Synthesis of 4-triazolopyrimidinone nucleotide and its application in synthesis of 5-methylcytosine-containing oligodeoxyribonucleotides.

5'-0-Dimethoxytritylthymidine (2) was phosphorylated and base-modified simultaneously to yield the 4-triazolopyrimidinone nucleotide (3). Coupling between (3) and other common deoxyribonucleotides gave a fully protected nonamer (4). Deblocking under different conditions yielded the nonamer as phosphodiester with concomitant conversion of 4-triazolopyrimidinone to 5-methylcytosine (aqueous ammonia) or thymine (N1,N1,N3,N3-tetramethyl-guanidinium syn-4-nitrobenzaldoximate solution).     

Nutritional and karyotypic characterization of a haploid cell culture ofDaucus carota

Summary The purpose of this study was to optimize growth conditions for a strain of haploid carrot callus and to follow its karyotypic changes in a long span of time. The strain has been maintained in liquid suspension since September 1977. It has remained predominantly haploid in its karyotype since that time. The original explant was initiated and subsequently subcultured in Gamborg's B5 medium. The components of the B5 medium were omitted one at a time and sequentially added back to determine their minimum, optimum, and maximum nontoxic concentrations. These changes were made in the original formula: the addition of an organic buffering agent and an increase in the iron and other micronutrient concentrations. Using this slightly modified B5 medium, we assessed the effect on growth by single additions of amino acids, different carbon sources, growth regulators, and vitamins. No improvement in plating efficiency resulted from addition of any of these compounds. We conclude that there are factors limiting the plating efficiency of the haploid cells other than these tested, or that single additions will not make a discernible difference, or that growth promoting factors cannot be exogenously supplemented to cultured cells.     

Purification of chicken brain choline acetyltransferase

Chicken brain choline acetyltransferase was purified to homogeneity using ammonium sulfate fractionation, followed by chromatography on DEAE-Sephadex (A-25), hydroxyapatite, Sephadex G-150, immunoabsorption and Sepharose-CoA columns. A purification of 3500-fold was achieved and the final preparation had a specific activity of 2:32 μmol acetylcholine formed per minute per milligram protein. The purified chicken choline acetyltransferase migrated as a single band on polyacrylamide gel electrophoresis in the presence and absence of sodium deodecyl sulfate. The native enzyme, with a molecular weight of 67,000 daltons, consists of two subunits of identical molecular weight. Chicken choline acetyltransferase has a sharp pH optimum of 7.4. It is activated by sodium chloride and potassium chloride but inhibited by cupric ion and N-ethylmaleimide.     

Specificity of two isolated wheat carboxypeptidases

The substrate specificity of two purified carboxypeptidases from germinated wheat has been examined. Both enzymes were active on a wide variety of carbobenzoxy substituted peptides but inactive with unsubstituted dipeptides. Neither enzyme was active upon endoprotease or amidase substrates and only low levels of esterase activity were evident. In time course studies, both enzymes gave rapid non-specific sequential release of amino acids, including proline, from the carboxyterminal of proteins and polypeptides of known amino acid sequence.     

Sesquiterpenes of thalloid liverworts of the genera Conocephalum,Lunularia, Metzgeria and Riccardia

Thalloid liverworts of orders Metzgeriales and Marchantiales elaborate essential oils distinguishable from those of the Jungermanniales by the absence of β-barbatene and anastreptene. Riccardia sinuata elaborates a novel tricyclic exomethylene sesquiterpene of as yet undetermined structure. Conocephalum conicum elaborates cadinene-type sesquiterpenes. β-Cadinene from the latter species is clearly enantiomeric to the same product from vascular plants.     

Ultraviolet light induced preferential cross-linking of histone H3 to deoxyribonucleic acid in chromatin and nuclei of chicken erythrocytes

Histones have been cross-linked to DNA in chicken erythrocyte nuclei and chromatin by using ultraviolet light irradiation at 254 nm. Following irradiation, cross-linked histone-DNA adducts were isolated and purified by hydroxylapatite chromatography, and the DNA component was subjected to acid hydrolysis. Of several hydrolysis techniques investigated, trichloroacetic hydrolysis of the DNA component of the adducts was found to be most effective. Histones isolated from hydrolyzed histone-DNA adducts were characterized by gel electrophoresis and fingerprint analysis. No histone-histone protein adducts were observed. All histone fractions have been shown to cross-link DNA in nuclei or chromatin by utilizing the technique employed, but with different propensities. The order of observed cross-linking, deduced from kinetic experiments, is H1 + H5, H3 greater than H4 greater than H2A much greater than H2B. The preferential binding of the core histone H3, as compared to the other core histones, is discussed in light of recent data concerning histone-DNA interactions and nucleosome structure. The use of the ultraviolet light technique as a conformational probe to study chromatin is also discussed.     

Association of neutral α-glucosidase with cytoplasmic granules of peritoneal macrophages

A neutral α-glucosidase (EC 3.2.1.20) activity was shown to be associated with granules which are sedimentable at 10 000 g after differential centrifugation of mouse peritoneal macrophage homogenates. When the post-nuclear supernatant was centrifuged in a sucrose density gradient, high activities for neutral α-glucosidase and β-glucuronidase (EC 3.2.1.31) were detected in the bottom fractions because of aggregation of the granules. Neutral α-glucosidase-containing granules were completely disaggregated by the addition of 20 units/ml of heparin and 10 mM Tris-HCl (pH 7.2), which caused only a partial disaggregation of β-glucuronidase-containing granules. The addition of a high concentration of heparin, Tris buffer, or KCl to the gradient gave the same patterns of disaggregation of the granules. Under the condition in which about 50% of the total β-glucuronidase activity was released into the medium, depending on phagocytosis, very little α-glucosidase was released. These observations suggested that neutral α-glucosidase may localize in non-lysosomal granules.     

Biochemical and Immunological Characterization of Collagenase in Tissues of Metamorphosing Bullfrog Tadpoles

Tissue inhibitor of metalloproteinases (TIMP, a specific inhibitor of collagenase) was found to inhibit thyroid hormone-induced tail regression, suggesting the important role of collagenase in this process. Collagenase was purified from culture media of back skin of tadpole of bullfrog, Rana catesbeiana . Anti-tadpole collagenase polyclonal antisera were obtained against the purified enzyme. The antibody inhibited the activity of tadpole collagenase. The antisera reacted to tissues of adult bullfrogs, tadpoles of african clawed frog, Xenopus laevis , and adult newts, Cynopus pyrrhogaster , and also reacted to human fibroblast collagenase. Immunoblot analyses suggested that tadpole collagenase lacks the procollagenase which is generally found in mammalian collagenases. Intense immunological stains were observed for the tissues of thyroid hormone-treated tadpoles as compared to those of untreated animals. Thyroid hormone increased amounts of collagenase not only in epidermal layer but also in mesenchymal tissues including fibroblastic cells.