[Column chromatography of nucleic acids].
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Biomedical subjects
Publications and source records attributed to L M Popa.
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An analysis is presented of the nature, number and relative molecular mass of the polypeptides of three influenza virus strains-A/Romania 1/73 (H3N2), A/Romania 1/75 (H3N2), and B/Romania 34/74-isolated during different epidemic outbreaks. The results obtained indicate some similarities between the protein subunits of the two influenza A virus strains (as concerns virus surface and nucleocapsid components) and point out the particular properties of the influenza B strain.
A simple and rapid method was worked out to evaluate the biotinylation level of the pBR322 and pSVK1H genetic cloning vectors, using gel electrophoresis. Avidin was used to slow down the migration of biotinylated DNA: the DNA migration speed diminished as the biotinylation level rose, due to DNA complexation. The highest level of biotinylation is characterized by the formation of a biotinylated nucleic acid-avidin complex with no electrophoretical mobility.
The 56 tissue samples were obtained from female patients with anogenital tumors, benign or malign, subsequently confirmed through pathomorphological analysis. The investigations were aimed at the discovering of HPV presence using hybridization techniques with biotinylated molecular probes and monitorisation through enzymatic reaction. The presence of ADN types 11, 16 and 18 was detected on about 52% of all the cases; the ADN HPV incidence was 57% among the patients with a neoplasm and/or cervix uteri papillomatosis diagnosis.
The biological activity of avidin was estimated by two different methods. The spectrophotometric method used the avidin titration with biotin in the presence of 4 hydroxiazobenzen-2'carboxilic acid as indicator. In the radioisotopic determination the titration with tritiated biotin was accomplished. Both methods led to the same results, but the spectrophotometric one is less avidin expensive and more rapid, being more convenient.
Data are presented on the separation and on some biochemical properties of an antigen to be used in the immunodiffusion (ID) test for detection of bovine leukemia virus. The antigen obtained proved to have the same efficiency in the ID test as a commercially available standard antigen. Experimental results suggest that the antigen is released into the culture fluid in the form of a glycoprotein molecule consisting of two subunits with a molecular weight of 60,000 linked by--SH bonds.
Investigations were conducted to establish the optimal conditions for silver staining of polypeptides separated by polyacryle-amide gel electrophoresis. The thickness of the gel layer was of 1.5 mm. The experiments showed that the modified Hankeshover and Dernick's technique (4) (replacement of the fixative for the electrophoretically separated polypeptides and extension of the bleaching by Farmer's reagent time) had the best sensitivity and reproducibility. This technique can be performed also on polyacryle-amide gel preparations already stained by the Coomassie Brilliant Blue method. To check the efficiency of this silver staining technique, the authors used it to detect the Sendai virus and B hepatitis virus surface antigen polypeptides.
The avidin-biotin system was adapted in view of the identification and dosage of the Sendai parainfluenza virus and of its antigens, using the method of double antibodies (biotinylated and nonbiotinylated) in ELISA type tests.
A study was conducted on the protective effect of some ribosomal preparations, isolated from chorionic-allantoic membranes of chicken embryos, infected or not with parainfluenza (Sendai) or influenza (AoPR8) virus, in mice experimentally inoculated with influenza virus strain AoPR8 adapted to the mouse. Results showed that the tested preparation, containing ribosomes and polysomes isolated from chorio-allantoic membranes of Sendai virus inoculated chicken embryos, ensure the mice complete protection against AoPR8 virus, if administrated before the control infection.
The effect of di- and tetravalent cis-diaminoplatinum chlorides on Sendai virus envelop HN glycoprotein was investigated. The partial inhibition of neuraminidase activity was greater in the case of the divalent platinum complex derivative.
The effect of some dissociation agents (SDS, beta-mercaptoethanol, urea, EDTA) on the rigid form of the Sendai virus nucleocapsid was studied. Polyacrylamide gel electrophoresis in the presence of lytic mixture (1% SDS, 2% beta-mercaptoethanol, 5 M urea, for 2 min at 100 degrees C) revealed two types of polypeptide subunits (mol. wts. 46,000 and 14,000), as well as the dissociation in the presence of 0.1% SDS only. The EDTA treatment leads to a disorganization of the protein part (10(-2) M) or of the nucleocapsid structure (5 x 10(-2) M).
This report focuses on two groups of cationic cancerostatics, anthracycline antibiotics and 1,4-benzoquinone-guanylhydrazone-thiosemicarbazone (ambazone), lining up biophysical and biochemical effects on the level of membranes and membrane constituents. The interaction of both drugs with multilamellar liposomes consisting of phosphatidylcholine used as a simple model membrane system could be ensured by means of steady state and nanosecond time-resolved fluorometric investigations. The biochemical effect on membranes is underlined by the inhibition of the neuraminidase activity of the Sendai virus, modification of the CAMP phosphodiesterase activity of leukemia L 1210 cells of mice and reduction of the lymphocyte blast transformation.
The report briefly reviews the strategies of utilisation of the avidin-biotin system and its variants, as well as the applications of this system in different fields, especially those linked to the diagnosis and the treatment of some viral and bacterial infections. Some of the techniques using the system are presented, as well as its advantages and the perspectives to ameliorate the methodologies and to enlarge the field of its applications.
A brief review is done of some of the most important studies realised by the "Stefan S. Nicolau" Institute researchers in the field of nucleic acids. It is worth mentioning among these the investigations about interactions between nucleic acids and some biologically active substances, the recombinant DNA, as well as the most recent works on DNA-DNA hybridization in the avidine-biotine system.
Liposomal particles may be more efficiently incorporated by cells through mechanisms still incompletely elucidated. This property allowed to use them as a vehicle for macromolecules. Research was conducted to obtain liposomal type particles of viral origin charged with various proteins (bovine serum albumin, ovalbumin, ribosomes, human 125I-immunoglobulin G) and to establish the distribution of proteins encapsulated in viral envelopes among various organs after inoculation to laboratory animals.
Total RNA was extracted from the chorioallantoic membrane (CAM) of Sendai virus-infected chick embryos by the original Kecskeméthy-Schäfer method and by a modification of this technique, including phenol treatment, in order to ensure complete removal of proteins. The purity of the total RNA extracted by the modified technique was ascertained by spectral analysis. Chromatography on oligo(dT)-cellulose of the total RNA led to the isolation of poly(A)-rich mRNA--the material retained by oligo(dT)-cellulose and eluted with the buffer of the lowest ionic strength. The ability of the poly(A)-rich mRNA thus obtained to stimulate incorporation of 14C-leucine into protein was demonstrated in a cell-free protein-synthesizing assay system.
Multilamellar and unilamellar phospholipid liposomes were prepared and investigated as regards their properties and the capacity of encapsulating biologically active materials, such as CrO4-(2-)ions, basic dyes, proteins, DNA, as well as Sendai virus particles. The efficiency of encapsulation ranged from 10% for CrO4-(2-)ions to about 80% for toluidine blue; it was found to depend not only on the type of encapsulated material, but also on the method used for liposome preparation and on liposome composition.