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R Kannagi

Publications and source records attributed to R Kannagi.

At least 109 records · Page 6Linked to original sources
PubMed1991

Calcium-induced intracellular cross-linking of lipocortin I by tissue transglutaminase in A431 cells. Augmentation by membrane phospholipids.

Covalently cross-linked multimers of lipocortin I are shown to be present in human epidermoid carcinoma A431 cells treated with epidermal growth factor or the calcium ionophore A23187. This intracellular cross-linking of lipocortin I is suggested to be mediated by the action of tissue transglutaminase, a Ca2(+)-dependent protein cross-linking enzyme. Cross-linking of lipocortin I competes with proteolytic digestion of the protein, and pretreatment of the cells with inhibitors for calpain (Ca2(+)-dependent intracellular protease) markedly enhanced the cross-linking of lipocortin I. Cross-linked lipocortin I is shown to be present in the soluble fraction of A431 cells as well as in the particulate fraction; a 34-kDa fragment of lipocortin I was solubilized successfully by plasmin digestion of the latter fraction. Immunofluorescence microscopy using specific antilipocortin-I antibody showed that cross-linked lipocortin I forms an envelope-like structure, which is not extracted with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) or Triton X-100. In vitro incubation of purified lipocortin I with tissue transglutaminase resulted in the formation of covalently cross-linked lipocortin I dimer, tetramer, and so on. Amine incorporation and cross-linking studies using lipocortin I and its N-terminal truncated derivatives indicated that the cross-linking site is localized within the plasmin-susceptible N-terminal 29 amino acids of lipocortin I. The cross-linking of lipocortin I is shown to be accelerated more than 10 times by the addition of phosphatidylserine vesicles, on which lipocortin I molecules are most likely aligned in a conformation suitable for cross-linking. Collectively, these findings suggest that an increase of intracellular calcium concentration results in the attachment of lipocortin I onto the plasma membrane phospholipids through the C-terminal domain of the molecule where the membrane-bound lipocortin I is cross-linked by the action of tissue transglutaminase through the N-terminal domain.

Annexins↗
PubMed1991

Genetic studies on experimental autoimmune gastritis induced by neonatal thymectomy using recombinant inbred strains between a high-incidence strain, BALB/c, and a low-incidence strain, DBA/2.

Thymectomy on day 3 after birth induced autoimmune gastritis (AIG) at the age of 2 months in 51-73% of BALB/c mice, and in only 3-5% of DBA/2 mice. AIG was detected by histological and serological (immunofluorescence staining for detecting anti-parietal cell autoantibody) examination. However, autoantibody was weakly positive in almost all of these DBA/2 mice when measured by ELISA using extract of murine gastric mucosa as the antigen. To investigate genetically the mechanism controlling the incidence of AIG, II recombinant inbred strains established by brother-sister mating of (BALB/c x DBA/2) F2 mice (C x D2 strains) were used. Among 26 markers tested, the Mls-1 locus on BALB/c chromosome 1 and the Hc locus coding a complement component (C5) on BALB/c chromosome 2 were found to be associated with high susceptibility to AIG. However, if one or both of the loci were of DBA/2 origin, mice showed medium or low susceptibility to AIG. For further analysis, F1, F2 and back-cross generations of these two strains were tested, but segregation of a single susceptibility or insusceptibility gene was not obtained. Taken together, it seems probable that two or more genes are involved in the induction mechanism of AIG. We did not detect C5 deposition in AIG lesions, nor complement-dependent cytotoxic antibody to parietal cells in serum from AIG mice. However, injection of irradiated spleen cells of DBA/2 mice into BALB/c mice thymectomized on day 3 augmented the incidence of AIG from 71 to 100%, but not that of oophoritis (33%). A relationship between Mls-1a determinants and the pathogenesis of AIG was further suggested from the fact that V beta 6 TcR-expressing T cells increased in number in AIG-bearing compared with normal BALB/c mice.

Animals↗
PubMed1991

[Carbohydrate auto-antigens in auto-immune hemolytic anemia].

Anti-erythrocyte antibodies which appear in the sera of patients with auto-immune hemolytic anemia frequently recognize carbohydrate auto-antigens. Most of cold agglutinins are known to recognize the Ii-antigens, and Donath-Landsteiner antibodies which appear in patients with paroxismal cold hemoglobinuria are almost exclusively directed to the P-antigen. These carbohydrate auto-antigens are strongly expressed in various tissues and organs other than erythrocytes, and behave as differentiation- or developmental-antigens, in both humans and mice. The study of nucleotide sequences of human and murine anti-Ii antibodies shows that these antibodies share a highly homologous antigen-binding site in their VH regions. These results indicate that the carbohydrate auto-antigens in autoimmune hemolytic anemia are evolutionally conserved developmental antigens, and suggest that the immunoglobulin genes which encode variable regions of the auto-antibodies directed to these antigens are also conserved evolutionally.

Anemia, Hemolytic, Autoimmune↗
PubMed1990

Gangliosides and sialoglycoproteins carrying a rare blood group antigen determinant, Cad, associated with human cancers as detected by specific monoclonal antibodies.

Two murine monoclonal antibodies, 2A3D2 and 2D11E2 (both IgM), which are directed to the gangliosides and sialoglycoproteins related to a rare blood group antigen, Cad, were obtained by using a ganglioside mixture prepared from human hepatocellular carcinoma cells (PLC/PRF/5) as the immunogen. These two monoclonal antibodies detected multiple ganglioside antigens present in the PLC/PRF/5 cells, and the major antigenic ganglioside was characterized as IV4GalNAc beta-GD1a, which has the carbohydrate structure GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3GalNAc beta 1---- 4(NeuAc alpha 2----3)Gal beta 1----Cer. The two antibodies also reacted with GM2 (GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer) and a Cad-active lactoseries ganglioside (IV4GalNAc beta-sialosylparagloboside, GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4GlcNAc beta 1---- 3Gal beta 1----4Glc beta 1----Cer), which have carbohydrate structures related to IV4GalNAc beta-GD1a. Beside gangliosides, both antibodies recognized the carbohydrate determinant carried by glycophorin A on very rare Cad-positive human RBC; the structure of which is GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3(NeuAc alpha 2---- 6)GalNAc alpha 1----Ser/Thr. From these findings, it is clear that monoclonal antibodies 2A3D2 and 2D11E2 both recognize the nonreduced carbohydrate terminus composed of three sugar residues, GalNac beta 1----4(NeuAc alpha 2----3)Gal beta 1----R, and are useful for detecting the Cad-related antigen in cells and tissues. By using these monoclonal antibodies, it was revealed that many cultured human hepatocellular carcinoma cell lines and cancer tissues taken from patients with hepatocellular carcinoma contain both Cad-active glycoprotein antigens and related gangliosides, while normal liver tissues contain no appreciable amount of either species of antigen. The Cad-active glycoprotein antigens in cultured human hepatocellular carcinoma cells appeared as triplet bands having molecular weights of 92,000, 75,000, and 61,000, under either reducing or nonreducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Essentially the same triplet proteins were observed in as many as 4 of 9 cases (44%) of cancer tissue from patients with hepatocellular carcinoma, but not in neighboring cirrhotic tissues or normal livers tissues. These results suggest that the rare blood group antigen Cad is associated with human cancers, especially hepatocellular carcinoma.

Animals↗
PubMed1990

Northern hybridization analysis of VH gene expression in murine monoclonal antibodies directed to cancer-associated ganglioside antigens having various sialic acid linkages.

The expression of the VH genes in 46 murine hybridoma cells that secrete mAb directed to the cancer-associated carbohydrate Ag, especially acidic glycolipids such as gangliosides and sulfated glycoplipids, was analyzed by Northern hybridization of poly(A)+ RNA of hybridoma with cDNA probes for nine VH gene families. Different hybridomas tended to express VH genes of the same family when the cognate Ag had the same or similar carbohydrate structures; i.e., the VH genes of the J558 family (group 1) were preferentially expressed in the mAb directed to various gangliosides that have NeuAc alpha (or NeuGc alpha) 2-3 and/or 2-8 linkage (71%), the most common linkage of sialic acid residues in the gangliosides of higher animals, and the hybridomas directed to sulfated glycolipids also expressed mainly the VH genes of the J558 family (80%). In contrast, the five mAb directed to various gangliosides with NeuAc alpha 2-6 linkage were exclusively encoded by the VH genes of Q52 family (group 2, 100%), and three antibodies directed to gangliosides with a NeuAc alpha 2-9 linkage all expressed genes of J606 family (group 6, 100%). The VH family usage was largely correlated with the linkage of sialic acid residues in the cognate carbohydrate Ag, but was not correlated at all with the difference in the fine specificities toward the core neutral carbohydrate chain, to which the sialic acid residues were attached. These findings suggest that the VH gene family in these anticarbohydrate antibodies is selected, depending primarily on the linkage of the sialic acid residues in carbohydrate Ag; these residues form the immunodominant sugar residue in the respective antigenic determinant.

Animals↗
PubMed1990

Accumulation of highly acidic sulfated glycosphingolipids in human hepatocellular carcinoma defined by a series of monoclonal antibodies.

An accumulation of sulfated and very complex, highly acidic glycolipids was observed in cultured human hepatocellular carcinoma cells. Among the cells tested, PLC/PRF/5 cells contained a significant amount of very complex sulfated acidic glycolipids, and HepG2 cells were characterized as having a large amount of relatively simple sulfated glycolipids. Several monoclonal antibodies (all IgM) directed to these sulfated and highly acidic glycolipids were established. Among them, 49-D6 and 7-E10 were both directed to SM3 (LacCer-II3-sulfate), a relatively simple sulfated glycolipid, and 34-A4 was directed to SD1a (GgOse4Cer II3,IV3-disulfate) and more complex sulfated glycolipids. The other four antibodies, 26-A10, 34-B9, 79-C8, and 16-E10, reacted with unknown highly acidic glycolipids, which were eluted in 0.9-2.7 M ammonium acetate in DEAE chromatography, indicating that these antigenic glycolipids were far more acidic than the usual glycolipids described until now. Analysis of the glycolipids extracted from the hepatocellular carcinoma tissues and cirrhotic livers of patients and from a normal liver with these monoclonal antibodies revealed that sulfated glycolipids having simple carbohydrate structures such as SM3 accumulated significantly in the cirrhotic liver (2 of 4 cases) as well as hepatocellular carcinoma tissue (15 of 17 cases, 88%), and more complex sulfated glycolipids and highly acidic glycolipids were much more specific to hepatocellular carcinoma tissues (10 of 17 cases, 59%) compared to the cirrhotic liver (0 of 4 cases).

Antibodies, Monoclonal↗
PubMed1990

The abnormal occurrence and the differentiation-dependent distribution of N-acetyl and N-glycolyl species of the ganglioside GM2 in human germ cell tumors. A study with specific monoclonal antibodies.

Human primary germ cell tumors were analyzed for the presence of the ganglioside GM2 using three specific monoclonal antibodies which can distinguish the molecular species of the sialic acid moiety: the antibody MK1-16 is specific for N-acetyl GM2, MK2-34 is specific for N-glycolyl GM2, and MK1-17 detects both N-acetyl and N-glycolyl GM2. When the occurrence of the GM2 antigen was tested in 107 cases of human germ cell tumors by the immunohistochemical technique using these antibodies, seminoma was characterized as having the highest frequency of N-acetyl GM2 (89.4%, 42 of 47 cases) among germ cell tumors, followed by embryonal carcinoma (40.0%), and teratocarcinoma (26.6%). Compared with this, yolk sac tumors and choriocarcinoma had a much lower positive incidence of the N-acetyl GM2 antigen. On the other hand, the N-glycolyl GM2 antigen was not found at all in 47 cases of seminoma (0%), and the positive incidence was very low in embryonal carcinoma (6.6%), although considerably higher incidences were obtained with choriocarcinoma (25.0%), yolk sac tumor (22.2%), and teratocarcinoma (13.3%). The presence and molecular species of the GM2 antigens in these human germ cell tumors were also ascertained chemically by the thin-layer chromatography (TLC) immunostaining of the ganglioside fractions prepared from primary germ cell tumors. These results indicate that seminoma specifically contains N-acetyl GM2 and no N-glycolyl GM2, suggesting that N-acetyl GM2 could be a good marker for seminoma. On the other hand, non-seminomatous germ cell tumors were characterized by the presence of N-glycolyl GM2, one of the Hanganutziu-Deicher antigens (H-D antigens). Moreover, the positive occurrence of N-glycolyl GM2 correlated very well with the degree of differentiation of non-seminomatous germ cell tumors, i.e., the differentiated tumors such as yolk sac tumors, choriocarcinoma, and teratocarcinoma had a higher positive incidence of N-glycolyl GM2 type H-D antigen but a lower positive incidence of N-acetyl GM2 when compared with embryonal carcinoma, the most undifferentiated tumors among non-seminomatous germ cell tumors.

Antibodies, Monoclonal↗
PubMed1990

Production of monoclonal antibody to human esophageal cancer cell line.

The immunization of Balb/C mice with esophageal cell line KYSE-50 established from poorly-differentiated esophageal squamous cell carcinoma, resulted in obtaining the monoclonal antibody KYMN-28-5. This monoclonal antibody is of the IgM class and recognizes a carbohydrate antigen contained in glycoproteins with molecular weights of 53 and 56K, and in neutral glycolipids extracted from teratomas. Tissue staining revealed that this monoclonal antibody reacts strongly with malignant tumors but only weakly, or not at all, with normal tissues, apart from squamous epithelial tissue. KYMN-28-5 is thus a useful tumor marker which will improved the accuracy of serological diagnosing squamous cell carcinoma when combined with the measurement of SCC antigen.

Animals↗
PubMed1990

[Immunoglobulin genes encoding antibodies directed to oncodevelopmental carbohydrate antigens].

We investigated the immunoglobulin genes which encode the variable region of the monoclonal antibodies directed to the onco-developmental carbohydrate antigens such SSEA-1, fucosyl SSEA-1, SSEA-3 and SSEA-4. The VH region of these antibodies was preferentially encoded by the gene members of the X24, VH7183 and Q52 families, the families which are known to be located at the 3'-end region of the murine germ line VH gene. This result is interesting particularly when considering that the members of the 3'-end VH families are known to be preferentially expressed in embryonic B lymphocytes by an intrinsic genetic program. The comparative study of the nucleic acid sequences of mRNAs encoding these antibodies and the sequences of the corresponding germ line VH genes disclosed that the sequences encoding the antibodies contain no mutation from the germ line VH genes, or contain only a few somatic mutations, which are thought to be insignificant for the reactivity of the antibodies to the nominal antigens. These results imply that some of the embryonic B lymphocytes that express the unmutated germ line VH genes of the 3'-end families can be reactive with embryonic carbohydrate antigens, albeit rearranged with appropriate D-JH gene segments, and coupled with proper light chains. The VH region of the syngenic monoclonal anti-idiotypic antibodies directed to these anti-carbohydrate antibodies were also encoded preferentially by the members of the 3'-end VH families. We propose here that a part of the virgin embryonic B lymphocytes, which express the antibody encoded by the gene members of the 3'-end VH families at the cell surface, will be stimulated by the embryonic carbohydrate antigens which are abundantly present in the internal milieu of the embryo. The clonally expanded B lymphocytes, in turn, will facilitate the proliferation of other populations of embryonic B lymphocytes expressing the corresponding anti-idiotypic antibodies, which are also encoded by the gene members of the 3'-end VH families. This process will lead to the formation of the primitive immune idiotype network system directed to the embryonic carbohydrate auto-antigens in the embryo, which is rarely exposed to the external antigens.

Animals↗
PubMed1990

Gene expression for calpain isozymes in human hematopoietic system cells.

Calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine endopeptidase) is known to exist in two forms of isozyme. Calpain I requires low (or microM)-Ca2+ for activation and calpain II requires high (or mM)-Ca2+. Both isozymes consist of one heavy (approx.80 kDa) and one light (approx. 30 kDa) subunit each. The heavy subunits of isozymes I and II are different genetic products, while the light subunits are identical. Antibodies respectively specific for the heavy subunits of pig calpains I and II were raised in rabbits, and the affinity-purified IgG proteins were used for Western blot analysis. When 23 human hematopoietic system cells were examined for the degree of their expression of the genes for calpains I and II, all of them were found to contain calpain I of detectable amounts in their cytosolic fluid. By contrast, only nine cell-line cells were positive in calpain II, and they were, without exception, the lineage which had been infected with HTLV-I, the retrovirus responsible for human adult T-cell leukemia. The enhanced production of calpain II in HTLV-I infected T-cells was also confirmed by running chromatographic analyses on the homogenates of these cells, and comparing them with those of uninfected T-cells. When YT-C3 cell, which is an uninfected, natural killer-like cell, was transfected with HTLV-I gene, the resulting transformed stable cells, YT-4 and YT-5.1, were found to produce increased amounts of calpain II concomitant with that of interleukin (IL)-2 receptor protein. These results suggest that the gene expression for calpain isozymes may vary during the course of differentiation of T-lymphocytes. The mechanism of regulation of calpain isozyme genes and the biological significance of the variation in expression during differentiation still remain unanswered.

Animals↗
PubMed1990

[Clinical reference values for laboratory hematology tests calculated using the iterative truncation method with correction: Part 1. Reference values for erythrocyte count, hemoglobin quantity, hematocrit and other erythrocyte parameters including MCV, MCH, MCHC and RDW].

Age and sex dependent differences in the clinical reference values for erythrocyte count (RBC), hemoglobin quantity (Hb), hematocrit (Ht) and other erythrocyte parameters including MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration) and RDW (red cell distribution width), were calculated by the iterative truncation method with correction (Usui's method) using the results from tests on 6,300 patients' specimens obtained at Kyoto University Hospital. For RBC, Hb and Ht, the data obtained from the individuals below 13 years old showed the normal or sometimes log-normal distribution, but adjustment by the Xn-type variable transformation was often necessary to obtain the normal distribution for the data taken from the populations containing individuals over the age of 14. For the clinical reference values of RBC, Hb and Ht, no sex difference was observed below the age of 12. The values for males were significantly higher than those of females in the age range 13-79, and the values showed no significant sex-dependent difference at ages above 80. In females, age-dependent change of values for RBC, Hb and Ht was less prominent than in males; especially the upper limit values for females were very stable for all ages. MCV and MCH gradually increased with age both in males and females, and the MCHC remained constant in all age populations of male and female. The reference value for RDW was generated by the percentile method instead of the iterative truncation method because of the strong deviation in the distribution pattern, and the RDW values showed a gradual increase with age in both males and females.

Age Factors↗
PubMed1989

Cross-linking of lipocortin I and enhancement of its Ca2+ sensitivity by tissue transglutaminase.

The stimulation of human epidermoid carcinoma A431 cells with the calcium ionophore A23187 resulted in the formation of high-molecular-weight lipocortins I, having apparent molecular weights of 75 kDa and 160 kDa as detected with specific anti-lipocortin I antibody. These immunoreactive proteins were identified to be covalently cross-linked multimers of lipocortin I, since essentially the same cross-linked multimers were observed when purified lipocortin I was incubated with tissue transglutaminase (TGase) in vitro. Classical amine substrates for TGase, such as dansylcadaverine and putrescine, were also incorporated stoichiometrically into lipocortin I. Cross-linking or amine incorporation was not observed with lipocortin II. Des 1-26 lipocortin I did not serve as a substrate for TGase, indicating that the N-terminal region of lipocortin I plays an important role in the formation of lipocortin I multimers. The cross-linking of lipocortin I by TGase resulted in a remarkable enhancement of calcium sensitivity for phospholipid binding; i.e., the free calcium concentration required for the cross-linked lipocortin I to attain 50% maximal binding to phosphatidylserine vesicles was as little as 3 microM, while that required for intact monomeric lipocortin I was 20 microM.

Annexins↗
PubMed1989

Enhancement of calcium sensitivity of lipocortin I in phospholipid binding induced by limited proteolysis and phosphorylation at the amino terminus as analyzed by phospholipid affinity column chromatography.

A phospholipid column was prepared by coating siliconized porous glass beads with phospholipids. The analysis of the Ca2+ requirement of lipocortin I and its derivatives in the binding to phospholipids was carried out with this column. The Ca2+ concentration required for 50% binding to the phospholipid column at room temperature was about 30 microM for lipocortin I, while that was reduced to 15 microM when lipocortin I was phosphorylated by the epidermal growth factor receptor/kinase, and a further reduction in the Ca2+ requirement was observed with proteolytic cleavage at the N-terminal region. Cathepsin D and calpain I (low calcium-requiring form of calcium-activated neutral protease) rapidly cleaved human placental lipocortin I at Trp-12 and Lys-26, respectively. These N-terminal-truncated proteins required only 5 microM Ca2+ for 50% binding to the phospholipid column. This enhancement of Ca2+ sensitivity by limited proteolysis was also observed for porcine lung lipocortin I. Essentially the same results were obtained when the Ca2+ sensitivities of the modified lipocortins I were analyzed using dispersed phospholipid vesicles instead of the phospholipid affinity column. Equilibrium dialysis indicated that the release of the N-terminal region markedly increased the affinity of lipocortin I for Ca2+ in the presence of phosphatidylserine, without any appreciable change of the number of Ca2+-binding sites. Limited proteolysis by endogenous proteases such as calpain may be an important regulatory mechanism for the Ca2+ sensitivity of lipocortin I in phospholipid binding.

Amino Acid Sequence↗