Immunological analysis of glycolipids on cell surfaces of cultured human tumor cell lines: expression of lactoneotetraosylceramide on tumor cell surfaces

Glycolipids of human cell lines of colonic adenocarcinoma (Colo 205 and BM 314), gastric tumor (AZ 521 and KATO-III), and lung tumor (A 549) were studied by the immunohistochemical fluorescence technique, flow cytometric analysis and immunostaining on thin layer chromatoplates with antibodies against gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), fucogangliotetraosylceramide (Fuc-Gg4Cer), blood group B active lipid, globopentaosylceramide (Gb5Cer) and lactoneotetraosylceramide (nLc4Cer). Anti-nLc4Cer antibody was the only antibody which reacted with all the tumor cell lines used. The glycolipid fractions of each cell line separated by Iatrobeads column chromatography were immunostained with the six antibodies mentioned above on thin layer plates. The presence of nLc4Cer was detected in all cell lines. On the other hand, Gg4Cer was detected in gastric tumor cell lines, and Gg3Cer was detected in AZ 521. Based on these results, the tumor cell lines were analyzed by flow cytometry using anti-nLc4Cer antibody. About 70% of total cells in each cell line were separated as nLc4Cer-expressing cells. The present findings, together with the occurrence of nLc4Cer in ascitic fluids of cancer patients (Taki, T., Kojima, S., Seto, H., Yamada, H., & Matsumoto, M. (1984) J. Biochem. 96, 1257-1265), suggest that nLc4Cer may be a tumor-associated lipid.     

Cordycepin analogs of ppp5'A2'p5'A2'p5'A (2-5A) inhibit protein synthesis through activation of the 2-5A-dependent endonuclease

Analogs of the triphosphate 2'-5'-linked adenylate trimer (ppp5'A2'p5'A2'p5'A, called 2-5A) which contain 3'-deoxyadenosine (cordycepin) instead of adenosine either in positions one and two, or in all three positions, are 10-100-fold less potent than is parent 2-5A in inhibition of protein synthesis in intact cells, when utilizing calcium co-precipitation techniques to introduce the 5'-triphosphate oligonucleotides into the cells. That the inhibition of protein synthesis was a consequence of activation of the 2-5A-dependent endonuclease by the 3'-deoxyadenosine analogs of 2-5A was demonstrated in obtaining the ribosomal RNA cleavage pattern that is characteristic of endonuclease activation by parent 2-5A. Additional results (i.e. lack of activity by the dimer species ppp5'(3'dA)2'p5'-(3'dA) or the monomer 3'dA) as well as kinetic analysis both in intact cells and in cell-free extracts provided further evidence that the inhibition of protein synthesis observed with these 3'-deoxyadenosine 2-5A analogs was not due to their degradation to the antimetabolite monomer unit 3'-deoxyadenosine.     

Electron microscopical findings with special reference to cancer in rats caused by inhalation of nickel oxide

A special exposure system was used for the inhalation of nickel oxide (NiO) aerosol by Wistar male rats. The median aerodynamic diameter and the geometric standard deviation were 1.2 μm and 2.2, respectively. A histopathological study of the rats was performed immediately, and at intervals of 12 and 20 mo after a 1-mo expsoure to NiO. Electron microscopy showed that localization of NiO particles was restricted to the lungs and that each particle had been engulfed by the alveolar macrophages. Type II pneumocytes and nonciliated bronchiolar epithelial cells (Clara cells), as well as numerous tubular myelin (surfactant) in the alveoli were prominent. In rats dissected after 12 mo, clusters of NiO particles were still present within the terminal bronchioli, alveolar walls, and lysosomes of the alveolar macrophages. Pools of tubular myelin were observed in the peribron-chial lymphatics. The Clara cells, which project into the lumen of bronchioli, showed active secretion and were filled with smooth en-doplasmic reticulum (SER) in the apical cytoplasm. In the experimental group sacrificed after 20 mo, one rat had papillary adenocarcinoma and two rats showed adenomatosis in the peripheral portion of the lung, but none in the upper respiratory tract.     

C-banding analysis of six species of lung flukes, Paragonimus spp. (Trematoda: Platyhelminthes), from Japan and Korea

We examined C-banded karyotypes of six species of lung flukes from Japan and Korea; diploid and triploid Paragonimus westermani, P. miyazakii, P. ohirai, P. iloktsuenensis and P. sadoensis, with special reference to their karyotypic diversification. C-band analysis between the diploid and the triploid westermani revealed that two of three homologues of the triploid resembled those of the diploid in C-band pattern, while the remaining chromosome showed a different pattern from any species examined here. This karyological evidence indicates that the triploid is allotriploid probably induced by interspecific hybridization between the diploid westermani and an unknown species; we, therefore, suggest that the triploid westermani is an independent species and synonymous with P. pulmonalis (Miyazaki 1978). As the morphologically similar three species, ohirai, iloktsuenensis and sadoensis, had the same C-band polymorphism in chromosome No. 4, these species are classified as the local races of P. ohirai. Paragonimus miyazakii has one common C-band (5q) with the diploid westermani, but other bands (1q, 4q, 6q, 7p and 7q) are different. From these observations, the six species examined are phylogenetically divided into three groups: (1) westermani group containing diploid and triploid (= pulmonalis) species, (2) miyazakii and (3) ohirai including two geographic races, iloktsuenensis and sadoensis.     

Macrophage Activation by Tetrahymena pyriformis. I. Active Subcellular Fractions of Tetrahymena

Experiments were carried out to determine what subcellular fractions of Tetrahymena pyriformis could, after inoculation into mice, activate macrophages to kill Toxoplasma gondii in vitro. Peritoneal macrophages from mice inoculated intraperitoneally with cilia, pellicles, mitochondria, and microsomes exhibited strong toxoplasmacidal activity and had an enhanced capacity to release hydrogen peroxide (H₂O₂) by stimulation of a membrane-active agent as compared with resident macrophages. In contrast, macrophages from mice inoculated with macronuclei and postmicrosomal supernatant showed no toxoplasmacidal activity and a low level of H₂O₂ release. Similar dose response was observed on the active subcellular fractions with regard to the degree of macrophage activation. Treatment of the active subcellular fractions with heating and trypsin markedly reduced their activity.     

Endogenous infection in mice with streptozotocin-induced diabetes. A feature of bacterial translocation

Slc:ddY mice that received a single intraperitoneal injection of 200 mg/kg streptozotocin (STZ) were examined for persistency of diabetes (changes of indigenous bacterial floras, and bacterial translocation. Significant diabetes (increase in plasma glucose and decrease in insulin) was recognized 2 weeks after the injection, and persisted for 12 weeks. The numbers of aerobic gram-negative bacilli, staphylococci (including micrococci), and streptococci in caecal and oral floras were significantly increased, but the numbers of anaerobic bacteria in caecal flora were hardly changed. Bacterial translocation of indigenous bacteria to the mesenteric lymph node, lung, or kidney was detectable in some mice 2 weeks after the injection. The incidence of bacterial translocation in these STZ-treated mice then increased; infection caused by several organisms, e.g., Klebsiella pneumoniae, Staphylococcus epidermidis, streptococci, or Lactobacillus sp., occurred in lung, liver, spleen, kidneys, and mesenteric lymph node. No indigenous bacteria were cultured from these organs of control mice. This endogenous infection may have been due to the over population of several bacterial strains caused by disruption of indigenous floras along with depression of immunological function.     

Primary structure of heat-stable enterotoxin produced by Yersinia enterocolitica

A heat-stable enterotoxin was isolated and purified from the culture supernatant of $\text{Yersinia enterocolitica}$ by reversed-phase high-performance liquid chromatography. The amino acid sequence of the purified toxin was determined to be as follows: Gln-Ala-Cys(X)-Asp-Pro-Pro-Ser-Pro-Pro-Ala-Glu-Val-Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys (X: not determined). The C-terminal sequence containing 6 half-cystine residues was highly homologous to that of heat-stable enterotoxin of enterotoxigenic $\text{Escherichia coli.}$     

Synthesis and biological activity of tubercidin analogues of ppp5'A2'p(5'A2'p)n5'A

A series of tubercidin (7-deazaadenosine) analogues of 2-5A of the general formula p5'(c7A)2'p[5'(c7A)-2'p]n5'(c7A) (n = 0-5) were prepared by lead ion catalyzed polymerization of the 5'-phosphoroimidazolidate of tubercidin. Through the corresponding imidazolidates, these oligonucleotide 5'-monophosphates were converted to the 5'-triphosphates. All reported structures were corroborated by enzyme digestion and 1H or 31P nuclear magnetic resonance. When evaluated for its ability to bind to the 2-5 A-dependent endonuclease of mouse L cells, the tubercidin analogue of trimeric 2-5A, namely, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), and the corresponding tetramer were bound as effectively as 2-5A itself; nonetheless, it and the corresponding tetramer, ppp5'-(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), failed to stimulate the 2-5A-dependent endonuclease as judged by its inability to inhibit translation in extracts of mouse L cells programmed with encephalomyocarditis virus RNA and to give rise to ribosomal RNA cleavage in the same cell system under conditions where 2-5A showed activity at 10(-9) M. The trimer, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), was an antagonist of 2-5A action in the L cell extract. In the lysed rabbit reticulocyte system, both the trimeric and tetrameric tubercidin 2-5A analogues were bound to the 2-5A-dependent endonuclease as well as 2-5A, but in this case, the tetramer triphosphate, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), was just as potent an inhibitor of translation as 2-5A tetramer triphosphate. Moreover, this inhibition was prevented by the established 2-5A antagonist p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunologic characterization and molecular profile of carcinoembryonic antigen detected by monoclonal antibodies

Four distinct monoclonal antibodies, which reacted with CEA preparations but not with nonspecific cross-reacting antigen or with nonspecific cross-reacting antigen 2, were established. Except for monoclonal antibody AS001 , all of these monoclonal antibodies immunoprecipitated molecular forms of 200K and 180K daltons that are not bridged by disulfide bonds. Immunodepletion experiments and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that these monoclonal antibodies recognized the same antigenic structure when 125I-CEA preparation was used. Monoclonal antibody AS001 is of particular interest, because this antibody reacted only with a 200K dalton molecule which is a part of the molecules recognized by the other three monoclonal antibodies. The rosette inhibition assay and the immunoprecipitation experiments suggest that each monoclonal antibody recognizes a different antigenic determinant. The antigenic determinants recognized by monoclonal antibodies YK013 and AS001 may be peptides in nature, whereas the determinants recognized by antibodies YK024 or AS005 might be carbohydrate. The radioimmunoassay with monoclonal antibody AS001 was established, and the results clearly indicate that the incidence of positivity for the sera from digestive tract cancer patients and from lung cancer patients obtained by monoclonal antibody AS001 was higher than that obtained by the polyclonal antibody. Monoclonal antibody AS001 was able to detect the corresponding antigen in the sera, which the polyclonal antibody failed to detect. This study therefore suggests that monoclonal antibodies may enhance and improve the diagnostic value in cancer patients with undetectable or lower CEA levels detected by conventional anti-CEA antibodies.