Specific targeting of human papillomavirus type 16 E7 oncogene with triple-helix forming purine oligodeoxyribonucleotides.

Molecular mechanical calculations (computer modelling), optical DNA melting experiments and co-migration assay were used to assess stable helix formation at homopurine-homopyrimidine-rich target sites present in the human papillomavirus type 16 E7 oncogene (positions 656-673 on the genome map). The target sequence, either present in the E7 oncogene obtained by PCR technique or prepared from oligodeoxyribonucleotides (ODNs), can be specifically recognised by different 17-merpurine ODNs designed to form antiparallel or parallel triple helices. These "in vitro" experiments realised with rather long purine ODNs having a high degree of specificity, open the way for "in vivo" tests focused on E7 oncogene targeting and suppression.

Specific targeting of human papillomavirus type 16 E7 oncogene with triple-helix forming purine oligodeoxyribonucleotides.

Molecular mechanical calculations (computer modelling), optical DNA melting experiments and co-migration assay were used to assess stable helix formation at homopurine--homopyrimidine-rich target sites present in the human papillomavirus type 16 E7 oncogene (positions 656-673 on the genome map). The target sequence, either present in the E7 oncogene obtained by PCR technique or prepared from oligodeoxyribonucleotides (ODNs), can be specifically recognised by different 17-merpurine ODNs designed to form antiparallel or parallel triple helices. These in vitro experiments realised with rather long purine ODNs having a high degree of specificity open the way for in vivo tests focused on E7 oncogene targeting and suppression.

Some new properties of DNA-YOYO-3 homodimer complexes revealed by electrophoresis and fluorescence lifetime measurements.

The DNA bis-intercalator oxazole homodimer (YOYO-3: 1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methy l- 2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene]-quinolinium tetraiodide) specifically alters the electrophoretic pattern of covalently closed circular (CCC) DNA molecules. Thus, YOYO-3 seems to remove the CCC DNA supercoils and induces the appearance of additional bands by changing the linking number. It also promotes an unusual "star" activity for the restriction enzyme Hind III. Fluorescence lifetime measurements indicate that YOYO-3 is capable of binding to DNA by bis- and mono-intercalation.

[The detection of human papillomaviruses in histological preparations by using dot-blot hybridization]. / Détection des papillomavirus humains sur des préparations histologiques à l'aide de l'hybridation dot-blot.

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 electrophoresis of biotinylated nucleic acids]. / Electrophorèse des acides nucléiques biotinylés.

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.

Estimation of avidin activity by two methods.

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.

[Research conducted at the Stefan S. Nicolau Institute of Virology of Bucharest in the area of nucleic acids and genetic engineering]. / Recherches conduites à l'Institut de Virologie "Stefan S. Nicolau" de Bucarest dans le domaine des acides nucléiques et du génie génétique.

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.

Cationic anticancer drugs and their modes of action.

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 effects of complex platinum compounds on the neuraminidase activity of the Sendai virus]. / Effets de quelques composés complexes du platine sur l'activité de la neuraminidase du virus Sendai.

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.

Some physico-chemical properties of the rigid form of the Sendai virus nucleocapsid.

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).

[The avidin-biotin system. Its application in virology]. / Le système avidine-biotine. Applications en virologie.

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.

[The protector effect of ribosomal preparations against experimental influenza infection in mice]. / Effet protecteur de quelques préparations ribosomales contre l'infection expérimentale à virus de la grippe chez la souris.

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.

[Adaptation of the avidin-biotin system for identifying and quantifying Sendai virus antigens]. / Adaptation du système avidine-biotine en vue de l'identification et du dosage des antigènes du virus Sendaï.

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.

[Study of the encapsulation and transport of several proteins to different organs by means of liposomal type particles of viral origin]. / Recherches sur l'encapsulation et le transport de quelques protéines vers différents organes par l'intermédiaire de particules du type liposomal d'origine virale.

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.

[Demonstration of viral proteins by silver staining]. / Mise en évidence des protéines virales à l'aide de la coloration par l'argent.

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.

Some physico-chemical and biological properties of egg-Sendai virus with altered haemagglutinin-neuraminidase protein.

Some physico-chemical and biological properties of egg-cultivated Sendai (egg-Sendai) virus particles with altered haemagglutinin-neuraminidase (HN) protein were studied. These particles were obtained accidentally from purified egg-Sendai virus stored at +4 degrees C. Egg-Sendai with altered HN protein was not infectious for cultured cells, but could still infect embryonated eggs. Though egg-Sendai virus with altered HN protein and glycoproteins had no haemagglutination activity, it unexpectedly possessed a relatively high neuraminidase (NA) activity representing about 70% of the activity of normal particles. Since polyacrylamide gel and rocket immunoelectrophoresis indicated that the altered particles contained only F protein, possibly the native HN was converted into a molecule of the same molecular mass as F protein.

Effect of some anthracycline antibiotics on the neuraminidase activity of Sendai virus and its isolated glycoproteins.

Anthracycline antibiotics violamycin BI (VBI) and adriamycin (Adr) strongly inhibited the neuraminidase (NA) activity of Sendai virus and of its isolated glycoproteins, occurring either in solution or associated in liposome-like particles. NA did not exhibit the classical Michaelis-Menten kinetics: the plot of reaction velocity versus substrate concentration was sigmoidal, while Hill's plotting indicated that the enzyme has at least two binding sites for the substrate. The reaction kinetics obtained suggested that Sendai virus NA acts as an allosteric enzyme, when its material support, the HN is located on the virus surface. The inhibitory effect of the anthracycline antibiotics in question on the NA activity seemed to be similar to that of negative effectors.

Isolation of poly(A)-rich mRNA from the chorioallantoic membrane of Sendai virus-infected chick embryos.

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.

Preliminary data on the encapsulation of biologically active materials in liposomes.

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.

Research in the field of nucleic acids performed in the "Stefan S. Nicolau" Institute of Virology.

A review is made of the research in the field of nucleic acids performed in the "Stefan S. Nicolau" Institute of Virology. The results obtained as regards the infectivity of viral nucleic acids, the oncogenic capacity of nucleic acids extracted from tumors, the isolation, characterization, physicochemical and biological activity of viral and cellular nucleic acids, as well as some achievements in recombinant DNA technology, are briefly presented.