Reiji Kannagi

Adhesion of human cancer cells to vascular endothelium mediated by a carbohydrate antigen, sialyl Lewis A.

Recently the lectin-like domain on ELAM-1 (endothelial leukocyte adhesion molecule-1) was shown to recognize a carbohydrate antigen, sialyl Lewis x. In this paper we demonstrate, by a series of inhibition experiments utilizing specific monoclonal antibodies and pure glycolipid preparations, that the sialyl Lewis a antigen serves as a specific ligand for ELAM-1 as well as sialyl Lewis x and plays a significant role in the ELAM-1-mediated binding of human cancer cells to activated endothelial cells.

Effect of different physical states of phospholipid substrates on partially purified platelet phospholipase A2 activity

Partial purification of alkaline phospholipase A2 (EC 3.1.1.4) from rabbit platelets was carried out and the effect of different physical states of the substrate phosphatidylcholine on the activity was investigated. (1) The enzyme was purified about 1020-fold by means of Sephadex gel chromatography after extraction from a particulate fraction of rabbit platelets, followed by CM-cellulose chromatography, and had a molecular weight of approx. 12 000 as determined by gel chromatography. (2) The activity of the purified enzyme was enhanced by the addition of detergents. Sodium deoxycholate and sodium cholate markedly stimulated the activity, and the effect of these substances was observed well below the critical micelle concentrations. Triton X-100 stimulated the activity moderately, and the activation was observed only above the critical micelle concentration. (3) The addition of negatively charged phospholipids to the substrate egg phosphatidylcholine induced a moderate activation of hydrolysis. (4) The addition of long-chain cation to the substrate induced an inhibition of the activity, whereas the addition of long-chain anion activated the hydrolysis of egg phosphatidylcholine, but did not activate the hydrolysis of phosphatidylcholine in the total lipid extract of rabbit platelets. (5) Hydrolysis of dimyristoyl phosphatidylcholine increased in the temperature region of the phase transition of the substrate. Addition of cholesterol at the concentration of 20 mol% diminished the effect of phase transition. (6) Release of [1-14C]arachidonic acid from an equimolar mixture of egg phosphatidylcholine with dipalmitoyl or distearoyl phosphatidylcholine was activated at the temperature of 0 degrees C or 20 degrees C, respectively. From these results, we suggest that platelet phospholipase A2 can be activated to release fatty acids from the platelet phospholipids at the domains within membranes, where exist the structural irregularities and/or accumulation of negative charge within the bilayers.

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.

Transglutaminase activity during the differentiation of macrophages.

Summary Transglutaminase (R-glutaminyl-peptide:amine γ-glutamyltransferase, E.C. 2,3,2,13) activity during the differentiation of murine leukemia cell lines (M1 cells) was investigated. Ml cells contained a significant transglutaminase activity of the tissue type. During the course of differentiation into mature macrophage-like M1 + cells induced with a protein inducer, the enzymatic activity was stimulated more than ten times as much as in the original undifferentiated Ml − cells. A remarkable enhancement of enzymatic activity was also observed when lipopolysaccharide was utilized as an inducer of differentiation. The enzymatic activity of undifferentiated M1 − cells was eluted at the region of M.W. ca. 80,000 as a single and symmetrical peak on Sepharose 4B column chromatography. By contrast, most of the activity in differentiated Ml + cells was eluted at the void volume under the same condition, though some activities were eluted at the same region as in Ml − cells. These data suggest that most of transglutaminase activity exists in the form of a high-molecular-weight complex with some cellular components in differentiated cells. The possible physiological significance of the enzyme in macrophage functions was discussed.

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.

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.

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.

Tissue distribution of 2‐3 and 2‐6 sialyl lewis A antigens and significance of the ratio of two antigens for the differential diagnosis of malignant and benign disorders of the digestive tract

The authors investigated the tissue distribution of two kinds of sialylated derivatives of Lewis A (Lea) antigen in patients with cancers of the digestive system using specific monoclonal antibodies, and evaluated the significance of determining the 2‐3 and 2‐6 sialylated Lea antigen levels for the diagnosis of cancer. In most specimens from patients with cancers of the pancreas, biliary tract, stomach, and colon, the 2‐3 sialylated Lea antigen was strongly expressed in cancer cells. However, 2‐6 sialylated Lea antigen was less frequently expressed in cancer cells. The former is therefore more specific to cancer than the latter. Also, the serum level of the 2‐3 sialylated Lea antigen was significantly higher than that of the 2‐6 counterpart in patients with cancers of pancreas, biliary tract, stomach, and colon. The resulting ratio of serum 2‐3/2‐6 sialylated Lea antigens was frequently high in patients with malignancy and was low in patients with benign disorders of these digestive organs. Therefore, the 2‐3/2‐6 sialylated Lea antigen ratio is a useful for the differential diagnosis of malignant disorders in these organs. However, liver disorders were found to be exceptional in that both antigens were mostly absent in hepatocellular carcinoma (HCC) cells in immunohistologic examination, as well as in nonmalignant parenchymal liver cells. Only the epithelial cells of the intrahepatic bile ducts expressed the 2‐6 sialylated Lea antigen strongly, and expressed the 2‐3 sialylated Lea antigen moderately. The levels of both antigens were sometimes high in patients with liver disorders, but the ratio always remained low in patients with HCC as well as benign liver disorders such as cirrhosis or chronic hepatitis. The sialylated Lea antigens, which sometimes accumulate in the sera of patients with HCC, were concluded to originate from the epithelial cells of the proliferating small bile ducts, and those serum antigens cannot be considered as evidence for the presence of liver cancer cells.

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.

Phospholipid-deacylating enzymes of rabbit platelets.

Abstract Phospholipase activities in sonicates of rabbit platelets were studied as these activities may be candidate enzymes for the release of arachidonic acid from platelet membrane phospholipids. Platelets contained phospholipases A1 (EC3.1.1.32), A2 (EC3.1.1.4), and lysophospholipase (EC 3.1.1.5) activities that can be distinguished with respect to optimum pH, dependence on calcium ion, heat lability, and response to detergent. Phospholipase A1 activity had an optimum pH of 4.5, and calcium ion did not affect the activity. Lysophospholipase activity was optimal at pH 8.5, relatively heat labile, and strongly inhibited by sodium deoxycholate. Calcium ion and EDTA had little effect on the activity. Platelets contained relatively high phospholipase A2 activity compared with the above-mentioned enzymes. The activity was optimal at pH 9.5 and relatively heat stable in acidic medium. Calcium ion is specifically required for the activity and EDTA strongly inhibited the activity. The addition of sodium deoxycholate strongly enhanced the activity. Because of its high activity and calcium ion dependency, phospholipase A2 with an optimum in alkaline pH range seemed worthy of further study. The enzyme was recovered mostly in a particulate fraction, preferred phosphatidylethanolamine to phosphatidylcholine, and showed no strict acyl chain specificity. Once sonicated, thrombin and other platelet activating agents and cyclic nucleotides had no direct effect on the activity.