Activation of 2-5A-dependent RNase by analogs of 2-5A (5'-O-triphosphoryladenylyl(2'----5')adenylyl(2'----5')adenosine ) using 2',5'-tetraadenylate (core)-cellulose

A variety of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) analogs were assayed for their abilities to activate murine 2-5A-dependent RNase (subsequently "the nuclease") using a recently developed method. This technique consists of immobilizing and partially purifying the nuclease using core-cellulose [A2'p)3A-cellulose) and then monitoring the breakdown of poly(U)-3'-[32P]Cp into acid-soluble fragments. Several 5'-adenosinecapped analogs of 2-5A (containing a tetra-, tri-, or diphosphate) were analyzed, and it was found that reducing the number of phosphoryl groups between the 5' to 5'-diadenosine linkages resulted in a progressive loss of activity. Because A5' pppp(A2'p)3A was a potent activator of the nuclease yet stable during the assay these results suggested that a free 5'-phosphoryl group may not be required for the activation of the nuclease. A number of 8-bromoadenosine-substituted analogs of 2-5A were also studied. Curiously, the brominations decreased the activities of the 5'-di- and triphosphorylated molecules while substantially increasing the activities of the 5'-monophosphorylated species. The results indicated that a tri- or diphosphate moiety on the 5'-end of 2-5A or the presence of ATP is not absolutely required for the nuclease to be active. Furthermore, the ATP analog, beta, gamma-methylene ATP, did not inhibit the activity of the nuclease. Finally, a 3',5'-phosphodiester linkage isomer of 2-5A and a 3'-deoxy (cordycepin) analog of 2-5A were tested, and both were found to be completely without activity.     

Antibody-nucleic acid interactions. Monoclonal antibodies define different antigenic domains in 2',5'-oligoadenylates

To define the epitopes involved in binding anti-oligonucleotide antibodies, several hybridomas producing monoclonal antibodies directed against 2',5'-oligoadenylate were established. A solid-phase enzyme-linked immunoassay that employed microtiter wells coated with Ficoll-2',5'-oligoadenylate conjugates proved useful in screening and characterizing hybridoma supernatants. Control experiments demonstrated that the conjugates were irreversibly adsorbed to polystyrene wells under the conditions employed in the assay. Reactivity of monoclonal antibodies with numerous analogues of 2',5'-oligoadenylate was measured by using a competition assay. Several monoclonal antibodies originating from different mice immunized with the same or different immunogens possessed distinctive fine specificities. At least one 2',5'-phosphodiester bond was important in forming each epitope, suggesting that the ribose phosphate backbone is a critical element in defining an antigenic domain of an oligonucleotide. The purine bases were also important, and modification of the bases had varied effects on the extent of antibody recognition. The length of the oligonucleotide and the nature of the termini were also of some importance. In several instances the modification created by linkage of 2',5'-oligoadenylate to carrier protein also contributed to the determinant. The monoclonal antibody most specific for 2',5'-oligoadenylates was relatively insensitive to ionic strength. In contrast, a monoclonal antibody with a 2',5'-oligopurine specificity appeared to bind 2',5'-oligoadenylate through one ion pair, whereas the binding of a monoclonal antibody with a low degree of base specificity appeared to bind through two ion pairs. The results demonstrated that 2',5'-linked oligoadenylate-protein complexes possess at least three distinct oligonucleotide-related antigenic surfaces that can be recognized with high apparent affinity by monoclonal antibodies. A model for the three epitopes is presented.     

Purification and characterization of cytosolic liver protein facilitating heme transport into apocytochrome b5 from mitochondria. Evidence for identifying the heme transfer protein as belonging to a group of glutathione S-transferases

The results of the present experiment are shown in terms of the transport of protoheme from mitochondria to apocytochrome b5 when fresh rat liver mitochondria, apocytochrome b5, and cytosol were incubated. The heme transfer protein was purified from rat liver cytosol up to approximately 133-140-fold with a 43% yield by the procedure discussed herein, including Sephadex G-75 and CM-cellulose column chromatography. The final preparation showed apparent homogeneity upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its native form was found to be a dimeric protein with a Mr = 45,000 which consists of a subunit with a Mr = 23,000. In the transporting system, the heme transfer depended on the concentration of mitochondria (donor), apocytochrome b5 (acceptor), and purified transfer protein, respectively. Omission of one of these components led to an almost complete loss of the transfer activity. The transport of mitochondrial protoheme was a rapid reaction which showed approximate linearity until 1.5 min and after that it became saturated. When the functional capacity was tested by the NADH-cytochrome c reductase system, the reconstituted cytochrome b5 expressed its complete original catalytic properties, as well as its characteristic absorption spectra for the hemoprotein. Furthermore, the detailed physicochemical and immunological characterization of the transfer protein provided evidence that the protein is identical with soluble glutathione S-transferase, which conjugates glutathione with a variety of electrophilic compounds. At least one of the glutathione S-transferase isozymes observed was identified as GST-C2, which comprises the subunit of Yb'Yb' by the immunoprecipitation reaction using various anti-glutathione S-transferase isozyme antibodies.     

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.     

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.     

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.