2',5'-Oligoadenylates and related 2',5'-oligonucleotide analogues. 1. Substrate specificity of the interferon-induced murine 2',5'-oligoadenylate synthetase and enzymatic synthesis of oligomers

The substrate specificity of the interferon-induced mouse L-cell enzyme, 2',5'-oligoadenylate synthetase, was determined with a number of nucleoside 5'-triphosphate analogues. Selected nucleoside 5'-triphosphates were converted to 2',5'-oligonucleotides with the following order of efficiency for the nucleoside: 8-azaadenosine greater than adenosine = 2-chloroadenosine greater than sangivamycin greater than toyocamycin greater than formycin greater than 3-ribosyladenine greater than ribavirin greater than tubercidin greater than adenosine 1-oxide greater than 2-beta-D-ribofuranosylthiazole-4-carboxamide greater than inosine = 1,N6-ethenoadenosine greater than guanosine greater than 8-bromoadenosine = uridine greater than cytidine. Adenosine 5'-((beta, gamma-imidotriphosphate) did not seem to be a recognizable substrate since no detectable product resulted. Either the 2',5'-oligoadenylate synthetase is not as specific as had been previously thought, or there may be more than one 2',5'-oligonucleotide synthetase. The 2',5'-oligonucleotide analogue products in which the adenosine of ppp(A2'P5')nA was replaced by the various nucleoside analogues were separated by DEAE-cellulose column chromatography and the chain length and number of 5'-phosphate residues analyzed by a rapid, efficient high-performance liquid chromatographic (HPLC) system involving ion-pairing C18 reversed-phase column chromatography. Separation of the 5'-mono-, 5'-di-, and 5'-triphosphorylated forms of the 2',5'-oligonucleotide analogue dimers, trimers, tetramers, and pentamers was readily achieved by this useful HPLC system. No 5'-nonphosphorylated forms were detected for any of the 2',5'-oligonucleotide analogue products.     

Effect of incubation media on the recovery of Escherichia coli K12 heated at 52 degrees C.

The exposure of exponentially grown Escherichia coli K12 to 52 degrees C for 30 min in Tris/Mg2+ buffer resulted in a considerable loss of viability when plated on tryptone agar. When such heated bacteria were held at 37 degrees C for 2 h in tryptone broth before plating on tryptone agar, there was a significant increase in viability. Thus, heat damage was repaired in tryptone broth but not on tryptone agar. Recovery was greater in tryptone broth than in synthetic medium. In tryptone broth, recA or polA mutants also recovered but a lex mutant did not. As a result of heating, the sensitivity of bacteria to ultraviolet radiation (u.v.), to mitomycin C and to plating on high salt medium was enhanced. After incubation for 2 h in tryptone broth at 37 degrees C, the bacteria regained their resistance to u.v. and mitomycin C and tolerance to high salt medium. Recovery of viability required RNA and protein synthesis, whereas recovery of u.v. resistance did not require protein synthesis. Heating for 30 min inhibited the release of acid-soluble material from DNA in all strains of E. coli used.     

Inhibition of deoxyribonucleic acid polymerases from human cells and from simian sarcoma virus by pyran.

Pyrans are co-polymers of divinyl ether and maleic anhydride. Four pyrans of various molecular weights more potently inhibited terminal deoxyribonucleotidyltransferase (EC 2.7.7.31) from a human cell line of acute lymphoblastic leukemia origin (Molt-4) than they did DNA polymerases alpha, beta and gamma from these cells and DNA polymerase from simian sarcoma virus. For example, the concentrations of one pyran required for 50% inhibition of terminal deoxynucleotidyltransferase, DNA polymerases alpha, beta and gamma and viral DNA polymerase were 0.9, 110, 125, 35 and 47 microgram/ml respectively. Quantitatively similar results were obtained with the other pyrans. Inhibition of these enzymes by pyran was dependent on the concentrations of both the bivalent cation and template/primer or initiator in assay mixtures, but not on the concentrations of the substrate (deoxyribonucleoside 5'-triphosphate), enzyme, or bovine serum albumin. These results suggested that pyran inhibited these enzymes by complexing bivalent cations, which caused a decreased affinity of template/primer or initiator for each enzyme and a decrease in enzyme activity.     

Anatomical studies of Ustilago galls on Hordeum vulgare L.

The present paper deals with the histopathological studies of galls of Ustilago hordei (Pers.) on Hordeum vulgare L. The gall formation in the present case is the result of both hyperplastic and hypertrophic activities of the infected host cells. Also the gall formation by U. hordei on. H. vulgare is being reported for the first time through present. communication.     

Demonstration of cross-reacting material in Tay–Sachs disease

Antibodies against placental hexosaminidase A and kidney alpha-subunits were raised in rabbits after cross-linking the antigens with glutaraldehyde. Anti-(alpha(n)-subunit) antiserum (anti-alpha(n)) precipitated hexosaminidase A but not hexosaminidase B, whereas anti-(hexosaminidase A) antiserum precipitated both hexosaminidases A and B. Specific anti-(hexosaminidase A) antiserum was prepared by absorbing antiserum with hexosaminidase B. Both anti-alpha(n) and anti-(hexosaminidase A) antisera precipitated the CR (cross-reacting) material from eight unrelated patients with Tay-Sachs disease. Immunotitration, immunoelectrophoresis, double-immunodiffusion and radial-immunodiffusion techniques were used to demonstrate the presence of CR material. The CR-material-antibody complex was enzymically inactive. Antiserum raised against kidney or placental hexosaminidase A, without cross-linking with glutaraldehyde, failed to precipitate the CR material, implying that treatment of the protein with glutaraldehyde exposes antigenic determinants that are hidden in the native protein. Since anti-(hexosaminidase B) antiserum did not precipitate the CR material during the immunoelectrophoresis of Tay-Sachs liver extracts, it is suggested that altered alpha-subunits do not combine with beta-subunits. By using immunotitration we have demonstrated the competition between the hexosaminidase B-free Tay-Sachs liver extract and hexosaminidase A for the common binding sites on monospecific anti-(cross-linked hexosaminidase A) antiserum. The amount of CR material in the liver samples from seven cases of Tay-Sachs desease was found to be in the same range as theoretically calculated alpha-subunits in normal liver samples. Similar results were obtained by the radial-immunodiffusion studies. The present studies therefore suggest that Tay-Sachs disease is caused by a structural-gene mutation.     

Fungi parasitising the eggs of certain fresh water fishes

By the present investigations the parasitic abilities of five isolates of Saprolegniaceous fungi have been established on the eggs of three fresh water fishesviz., Channa marulius Ham.,Channa gachua Ham., andNotoptenis notopterus Ham. These fungi areAchlya orion CokerAchlya prolifera (Nees) De Bary,Aphanomyces laevis Minden andSaprolegnia ferax (Gruith) Thuret.     

Purification and properties of glutathione peroxidase from human placenta.

Glutathione peroxidase (glutathione--H2O2 oxidoreductase; EC 1.11.1.9) was purified to homogeneity from human placenta by using (NH4)2SO4 precipitation, ion-exchange chromatography, Sephadex gel filtration and preparative polyacrylamide-disc-gel electrophoresis. Glutathione peroxidase from human placenta is a tetramer, having 4g-atoms of selenium/mol of protein. The molecular weight of the enzyme is about 85000 with a subunit size of about 22,000. Kinetic properties of the enzyme are described. On incubation with cyanide, glutathione peroxidase is completely and irreversibly inactivated and selenium is released as a low-molecular-weight fragment. Reduced glutathione, beta-mercaptoethanol and dithiothreitol protect the enzyme from inactivation by cyanide and the release of selenium. Properties of human placental glutathione peroxidase are similar to those of isoenzyme A reported earlier by us from human erythrocytes. The presence of isoenzyme, B, reported earlier by us in human erythrocytes, was not detected in placenta. Also selenium-independent glutathione peroxidase (isoenzyme II), which is specific for cumene hydroperoxide, was not present in human placenta.     

The artifactual nature of fluoride inhibition of reverse transcriptase and associated ribonuclease H

Surprisingly, the $\text{sn}$-1 configuration of 1-0-hexadecyl-2-acetyl-glycerylphosphorylcholine showed significant activity, 3.22 × 10^?9 M, when compared to the $\text{sn}$-3 enantiomer, 2.92 × 10^?10 M and a racemic mixture with a value of 7.2 × 10^?10 M. A methoxy substitution at the C-1 or C-2 position of octadecyl glycerylphosphorylcholine gave a derivative with high biological activity for stimulating serotonin release from rabbit platelets. A 1-0-dodecyl-2-methoxy analogue showed very low activity; also, a comparable series of 0-benzyl derivatives were inactive. Examination of 1-0-hexadecyl, 1-0-octadecyl- or 1-0-dodecyl-2-acetyl-$\text{sn}$-glyceryl-3-phosphorylcholine showed that the hexadecyl compound had three times the biological activity of the octadecyl and five times that of the dodecyl.