Anti-O-phosphotyrosine antibodies in human sera

Antibodies reactive with O-phosphotyrosine (PTYR) were detected in 60 out of 621 inpatients, with high frequencies in hematologic and lung malignancies, hepatic diseases, cerebrovascular diseases, and autoimmune diseases. Affinity-purified antibodies proved capable of recognizing PTYR-containing proteins in a human carcinoma cell line, A431, both by immunofluorescent staining and by immunoaffinity chromatography, but had no detectable affinity for phosphorylated serine or threonine, or for the nucleotides tested. In these respects, the antibodies observed in human sera were indistinguishable from anti-PTYR antibodies raised experimentally in rabbits or mice.     

Raman diagnosis of nucleic acid structure: sugar-puckering and glycosidic conformation in the guanosine moiety.

Observations of Raman spectra of various nucleic acids indicate that the guanine ring breathing frequency is sensitive to the internal rotation angle around the glycosidic bond and to the conformation of the five-membered ring of the ribose residue that is directly connected with the guanine residue in question. It is found that 682 cm-1 for C2'-endo-anti, at 665 cm-1 for C3'-endo-anti, and at 625 cm-1 for C3'-endo-syn. A DNA octamer d(GpGpApApTpTpCpC) shows, in its aqueous solution, a broad Raman band at 680 cm-1 with a tail at 670 cm-1. This fact suggests that the guanosine residues in this oligomer take primarily C2'-endo-anti conformation but an appreciable amount of fluctuation of the ribose ring structure towards C3'-endo is involved.     

Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells

Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.     

Cross-ligation and exchange reactions catalyzed by hairpin ribozymes.

The negative strand of the satellite RNA of tobacco ringspot virus (sTobRV(-)) contains a hairpin catalytic domain that shows self-cleavage and self-ligation activities in the presence of magnesium ions. We describe here that the minimal catalytic domain can catalyze a cross-ligation reaction between two kinds of substrates in trans. The cross-ligated product increased when the reaction temperature was decreased during the reaction from 37 degrees C to 4 degrees C. A two-stranded hairpin ribozyme, divided into two fragments between G45 and U46 in a hairpin loop, showed higher ligation activity than the nondivided ribozyme. The two stranded ribozyme also catalyzed an exchange reaction of the 3'-portion of the cleavage site.     

Physical effects of negative air ions in a wet sauna

 The physical effects of negative air ions on humans were determined in an experimental sauna room equipped with an ionizer. Thirteen healthy persons took a wet sauna bath (dry bulb temperature 42° C, relative humidity 100%, 10 min exposure) with or without negative air ions. The subjects were not told when they were being exposed to negative air ions. There were no differences in the moods of these persons or changes in their blood pressures between the two saunas. The surface temperatures of the foreheads, hands, and legs in the sauna with negative ions were significantly higher than those in the sauna without ions. The pulse rates and sweat produced in the sauna with ions were singificantly higher than those in the sauna without ions. The results suggest that negative ions may amplify the effects on humans of the sauna. Received: 31 March 1995 / Revised: 25 July 1995 / Accepted: 26 July 1996     

Studies on transfer ribonucleic acids and related compounds. XL. Synthesis of an eicosaribonucleotide corresponding to residues 35-54 of tRNAfMet from E. coli.

An E. coli tRNAfMet fragment [C-A-U-A-A-C-C-C-G-A-A-G-G-U-C-G-U-C-G-G (bases 35-f54)] containing the anticodon triplet has been synthesized by the phosphotriester method involving protected oligonucleotide blocks. Di- or tri-nucleotide blocks were prepared by condensation of 2'-O-(o-nitrobenzyl) nucleotide derivatives and used for the synthesis of pentanucleotide blocks. The 5'-hydroxy, heterocyclic amino and internucleotide linkage were protected with monomethoxytrityl, acyl and p-chlorophenyl groups, respectively. The 3'-phosphates of the pentanucleotides, except for the GUCGG block where 2'-O-benzoyl 3'-O-(o-nitrobenzyl) N-isobutyrylguanosine was used, were protected with p-chlorophenyl and anilido groups. The anilido groups were removed by treatment with isoamyl nitrite and the 3'-phosphodiesters of resulting pentamers were activated with mesitylenesulfonyl nitrotriazolide to give protected decanucleotides in yields of 61-89%. The two decanucleotides were condensed similarly to yield the protected eicosanucleotide in a yield of 59%. The product was deblocked and purified by ion-exchange chromatography on DEAE-Sephadex A-25 and characterized by enzymatic hydrolysis after labelling the 5'-end by phosphorylation using polynucleotide kinase and [gamma-32P]ATP.     

Non-cognizable ribonucleotide, 2''AMP, binds to a mutant ribonuclease T1 (Y45W) at a new base-binding site but not at the guanine-recognition site.

Complex of a mutant ribonuclease T₁ (Y4SW) with a non-cognizable ribonucleotide, 2′AMP, has been determined and refined by X-ray diffraction at 1.7 Å resolution. The 2′AMP molecule locates at a new base-binding site which is remote from the guanine-recognition site, where 2′GMP was found to be bound. The nucleotide adopts the anti conformation of the glycosidic bond and C3′-exo sugar pucker. There exists a single hydrogen bond between the adenine base and the enzyme, and, therefore, the site found is apparently a non-specific binding site. The results indicate that the binding of 2′AMP to the guanine-recognition site is weaker than that to the new binding site.     

An endonuclease activity in human polymorphonuclear neutrophils that removes 8-hydroxyguanine residues from DNA+

An endonuclease that specifically removes 8-hydroxyguanine (oh8Gua) from DNA has been isolated from Escherichia coli. As the amount of oh8Gua produced in DNA of X-ray-irradiated mice is known to decrease with time after irradiation, an attempt was made to find a similar activity in human polymorphonuclear neutrophils (PMNs) using a synthetic dsDNA containing oh8Gua as a substrate. The PMN enzyme was isolated free of other DNases, and found to cleave the substrate DNA simultaneously at 2 sites, the phosphodiester bonds 5' and 3' to oh8Gua, producing free hydroxyl and phosphate groups, respectively. The enzyme showed almost no activity on DNAs containing other kinds of modified base tested or mismatched DNA. Thus human cells also contain an endonuclease that specifically removes oh8Gua residues from DNA.