The frequency of marker changes in sugarcane plants regenerated from callus culture

This study investigates the frequency of apparent and permanent expression of marker change following two types of tissue culture, conventional callus and direct regeneration cultures, and for two markers it relates this frequency to that following breeding. Each clone was used for only one marker. After conventional callus culture, plants of the sugarcane clone Arundoid B, a clone having a growth habit with shortened internodes and leaves, were freed of this marker at a rate of 1 in 172 plants. Marker remission in a second clone with a leaf blotch was enhanced in the presence of a mutagen. Callus culture alone gave a remission rate of 1/280 plants, while treatment of callus with ethyl methanesulfonate gave a remission rate of 1/42 plants. Of two markers subjected to vegetative and sexual transmission, the first, a leaf marker, was stable in callus culture with no remissions; crossing with non-marker parents produced progeny with 54% lacking the marker. The second, a stalk marker (multibud), showed epigenetic effects during two generations of vegetative propagation; plants lacking the multibud marker produced vegetative progeny in which the marker reappeared. Nine crosses to nonmarker parents produced progeny of which an average of 29% had the marker. The use of stalk chimeras as markers demonstrated that passage through conventional callus or direct regeneration culture resulted in the loss of the donor phenotype in all plants regenerated. Phenotypic variation in plants derived from callus culture appears to arise from several sources; chimeral segregants, epigenetic transients, and mutational variants.     

Monoclonal antibodies against hepatitis B virus surface antigen: preparation and characterization

Hybridomas secreting HBsAg antibodies were obtained by fusing murine myeloma cell line P3-X63-Ag8 to spleen cells of BALB/c mice sensitized with HBsAg. The surface antigen used for immunization of mice was prepared by purification from pooled human plasma specimens. Resulting monoclonal antibodies were detected by the SPRIA method. Clones producing highest anti-HBs titres were used to prepare mouse ascitic fluids. Monoclonal antibodies in ascitic fluid reached a titre of 10(6) to 10(7) at a protein concentration of 1 mg per ml. Two of the prepared monoclonal antibodies, HBS-01 and HBS-02, both belonging to IgG1 subclass of immunoglobulins, were selected for further study in order to assess their potential useability in the commercial ELISA kit. The pI values for HBS-01 ranged from 6.60 to 6.85, for HBS-02 from 5.6 to 6.1. In solid phase ELISA test the use of HBS-01 antibody improved accuracy of the assay by increasing its detection sensitivity for HBsAg subtypes adw and ayw in the reference serum; this sensitivity was evidently much better than that seen with the commercially available rabbit polyclonal anti-HBsAg antibody. The monoclonal antibody HBS-01 is specific to the determinant "a", which makes it suitable for use in ELISA test aimed at HBsAg detection. The antibody HBS-02 showed a markedly better reaction with HBsAg subtype adw than subtype ayw and can thus be used with advantage for their discrimination.     

Cultivation of Tuleniy virus (strain Murman) in vitro.

In vitro culturability of Murman strain of Tuleniy flavivirus isolated recently in the northern regions of the USSR was studied. Stable PS pig kidney line was found suitable as a primary sensitive cell substrate for the isolation, proliferation and serial propagation of the virus. The pronounced pathogenicity of the virus to PS cells permits the testing of its infective activity comparable with i.c. titrations on mice, VNT in vitro and the plaquing technique. PS line is suitable for the demonstration and identification of the virus antigen and/or for the study of reproduction of the virus on cellular level using the technique of immunofluorescence.     

3H] norepinephrine binding by rat glial cells in culture. Lack of correlation between binding and adenylate cyclase activation.

Subcellular fractions prepared from rat glial cells in culture (clonal line c6) were used in an attempt to characterize the adrenergic receptor involved in adenylate cyclase activation. Both [3H] norepinephrine binding and enzyme activation were measured under identical experimental conditions. Binding sites for norepinephrine could be detected; their main characteristics were: apparant Km: 4 - 10-6 M, macimal capacity: 20 pmol/mg protein.Their stereospecificity towards structually related drugs was found to be different from the stereospecificity of the receptor involved in adenylate cyclase activation. Thus, 3-methoxydopamine (a competitive inhibitor of norepinephrine for adenylate cyclase activation), phenylephrine (a partial adrennergic agonist) and the blocking agent propranolol were unable to compete with [3H] norepinephrine for binding. On the other hand, several molecules like dopa bearing a catechol group and which are unable to interact with the adenylate cyclase as agonists or competitive inhibitors strongly inhibited [3H] norepinephrine binding. As in several other systems so far studied, the presence on the glial cell's membrane of a large number of "catechol-binding sites" makes it difficult to characterize the beta-adrenergic receptor.     

Crystal structure of the invertebrate bifunctional purine biosynthesis enzyme PAICS at 2.8 Å resolution

Two important steps of the de novo purine biosynthesis pathway are catalyzed by the 5‐aminoimidazole ribonucleotide carboxylase and the 4‐(N‐succinylcarboxamide)‐5‐aminoimidazole ribonucleotide synthetase enzymes. In most eukaryotic organisms, these two activities are present in the bifunctional enzyme complex known as PAICS. We have determined the 2.8‐Å resolution crystal structure of the 350‐kDa invertebrate PAICS from insect cells (Trichoplusia ni) using single‐wavelength anomalous dispersion methods. Comparison of insect PAICS to human and prokaryotic homologs provides insights into substrate binding and reveals a highly conserved enzymatic framework across divergent species. Proteins 2013; 81:1473–1478. © 2013 Wiley Periodicals, Inc.     

Chromosomal variation in social voles: a Robertsonian fusion in Günther’s vole

The study reports on chromosomes in several populations of social voles from south-eastern Europe and the Middle East. The standard karyotypes of individuals of Microtus hartingi and Microtus guentheri originating from both south-eastern Europe and Asia Minor comprised 54 mostly acrocentric chromosomes. However, variation between populations was found in the amount and distribution of C-heterochromatin in certain autosomes and the sex chromosomes. Furthermore, a specific pattern of argyrophilic nucleolar organizer region distribution was recorded in different geographic populations. In a population from Asia Minor, a heterozygous centric fusion of two autosomes was found. The G-banded karyotypes of M. guentheri and Microtus socialis were compared, and tandem fusions of autosomes were suggested as possible mechanism of the divergence. The karyotypes of the nine currently recognized species of social voles are reviewed, and implications of chromosomal data for systematics are evaluated.     

Influence of the O-phosphorylation of serine, threonine and tyrosine in proteins on the amidic 15N chemical shielding anisotropy tensors

Density functional theory was employed to study the influence of O-phosphorylation of serine, threonine, and tyrosine on the amidic ¹⁵N chemical shielding anisotropy (CSA) tensor in the context of the complex chemical environments of protein structures. Our results indicate that the amidic ¹⁵N CSA tensor has sensitive responses to the introduction of the phosphate group and the phosphorylation-promoted rearrangement of solvent molecules and hydrogen bonding networks in the vicinity of the phosphorylated site. Yet, the calculated ¹⁵N CSA tensors in phosphorylated model peptides were in range of values experimentally observed for non-phosphorylated proteins. The extent of the phosphorylation induced changes suggests that the amidic ¹⁵N CSA tensor in phosphorylated proteins could be reasonably well approximated with averaged CSA tensor values experimentally determined for non-phosphorylated amino acids in practical NMR applications, where chemical surrounding of the phosphorylated site is not known a priori in majority of cases. Our calculations provide estimates of relative errors to be associated with the averaged CSA tensor values in interpretations of NMR data from phosphorylated proteins.