homogeneity of the anti-carbohydrate antibodies.

Animals↗
PubMed1989

[Clinical application of cancer-associated carbohydrate antigens in the diagnosis and therapy of human cancers].

Cell surface carbohydrates are important cancer-associated antigens. The carbohydrate antigens which have been proved to be useful for the serum diagnosis of human cancers are classified from their biochemical primary structure into three categories; the type 1 chain antigens, type 2 chain antigens, and core structure-associated antigens. The SPan-1 antigen, the newly-introduced tumor marker for human pancreas cancers, seems to be a type 1 chain antigen, judging from its distribution pattern among human cancers and its good correlation with other type 1 chain antigens. Another newly-introduced tumor marker, the CA54/61 antigen, which is specific for ovarian cancers, seems to belong to the family of the core-structure-associated antigens, since both antibodies (MA54 and MA61) used for the detection of the CA54/61 antigen are shown to recognize the antigen molecule which is closely related to sialyl Tn antigen, the well-known carbohydrate core structure of mucins. There are two approaches for the therapy of human cancers employing carbohydrate antigens as target molecules. One is the direct application of the specific monoclonal antibodies (alone, or in the drug-conjugated forms) in cancer patients. In this case, special attention is paid to the use of murine antibodies in human patients. The other approach is to induce specifically an effective immune response against the cancer-associated carbohydrate antigens. For this approach, further methodological development is necessary to determine how to obtain an effective immune response to carbohydrate antigens, since they have long been known as very poor immunogens which are T-independent.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1989

[Monoclonal antibodies directed to human lung cancer].

Recently many monoclonal antibodies are applied in the clinical diagnosis of human cancers. Most of the antigens recognized by these monoclonal antibodies are carbohydrate in their chemical nature. Among these carbohydrate antigens, the sialyl SSEA-1 antigen is particularly useful for the diagnosis of lung cancers, as the elevated serum level of the sialyl SSEA-1 antigen is frequently observed in patients with adenocarcinoma of the lung (ca. 45-75%). Some of the other carbohydrate antigens, such as CA19-9, are also known to be useful for the diagnosis of lung cancers. These carbohydrate antigens are preferentially found in the sera of patients with adenocarcinoma of the lung, while the SCC antigen (TA-4) is closely associated with squamous cell carcinoma of the lung, and NSE (neuron-specific enolase) is frequently detected in the sera of patients with small cell carcinoma of the lung. The specific serum diagnosis of lung cancer of each histological type is now feasible because of the development of these appropriate tumor markers.

Animals↗
PubMed1988

Stage-specific expression of SSEA-1-related antigens in the developing lung of human embryos and its relation to the distribution of these antigens in lung cancers.

The localization of three carbohydrate antigens, Lex, Ley, and sialylated Lex-i, which are closely related to stage-specific embryonic antigen 1, in the lung of developing human embryos was investigated using specific monoclonal antibodies. In the 38-day-old embryo, when the primitive lung bud has appeared and developed into two lung sacs, only Ley antigen was specifically positive in the proliferating cells in the terminal portion of lung bud. In the 50-53-day-old embryos, the future bronchi were actively developing from the bronchial buds. At this stage, the Ley antigen was maximally expressed and the Lex antigen appeared in the bud cells. In the lung of the 12-week-old embryo, buds for the future bronchioles were lined by simple cuboidal epithelial cells, which were strongly positive for Lex antigen, weakly positive for Ley antigen, and still completely negative for sialylated Lex-i antigen. Sialylated Lex-i antigen finally appeared in 18-week-old embryos, in the cells of the terminal buds for the future alveoli. At this stage, the Lex and Ley antigens were already beginning to disappear and were only weakly positive in cells of terminal buds. At 20 weeks, only sialylated Lex-i antigen was weakly detected in the cells in the terminal buds; after 8 months, all three antigens were essentially not detected in the respiratory cells in most of the embryos examined in this study. Formation of bronchial glands was detected at 18 weeks, where the developing gland cells were specifically positive for sialylated Lex-i antigen. Ciliation of the bronchial epithelial cells started at 12 weeks and propagated thereafter. The ciliation was accompanied by the reappearance of Ley and Lex antigen in the epithelial cells. These findings collectively indicated that the three antigens all have a physiological significance as stage-specific developmental antigens of the human lung; those antigens were specifically present in the bud cells at each important step of the morphogenesis of the human lung, such as cells in the lung buds, bronchial buds, and terminal buds for the formation of the alveolus, and cells differentiating into bronchial gland cells. The three antigens gradually disappear in the later stage of development along with the maturation process of the lung. Stage-specific embryonic antigen 1 and related antigens are known to be associated with various human cancers, including lung cancers. We suggest that the expression of these antigens in the lung cancer cells is the result of the retrodifferentiation of the cancer cells to the stages of immature embryonic lung cells.

Adenocarcinoma↗
PubMed1988

Monoclonal antibodies directed to a disulfated glycosphingolipid, SB1a (GgOse4Cer-II3IV3-bis-sulfate), associated with human hepatocellular carcinoma.

Two murine monoclonal antibodies, 2H6G5 (IgM) and 4A9E10 (IgG3), were obtained by using either cultured human hepatocellular carcinoma cells (PLC/PRF/5) or the acidic glycolipid mixture prepared from the same cells as immunogens. The antigen in PLC/PRF/5 cell membranes recognized by both antibodies was identified as a disulfated acidic glycolipid, GgOse4Cer-II3IV3-bis-sulfate (SB1a). Both antibodies reacted specifically with SB1a, and no significant reactivity was noted with other sulfated glycolipids or gangliosides except that the antibody 2H6G5 showed a weak cross-reactivity with LacCer-II3-sulfate (SM3), another sulfated glycolipid which partly shares the same carbohydrate structure as SB1a. The SB1a antigen is a relatively minor glycolipid in PLC/PRF/5 cells, but it was strongly expressed at the surface of PLC/PRF/5 cells as ascertained by cytofluorometry using both antibodies. A significant amount of SB1a antigen was present in 3 of the acidic glycolipid fractions isolated from 15 human hepatocellular carcinoma tissues as well as in the acidic glycolipid fraction prepared from PLC/PRF/5 cells, while all the acidic glycolipid fractions prepared from cirrhotic livers and a normal liver were essentially negative for SB1a, as ascertained by both solid phase enzyme immunoassay and the thin-layer chromatography-immunostaining method. These results strongly suggest that the SB1a antigen as defined by these new monoclonal antibodies is associated with human hepatocellular carcinoma.

Animals↗
PubMed1988

Generation of two murine monoclonal antibodies that can discriminate N-glycolyl and N-acetyl neuraminic acid residues of GM2 gangliosides.

Since the preliminary analyses of the glycolipids of small cell carcinomas of the lung showed an increase of GM2 ganglioside, we generated new murine monoclonal antibodies directed to GM2 to identify the molecular species of the glycolipid. The monoclonal antibodies MK2-34 and MK1-16 (both IgM), which specifically detect N-glycolyl GM2 and N-acetyl GM2, respectively, were generated by immunizing mice with liposomes containing monophosphoryl lipid A, trehalose dimycolate, and the antigenic ganglioside. Among the glycolipid preparations extracted from the cancer tissues of 39 patients with lung cancer, a significant amount of N-acetyl GM2 was detected with MK1-16 antibody in 70% of the squamous cell carcinoma cases, 50% of the lung adenocarcinoma cases, 33% of the large cell carcinoma cases, and 100% of the cases of small cell carcinoma of the lung. On the other hand, N-glycolyl GM2 which was defined by the monoclonal MK2-34 was not found in any of the glycolipid fractions prepared from the lung cancer tissues examined in this study. Immunohistochemical studies of the lung cancer tissues with the MK1-16 antibody showed that the N-acetyl GM2 was present not only in small cell carcinoma tissues as one of the antigens related to tumors of neuroectodermal origin, but also in the squamous cell carcinoma and adenocarcinoma of the lung with a comparable frequency. The appearance of the N-acetyl GM2 antigen correlated well with the degree of differentiation of the cancer cells in patients with squamous cell carcinoma and adenocarcinoma of the lung.

Animals↗
PubMed1988

1H-n.m.r. analysis of type-2 chain lacto-gangliosides. Confirmation of structure of a novel cancer-associated fucoganglioside, alpha-NeuAc-(2----6)- beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-[alp ha-L- Fucp-(1----3)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta -D-Glc p- (1----1)-Cer (VI6NeuAcIII3FucnLc6Cer).

Neolacto-glycosphingolipids, substituted with alpha-NeuAc-(2----3)- and -(2----6)-linked D-Galp residues were analyzed by one- and two-dimensional 1H-n.m.r. spectroscopy at 500 MHz in 49:1 (v/v) di(2H3)methyl sulfoxide-deuterium oxide solution. For the simplest structures analyzed, nLc4Cer, IV3NeuAcnLc4Cer, and IV6NeuAcnLc4Cer, sialosylation-induced changes in shifts of terminal and subterminal core residues were interpretable in terms of existing conformational models. Chemical shifts for H-3e and H-3a of NeuAc characteristic for the type of linkage, were also determined. In addition, regularly reproducible shifts were seen for H-1 and other resonances of terminal and subterminal core residues of all structures tested. Chemical-shift correlations proved to be useful in elucidating the structure of a unique ganglioside bearing an internal beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1---- 3) residue ("X-trisaccharide") with an alpha-NeuAc-(2----6)-substituted terminal group.

Carbohydrate Sequence↗
PubMed1988

Quantitative and qualitative characterization of human cancer-associated serum glycoprotein antigens expressing epitopes consisting of sialyl or sialyl-fucosyl type 1 chain.

The levels of carbohydrate antigens having epitopes consisting of type 1 chain (R----Gal beta 1----GlcNAc beta 1----3Gal beta 1----R) in the sera of patients with various malignant and nonmalignant disorders have been investigated with the use of three monoclonal antibodies, N-19-9, FH-7, and FH-9. Serum levels of 2----3 sialylated Lea antigen and 2----6 sialylated Lea antigen, defined respectively by antibodies N-19-9 and FH-7, were found to be frequently high in patients with cancer of the digestive system, particularly pancreatic cancer. High levels of 2----3,2----6 disialylated Lc4 antigen, defined by antibody FH-9, were less frequent in cancer patients when compared with the other two antigens. In patients with nonmalignant disorders, especially renal and autoimmune diseases, serum levels of the two type 1 chain antigens defined by FH-7 and FH-9 were more frequently high than that defined by N-19-9. Molecular weights and other general biochemical characteristics of serum mucin carrying the type 1 chain determinants were not significantly different in cancer patients as compared with patients with nonmalignant disorders. However, the degree of glycosylation of the antigen, as assessed by its solubility in perchloric acid, showed significant differences; i.e., the mucin antigen carrying 2----6 sialylated Lea determinant in the sera of patients with nonmalignant disorders had the highest carbohydrate/protein ratio, followed by the mucin carrying the same determinant in the sera of cancer patients. Mucin antigen carrying 2----3 sialylated Lea antigen or 2----3, 2----6 disialylated Lc4 antigen in cancer patients had the lowest carbohydrate/protein ratio among the four groups tested. Thus, the carbohydrate/protein ratio in the type 1 chain mucin antigens in sera of normal subjects is higher than that in sera of cancer patients (P less than 0.05). This finding is in contrast to previous findings on the mucin antigens carrying the type 2 chain determinant (R. Kannagi et al., Cancer Res., 46: 2619-2626, 1986), in which the mucin antigen in cancer patients was found to have a much higher carbohydrate/protein ratio than that carrying the same antigenic determinants in patients with nonmalignant disorders.

Antibodies, Monoclonal↗
PubMed1988

Pig heart calpastatin: identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis.

Isolation and nucleotide sequencing of the complementary DNA for pig heart calpastatin have been completed. The amino acid sequence of 713 residues predicted from the nucleotide sequence contains five domains, each composed of approximately 140 amino acid residues. A unique N-terminal domain is followed by four mutually homologous domains. The best fit alignment of these four domains gives residue identities between any two domains of 22.5-36.0%. The analysis of the sequence similarities by several methods also suggests the existence of additional shorter repeats at intervals of 60-80 residues. The calculated molecular weight of pig calpastatin of 713 amino acid residues (Mr 77,122) is significantly lower than the value of purified pig heart calpastatin (Mr 107,000) estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The expression of the calpastatin genes in Escherichia coli and the detection of the translation products of 713, 366, and 140 amino acid residues by the specific anti-calpastatin antibody indicate that the products always migrate considerably slow on SDS-PAGE, giving an average of 1.53 for the ratio of the molecular weight estimated by SDS-PAGE to the value calculated from the amino acid sequences. It is most likely that the discrepancy in the molecular weight is caused by an anomalous behavior of calpastatin in SDS-PAGE.

Amino Acid Sequence↗
PubMed1988

Fucosylated type-2 chain polylactosamine antigens in human lung cancer.

Four specific monoclonal antibodies (MAbs) have been used to study distributions of fucosylated type-2 chain polylactosamine antigens, Lex, poly Lex, Ley and sialylated Lex-i antigens, in human lung cancer tissues and in the serum of patients with lung cancers. Radioimmunoassay frequently showed abnormally high antigen levels in the sera of 66 lung cancer patients tested. When histological typing was performed, high serum levels of the above 4 antigens were most frequently observed in patients with adenocarcinoma of the lung; i.e., after combining the results from the 4 antigens, 75% of the sera from patients with lung adenocarcinoma were positive (50% in the case of large-cell carcinoma, 30% in the case of squamous-cell carcinoma and 27% for small-cell carcinoma). Among the 4 antigens, the sialylated Lex-i antigen had the highest positive incidence, 58%, in the sera of patients with adenocarcinoma of the lung, compared to 33% for Ley, 29% for poly Lex, and 8% for Lex antigen. On the other hand, when the distributions of these antigens in the lung cancer tissues of 42 patients were studied by immunohistological techniques, the Ley antigen had the highest positive incidence, 100%, in lung adenocarcinoma tissues, poly Lex antigen had 86%, sialylated Lex-i antigen had 71%, and Lex antigen had 29%. In cancer tissues, the incidence of non-sialylated antigens, such as Ley, poly Lex and Lex antigens, often exceeds the positive incidence of the sialylated antigen, but the sialylated form of the antigen, such as sialylated Lex-i antigen, appears more often than the non-sialylated form in patients' sera.

Adenocarcinoma↗
PubMed1988

Significance of 2-3 and 2-6 sialylation of Lewis a antigen in pancreas cancer.

Distributions of sialylated derivatives of Lewis a (Lea) antigen in cancer tissue of the human pancreas and in the sera of patients with pancreas diseases have been studied; the significance of 2-3 and 2-6 sialylation of Lea antigens in pancreas cancer have been investigated using specific monoclonal antibodies. In most pancreas cancer tissue the 2-3 sialylated Lea antigen was found to be specifically distributed in cancer cells as determined by immunohistologic techniques, while a significantly smaller amount of the antigen was detected in surrounding nonmalignant pancreas tissue, which was infiltrated by cancer cells. Conversely, the 2-6 sialylated Lea antigen was abundantly present in nonmalignant pancreas tissue, while it was less frequently present in pancreas cancer cells. When the sera of 66 patients with pancreas diseases were examined for these sialylated Lea antigens, correlation studies showed that the levels of 2-3 sialylated Lea tended to be 44.1 times more than the levels of 2-6 sialylated Lea in the sera of cancer patients. The average ratio of 2-3 sialylated Lea to 2-6 sialylated Lea was 0.23 in the sera of patients with pancreatitis. These data collectively indicate that the 2-3 sialylation of Lea is remarkably enhanced, and the 2-6 sialylation of Lea antigen is suppressed in pancreas cancer. The determination of the 2-3 sialylated Lea to 2-6 sialylated Lea ratio in patients with pancreas diseases may be helpful for the differential diagnosis of pancreas cancer and nonmalignant pancreatic disorders.

Antibodies, Monoclonal↗
PubMed1988

Glycolipid-directed FH6 monoclonal antibody recognizes high molecular weight glycoprotein antigen carrying sialyl Lex-i determinant in the culture supernatant of PC-9 cells.

The properties of the antigen recognized by monoclonal antibody FH6 have been analyzed. FH6 was originally generated against a glycolipid, i.e. a difucoganglioside isolated from human colonic adenocarcinoma, and specifically reacts with sialyl Lex-i determinant. Several culture supernatants of human carcinoma cell line cells were found to have high levels of FH6-reactive antigen, and PC-9, a human lung carcinoma cell line was used for the analysis. A solid-phase sandwich radioimmunoassay was performed to detect the antigen. The antigenic activity was extractable in 0.6 M PCA or 7% TCA, and was sensitive to mild alkaline treatment and to Pronase digestion. Most of the antigen was eluted in the void volume of a Sepharose CL-2B column, which indicates that its molecular weight is greater than several million. It was eluted from a DEAE-cellulose column at a NaCl concentration in the range of 0.2-0.25 M. The immunoaffinity-purified antigen has a high carbohydrate content of more than 80%. These data indicate that the antigen recognized by FH6 in the culture supernatant of PC-9 is not a glycolipid, but a high molecular weight glycoprotein which could be referred to as a mucin, or a proteoglycan, which contains keratan-sulfate like glycosaminoglycan chains, as judged from the results of the glycosidase treatments.

Antibodies, Monoclonal↗
PubMed1988

Calpain activates two transglutaminases from porcine skin.

Two transglutaminases (TGase) with estimated molecular weight of 55,000 (55-K TGase) and 120,000 (120-K TGase) were partially purified from the cytosolic fraction of porcine skin (epidermis-rich preparation) using DEAE-cellulose and gel-filtration chromatographies. The enzyme activities of both transglutaminases were enhanced more than 20-fold by treatment with calpain (Ca2+-dependent cysteine proteinase) in the presence of Ca2+, and this enhancement was inhibited by adding EDTA, leupeptin, or an endogenous calpain-specific inhibitor protein (calpastatin). 55-K TGase was effectively activated by a smaller amount of calpain than was 120-K TGAse, while known activating reagents such as thrombin and dimethyl sulfoxide or heat treatment preferentially activated 120-K TGase. One of the physiological functions of calpain in the epidermis may be the activation of epidermal transglutaminases.

Animals↗