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Biomedical subjects

R Kannagi

Publications and source records attributed to R Kannagi.

At least 145 records · Page 8Linked to original sources
PubMed1988

Biochemical analysis of inhibitory effects of a lymphokine suppressive B-cell factor on the activation process of resting B cells.

Suppressive B-cell factor (SBF) is elaborated by FcR gamma (Fc receptor for IgG)-bearing small, resting B cells after the stimulation of immune complexes and is known to inhibit humoral immune responses by acting on resting B cells. In order to elucidate where and how SBF interferes with B-cell activation in the course of transmembrane signaling, we examined the effect of SBF on the several sequential events which B cells undergo after crosslinking surface immunoglobulin (sIg). Hyper-Ia expression, plasma membrane depolarization, and activation of phosphatidylinositol (PI) hydrolysis of resting B cells, all of which were induced by the stimulation with anti-mu antibody, were significantly suppressed by the pretreatment of cells with SBF. However, SBF had no effect on the intracytoplasmic cyclic AMP level of either activated or resting B cells. Another inhibitory effect of SBF on the activation process of resting B cells by anti-mu antibody was to suppress the transient elevation of intracytoplasmic free Ca2+ only in the initial phase after triggering with anti-mu antibody. This seems to be due to a decrease in the release of inositol triphosphate into the cytoplasm by suppressing the activation of PI hydrolysis. Considering all the data, the suppressive effect of SBF on the transmembrane signaling by sIg crosslinking is ascribed to the selective suppression of the activation of PI hydrolysis. This provides a concept on a molecular basis that feedback regulation of humoral immune response is, at least partly, regulated by SBF.

Animals↗
PubMed1988

Purification and characterization of calpains from pig epidermis and their action on epidermal keratin.

Two forms of Ca++-dependent cysteine proteinases, calpain I, requiring low Ca++ (microM concentration), and calpain II, requiring high Ca++ (mM concentration), were purified from the cytosolic fraction of pig epidermis. Calpains I and II were separated on DEAE-cellulose chromatography, and thereafter they were purified by separate but almost identical procedures, which included chromatographies on Sephacryl S-300, Blue Sepharose CL-6B, and DEAE Bio-Gel A. Purified calpains I and II required 10 and 450 microM Ca++ for half-maximal activation, respectively, and had an optimal pH of 7.0 to 8.0. Both enzymes were heterodimers and composed of one heavy subunit (83 kDa for calpain I and 80 kDa for calpain II) and one light subunit (29 kDa for both enzymes). The action of calpains I and II on keratin extracted from the same tissue was studied. Both enzymes rapidly cleaved keratin into small fragments. The cleavage depends on Ca++ and could be blocked by leupeptin and calpastation, an endogenous calpain-specific inhibitor, which was also found in the cytosolic fraction of pig epidermis and partially purified.

Animals↗
PubMed1988

Distribution of calpains and calpastatin in human blood cells.

The occurrence and molecular sizes of calpains and calpastatin in the lysates of human erythrocytes, platelets, lymphocytes/monocytes, and polymorphonuclear cells were studied by immunoelectrophoretic blot analysis. The basic uniformity among these cells of the 85-kDa and 83-kDa heavy subunits of low- and high-Ca2+-requiring calpains I and II, respectively, and of the 29-kDa light subunit was confirmed. Molecular diversity of calpastatin species, ranging from 70 kDa to 107 kDa, among different blood cells was also shown. The obtained data are consistent with those known for other animal tissues, thus settling hitherto uncertain or rather controversial issues on the distribution of calpains and calpastatin in human blood cells.

Blood Platelets↗
PubMed1987

Platelet factor XIII is activated by calpain.

The action of calpain (EC 3.4.22.17; Ca2+-dependent cysteine proteinase) on platelet factor XIII has been studied. Calpain I activated platelet factor XIII up to 76% of the maximum level observed with thrombin. Activation was accompanied by the limited proteolysis of the a subunit of platelet factor XIII to produce a 76 kDa fragment which was comparable to the proteolytic product by thrombin. Activation of platelet factor XIII by calpain was inhibited by EDTA, leupeptin, and endogenous calpain-specific inhibitor calpastatin. These findings suggest that calpain is responsible for the intracellular activation of platelet factor XIII.

Blood Platelets↗
PubMed1987

Repetitive region of calpastatin is a functional unit of the proteinase inhibitor.

A cDNA portion coding for one of the repetitive regions of pig heart calpastatin (107 kDa) was subcloned into E. coli plasmid pUC119 to express the portion of the proteinase inhibitor gene in bacteria. The expressed protein was a chimaeric protein whose calpastatin segment (130 amino acid residues) was fused with an amino-terminus portion (7 amino acid residues) of beta-galactosidase. The chimaeric protein could inhibit proteolytic activity of calpain (Ca2+-dependent cysteine proteinase), and maintained properties of the authentic calpastatin concerning inhibition specificity and heat stability. These findings led us to conclude that the repetitive region is a functional unit of the proteinase inhibitor.

Amino Acid Sequence↗
PubMed1987

Monoclonal antibodies directed to chemically synthesized lactogangliotetraosylceramide, a leukemia-associated antigen having a novel branching structure.

Murine leukemia cells (M1), in their undifferentiated state, have been characterized by the presence of cancer-associated lactoganglio-series glycolipids, one of which was identified as lactogangliotetraosylceramide (LcGg4) having a novel branching at the II-Gal of lactosylceramide through GlcNAc beta 1----3 and GalNAc beta 1----4 linkage, as shown below (Kannagi, R., Levery, S.B., and Hakomori, S. (1984) J. Biol. Chem., 259, 8444-8451): GalNAc beta 1----4 Gal beta 1----4Glc beta 1----1Cer GlcNac beta 1----3 Since this glycolipid is a very minor component, it has been difficult to obtain enough of the purified glycolipid for the preparation of a monoclonal antibody. We developed a method to chemically synthesize this glycolipid using a lactose unit, a ceramide unit, and two hexosamine donors as synthons and made the synthetic glycolipid available as an immunogen. The two monoclonal antibodies we obtained (YI328-18 and YI328-51, both IgG3) specifically recognized the novel branching structure and had no cross-reactivity with gangliotriaosylceramide or lactotriaosylceramide. Thus, the antibodies were found to be useful probes to detect lactogangliotetraosylceramide expressed in undifferentiated M1 leukemia cells, which disappears on induced differentiation. The results of this study indicate a new strategy to establish monoclonal antibody directed to novel minor glycolipid markers or their artificially designed analogs, employing chemically synthesized glycolipid antigens.

Animals↗
PubMed1987

High-performance liquid chromatographic assay of transglutaminase and its application to the purification of human erythrocyte transglutaminase and platelet factor XIII.

A high-performance liquid chromatographic method was developed for the assay of transglutaminase [EC 2.3.2.13] activity. Casein and dansylcadaverine were used as substrates and the reaction was stopped by adding an excess amount of EGTA. Casein-bound dansylcadaverine was separated from free dansylcadaverine by high-performance liquid chromatography on a TSK SW gel column on the basis of the differences in the molecular weight and hydrophobicity. The sensitivity was approximately 0.04 nmol of casein-bound dansylcadaverine in the assay mixture. With this assay method, human erythrocyte transglutaminase and platelet factor XIII were purified by successive chromatographies on DEAE-cellulose and Sephacryl S-300, which were common for both enzymes, followed by Blue Sepharose CL-6B and DEAE Bio-Gel A for erythrocyte transglutaminase or Phenyl-Sepharose CL-4B for platelet factor XIII. The purification factors and activity yields were 15,300-fold and 22% for erythrocyte transglutaminase and 43.8-fold and 33% for platelet factor XIII.

Blood Platelets↗
PubMed1987

Specific localization of tissue-type transglutaminase in adrenocorticotropin-producing cells of the human pituitary gland as demonstrated by immunohistochemistry.

The distribution of transglutaminases, the Ca2+-dependent protein cross-linking enzymes, in the human pituitary gland was investigated by immunohistological methods using specific antibodies. Tissue-type transglutaminase was specifically localized in ACTH-producing cells, and the cells producing GH, PRL, TSH, FSH, and LH contained no appreciable amount of the enzyme. No detectable plasma-type transglutaminase (coagulation factor XIII) was found in pituitary tissue. In a previous study we demonstrated that ACTH-producing cells contain very little Ca2+-dependent proteinases (calpain), but a remarkable amount of their inhibitor, calpastatin. Pituitary gland cells producing hormones other than ACTH contained calpains, but no detectable calpastatin. These results collectively suggest that intracellular substrate proteins in ACTH-producing cells are protected from Ca2+-dependent degradation and are substrates for Ca2+-dependent cross-linking catalyzed by the tissue-type transglutaminase. In other pituitary gland cells, conversely, the intracellular substrate proteins are more likely to undergo Ca2+-dependent degradation than cross-linking.

Adolescent↗
PubMed1987

Differentiation antigens defined by mouse monoclonal antibodies against human germ cell tumors.

We have developed two mouse monoclonal antibodies, M912-2A2 and M912-2G10, against cell surface antigens of a human infantile embryonal carcinoma cell line, MTE. The distribution of these antigens (designated as 2A2 and 2G10) was almost identical in human germ cell tumors in which they hallmarked yolk sac components and some tubular endodermal structures. Immunoelectron-microscopically, the antigens were located on the microvilli of MTE tumor cells. These antigens were not found on other common childhood tumors. In normal and fetal tissues they exhibited quite different distributions. In the kidney, 2A2 and 2G10 were present on the collecting tubules and proximal/distal tubules, respectively. Expression of both antigens was already observed in fetal kidneys of 10 weeks gestational age. In hematopoietic cells 2G10 was present only on granulocytes and on erythrocytes regardless of ABO blood group, whereas 2A2 was not present on any peripheral blood cells. Both antigens were equally expressed in testis and epididymis. Biochemically, reactivity of both antibodies was abolished with periodate treatment, suggesting their carbohydrate nature. Further biochemical characterization revealed that antibody to 2G10 reacts with the nonreducing terminal structure of type 2 carbohydrate chain, Ga1 beta 1-4G1cNAc, common to nLc4 (paragloboside), nLc6 (neolactohexaose), and Y4 neutral glycolipids of O-type erythrocytes. These data illustrate the complexity of carbohydrate antigens on yolk sac components of human germ cell tumors and provide a basis for the study of primitive endodermal and yolk sac differentiation in these tumors.

Animals↗
PubMed1986

Evidence for the repetitive domain structure of pig calpastatin as demonstrated by cloning of complementary DNA.

A clone of complementary DNA (cDNA) coding for pig heart calpastatin was isolated using synthetic oligonucleotide probes. The amino acid sequence deduced from the nucleotide sequence revealed the occurrence of a repetitive sequence at the interval of 140 amino acids, substantiating the multidomain structure of calpastatin. A portion of the sequence of 251 amino acid residues predicted for pig heart calpastatin (107 kDa) was found to be identical with that of a peptide fragment derived from pig erythrocyte calpastatin (68 kDa) and sequenced by Edman degradation.

Amino Acid Sequence↗
PubMed1986

Two different molecular species of pig calpastatin. Structural and functional relationship between 107 kDa and 68 kDa molecules.

Calpastatin, the inhibitor protein acting specifically on calpain (EC 3.4.22.17; Ca2+-dependent cysteine proteinase), is known to be widely distributed in mammalian and avian cells. Two different molecular species of calpastatin were isolated and purified to homogeneity from pig heart muscle and from pig erythrocytes, and shown to be of 107 kDa and 68 kDa respectively on SDS/polyacrylamide-gel electrophoresis. Both calpastatins had very similar amino acid compositions when expressed as mol per cent of the residues, differed by only 0.1 pH unit in their isoelectric points, and showed immunological cross-reactivity. One molecule of the 107 kDa species could bind approx. 8 calpain molecules, whereas the 68 kDa inhibitor could bind approx. 5 calpain molecules. These findings suggest similar protein structures of the 107 kDa and 68 kDa calpastatins, each being composed of extended multidomains, with unit inhibitor domains aligned along the polypeptide chain of the molecule. The present study does not conclude, however, whether or not the 68 kDa calpastatin found in erythrocytes is a derived product from the 107 kDa species, which is present as such in heart muscle.

Amino Acids↗