Establishment of a nucleoside-specific suppressor T cell line from SLE prone (NZB/NZW)F1 mice

A long-term cultured suppressor T cell line (GTS-124) was established from an autoimmune mouse strain, (NZB X NZW)F1, by a two-part procedure: a) B/W F1 mice were made tolerant to guanosine (G) by administration of a tolerogen, the G-modified copolymer of D-glutamic acid and D-lysine (G-D-GL); and b) the spleen cells obtained from tolerant mice were repeatedly stimulated with mitomycin C-treated G-modified syngeneic spleen cells. The GTS-124 cells suppressed the secondary in vitro response to G-keyhole limpet hemocyanin (G-KLH) but did not suppress the response to unrelated antigens, sheep erythrocytes (SRBC), or trinitrophenyl-KLH (TNP-KLH). The expression of Thy-1 antigen on the cell surface of GTS-124 was demonstrated by flow cytometry. Growth of GTS-124 cells was dependent on IL 2. To determine whether GTS-124 cells could suppress the response to nucleosides other than G, KLH coupled with four nucleosides (adenosine [A], G, cytidine [C], and thymine riboside [T]) collectively (AGCT-KLH) was first used as the antigen in the assay system. The PFC response to the individual nucleosides (anti-A, -G, -C, and -T PFC) were effectively inhibited by GTS-124 cells, suggesting that the GTS-124 cells mediated cross-suppression toward all four nucleosides. A more stringent cross-suppression test was conducted by using only the T moiety bound to KLH (T-KLH) as antigen. The results showed that GTS-124 cells were capable of suppressing the T-specific response. The cross-suppression could be seen after repeated selection on a G-BSA-coated dish. These results provide direct evidence that the suppressor T cells induced by in vitro stimulation with G-modified self can indeed suppress the response to nucleosides other than G.     

Oxygen-binding characteristics of Potamilla chlorocruorin

Accurate oxygen equilibrium curves of chlorocruorin of a marine polychaete annelid, Potamilla leptochaeta, were determined under a variety of experimental conditions. Like chlorocruorins from other species Potamilla chlorocruorin exhibited a low oxygen affinity, a large Bohr effect, and high cooperativity compared to those of human hemoglobin. However, in contrast to chlorocruorins from other species, the shape of the oxygen equilibrium curve for Potamilla chlorocruorin varied dramatically upon changes of pH or temperature. As observed in hemocyanins and annelid hemoglobins, cations, especially divalent ones such as Mg2+ and Ca2+, caused marked increase in oxygen affinity and cooperativity of Potamilla chlorocruorin. This finding together with the determination of cations in Potamilla blood has made clear the physiological role of chlorocruorin as an oxygen carrier. A graphical analysis based on the Monod-Wyman-Changeux allosteric model indicated that the number of sites for oxygen binding involved in heme-heme interactions is six, defining the functional unit of chlorocruorin molecule.     

An oxygen-evolving complex with a simple subunit structure - ‘a water-plastoquinone oxidoreductase” - from the thermophilic cyanobacterium Synechococcus sp.

An oxygen-evolving complex has been highly purified from the thermophilic cyanobacterium Synechococcus sp. The complex, which reproducibly showed 5 major polypeptide bands of 47, 40, 35, 30 and 9 kDa on SDS-polyacrylamide gel electrophoresis and contained 3.2 Mn per Q_A, had an oxygen-evolving activity of 300–400 μmol/mg chl per h in the presence of 5 mM MnCl₂; or CaCl₂. The complex most likely represents a minimum functional unit of the photosynthetic oxygen evolution.     

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.     

The differentiation of HL-60 cells in the synthetic medium induced by GM3 ganglioside

GM3 ganglioside, added exogenously to a promyelocytic leukemia cell line (HL-60 cells) in serum-free synthetic medium, induced differentiation into macrophage-like cells. Macrophagic morphology and function of differentiation-induced cells were determined by cell growth behavior, May-GriJnwald-Giemsa staining, activities of nonspecific esterase, phagocytosis and nitroblue tetrazolium (NBT) reduction. GM3 ganglioside may play a role in triggering differentiation of HL-60 cells into macrophage-like cells.     

In vitro establishment of human fibroblasts of lysosomal diseases,GM1-gangliosidosis and Sandhoff disease,by transformation with origin-minus SV40 DNA

The permanent human cell lines preserving defects of lysosomal enzymes, G_M1-1019-SV and SA-1077-SV, were established from the respective fibroblasts from patients with G_Ml-gangliosidosis and Sandhoff disease by transfection with replication origin-minus simian virus 40 DNA. These ceils grow rapidly without entering senescence during more than 120 population doublings. The activity of -galactosidase in G_M1-1019-SV and of B-N-acetylhexosaminidase in SA-1077-SV was respectively 40- and 180-fold lower than that of normal fibroblasts.     

Formation of giant protoplasts from protoplasts of Pleurotus cornucopiae by the cell wall lytic enzyme

Summary When purified protoplasts of Pleurotus cornucopiae IFO9614 were incubated with a mixture of cell wall lytic enzymes, they were found to increase their size. Their average diameter increased from 4.3 m to 31 m after 65 h incubation at 24° C. The presence of cellulase ONOZUKARS in the enzyme mixture had a significant effect on the formation of giant protoplasts. Regeneration frequency of giant protoplasts in a medium containing 0.5 M sucrose was 3.5%, approximately six times that of normal protoplasts.     

Effect of DN-1417, a thyrotropin releasing hormone analog, on dopaminergic neurons in rat brain

Acute and chronic effects of γ-butyrolactone-γ-carbonyl-histidyl-prolinamide (DN-1417) were investigated on motor activity, dopamine (DA) metabolites and DA receptors in various brain regions of rats. The motor activity, as measured with Automex recorder, was enhanced after a single injection with DN-1417 (20 mg/kg, IP), and the motor stimulating action persisted during 21 daily injections. Acute DN-1417 elevated both homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in 7 brain regions, prefrontal cortex polar, medial and lateral fields, nucleus accumbens, olfactory tubercles, amygdala and striatum. After chronic treatment for 7 days, the acute effect of DN-1417 on DA metabolites disappeared in all regions except for the striatum in which DN-1417 still increased HVA and DOPAC. The response of striatal DA metabolites was also observed after chronic treatment for 21 days. Chronic DN-1417 produced no significant change in ³H-spiperone binding in the prefrontal cortex, nucleus accumbens, olfactory tubercles and striatum, while striatal ³H-DA binding displaced by 30 nM spiperone was enhanced after chronic treatment. These results indicate that DN-1417 interacts with mesocortical, mesolimbic and nigrostriatal DA systems in the different modes of action. The lack of tolerance to motor hyperactivity, however, raises the question as to whether DN-1417-induced hyperactivity may be mediated by the activation of mesolimbic DA neurons. The involvement of nigrostriatal neurons in DN-1417-induced motor hyperactivity is suggested.     

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.