An investigation of the effect of antisense RNA gene on bovine leukaemia virus reproduction in cell culture.

A possible approach to control of bovine lymphoproliferative disease caused by bovine leukaemia virus (BLV) may be the development of an "antiviral information immunity" based on the effect of anti-sense RNA (asRNA). A numbers of constructs were obtained, under control of various promotors (herpesvirus thymidine kinase, T-antigen SV40 promoter), carrying as DNA against gene X, the expression product of which is a transactivator of viral transcription from the BLV LTR promotor. As a model system for the analysis of antiviral activity of constructs developed, cloned continuous cell lines of BLV-producing FLK cells were used. The level of BLV expression in cells transfected with the constructs was determined by various parameters. Differences were detected in different clones obtained from non-transfected cells, as well as variation between transfected clones, as measured by reverse transcriptase, competitive radio-immunoassay for BLV p24, the viral particle count on agar membrane, and the tumorigenicity for nude mice. The differences in inhibition of expression of BLV genes and their products may be explained in terms of the site of integration of asDNA and the number of integrated copies.

[Nature of the endogenous retrovirus-like particles of the rat liver]. / Issledovanie prirody éndogennykh retrovirusopodobnykh chastits pecheni krys.

Previously virus-like particles (VLP) with properties resembling retroviruses were isolated from the liver of Wistar rats. Molecular hybridization and CRIA test were used for further analysis of the VLP. The CRIA method showed that VLP preparation lacked antigenic determinants of the major internal protein of C-type virus. By the dot hybridization technique no homology was detected between VLP and Mo-MuLV DNA, however VLP RNA was found to be homologous to IAP (interstitial A particles) DNA of mice. VLP proviral DNA was detected in the rat genome by the blot hybridization technique. Thus, it was concluded that VLP resemble IAP. A possible role of rat IAP expression is discussed.

Reverse transcriptase of rat liver associated with the endogenous retrovirus related to the mouse intracisternal A-particles.

Reverse transcriptase activity was found in rat liver enclosed in virus-like particles. Through hybridization with DNA probes of A- and C-type retroviruses and with the help of electron microscopy the virus-like particles have been identified as endogenous retroviruses related to the mouse intracisternal A-particles. Blot hybridization revealed the provirus DNA in the rat genome. The enzyme was isolated from the virus-like particles, purified and characterized. The main properties of the enzyme resemble those of the mammalian retrovirus reverse transcriptase.

[Mechanism of enzymatic imprinting induced in rats by an early postnatal administration of galactose]. / Izuchenie mekhanizma fermentativnogo imprintinga, indutsirovannogo u krys rannim postnatal'nym vvedeniem galaktozy.

Administration of galactose into young rats within an early postnatal period led to alteration in activity of some enzymes involved in utilization of galactose (galactose-1-phosphaturidyl transferase, galactose-6-phosphate dehydrogenase etc) for a long period of the animals life. This stable alteration in activity of adaptive enzymes was characterized as the enzymatic imprinting. After administration of galactose into neonatal animals synthesis of RNA, matrix activity of chromatin, activities of DNA-dependent RNA polymerase and RNA-dependent DNA polymerase were shown to increase in liver tissue of these animals. These alterations are considered as a possible basis for the stable alterations in the genes expression. The elevated activities of DNA-dependent RNA-polymerase and reverse transcriptase were maintained within a long period of the animals life.

[Changes in the RNA-dependent DNA-polymerase activity of retrovirus-like particles in the rat liver during postnatal ontogeny]. / Izmenenie RNK-zavisimoi DNK-polimeraznoi aktivnosti retrovirusopodobnykh chastits pecheni krys v protsesse postnatal'nogo ontogeneza.

The RNA-dependent DNA-polymerase activity was studied in the postmicrosomal fraction and in the microsomal sediment of the liver of the newborn and adult Wistar rats. In the microsomal sediment of 4-6 day old rats the RNA-dependent DNA-polymerase activity was approximately by one order of magnitude higher than in that of 2 week old and adult rats. In the postmicrosomal fraction of 3 day old rats the RNA-dependent DNA-polymerase activity was also higher, but only by 30-35%, than in that of animals of the other age groups. Particles with density of 1.17 g/ml were found in the microsomal sediment of all studied animals. The characteristic morphology, sensitivity of the particle RNA-dependent DNA-polymerase activity to RNAse and the presence of reverse transcriptase allow for these particles to be referred to as retroviruses. A suggestion is put forward that the high intensity of reverse transcription during the early postnatal period can be due to still continuing processes of cell differentiation and enzymatic imprinting.

[Relation of RNA-dependent DNA-polymerase from the rat liver with virus-like particles]. / Sviaz' RNK-zavisimoi DNK-polimerazy iz pecheni krys s virusopodobnymi chastitsami.

RNA-dependent DNA-polymerase activity was found in the 165 000 g supernatant and pellet of the postmitochondrial rat liver fraction. Further fractionation of the 165 000 g pellet in the linear sucrose gradient (20-50%) showed that RNA-dependent DNA-polymerase activity was distributed between fractions with densities 1.18-1.19 g/ml and 1.09-1.1 g/ml. In the fractions with 1.18-1.19 g/ml density the enzymic activity could be detected only after Triton X-100 treatment and disappeared after the incubation with pancreatic ribonuclease A. Triton X-100 treatment of the 165 000 g supernatant and the fractions with density 1.09-1.1 g/ml did not increase further the enzymic activity. Electron microscopy revealed in the 1.18 g/ml fraction virus-like particles resembling retroviruses of A and C type. In the light peak "non-mature" virus-like particles were found. The 165 000 g supernatant devoid of virus-like particles contained free RNA-dependent DNA-polymerase activity. The virus-like particles of both types seem to be endogenous rat retroviruses serving as a source of the particular and free reverse transcriptase in the rat liver.

[RNA-dependent DNA polymerase from rat liver]. / RNK-zavisimaia DNK-polimeraza iz pecheni krys.

RNA-dependent DNA-polymerase was isolated from rat liver, and its characteristics were studied. Wistar rat livers were homogenized in the disruptive buffer, centrifuged at 100,000 g and the supernatant was freed of the nucleic acids by DEAE-cellulose (DE-23) chromatography. The further chromatography of the eluate on DEAE-cellulose (DE-52) and phosphocellulose P-11 resulted in the obtaining of 300-400-fold purified RNA-dependent DNA-polymerase. Even more purified enzyme 1500-fold was isolated from the 165,000 g pellet of postmitochondrial rat liver fraction. The main properties of the purified enzyme are characteristic for the retroviral reverse transcriptase. The enzyme catalyzes DNA synthesis when poly(A)+mRNA is used as a template-primer. Its sedimentation constant amounts to 4.6 S. Mg2+ is preferable to Mn2+ as an activator of the enzyme. The optimal pH is 7.8. Among the products of the enzymic reaction hybrid RNA X DNA molecules were identified.

[Appearance of new transplantation antigens in tumor cells treated with RNA]. / Poiavlenie novykh transplantatsionnykh antigenov v opukholevykh kletkakh, obrabotannykh preparatami RNK.

Immunization of mice with the Wistar rat liver tissue increased their resistance to the subsequent intramuscular transplantation of Krebs-2 tumour cells preincubated with the rat liver RNA and did not affect the transplantability and growth of the same untreated tumour cells. The growth of the tumour cells pretreated with allogenic RNA did not differ from the growth of the untreated tumour cells when the mice were immunized with material genotypically different from the RNA tissue-source. When the immunizing tissue and RNA source were genotypically identical, the mass of the tumours growing in three tested mice strains (A/He, CC57BR, BALB/c) was 40-50% less than that of untreated tumours and the enhancement effect was observed in C3Hf mice. It is suggested that RNA preparations induce the appearance of the transplant antigens in tumour cells similar to those of RNA donors tissues. The effect of RNA preparations was abolished by RNAse incubation.

Probable role of reverse transcription in learning: correlation between hippocampal RNA-dependent DNA synthesis and learning ability in rats.

The activities of RNA-dependent DNA polymerase and DNA-dependent DNA polymerase were measured in hippocampus of fast and slow learning Wistar rats. The RNA-dependent DNA polymerase activity in the hippocampus of fast learning rats exceeds two-fold that in the slow learning ones, while the rates of the DNA-dependent DNA polymerase activities are similar. A significant increase in RNA-dependent DNA polymerase only was found in the hippocampus of rats 20 min after training for the conditioned food response before the trace consolidation registered 40 min after the training session. The data obtained are consistent with the suggestion that reverse transcription plays an important role in memory consolidation.

[Use of neonatal induction of enzymes for the correction of experimental hereditary enzymopathies]. / Korrektsiia nasledstvennykh fermentopatii v éksperimente putem neonatal'noi induktsii fermentov.

Administration of enzyme-inducing agents into newborn animals resulted in stable changes of the activities of the relevant inducible enzymes for long periods of their life in adulthood. The phenomenon designated as enzymic imprinting was used for correction of inherited enzymopathies in animals. Neonatal administration of galactose into the W/ssm rats with inherited galactosemia stably decreased galactose transport into the erythrocytes, increased activities of hexose oxidizing enzymes and prevented development of cataracts and other galactosemia symptoms. Neonatal administration of the inducer of mixed function oxidases into the SWR/J mice with inherited hypercholesterolemia stably increased the activities of these cholesterol oxidizing enzymes and abolished the hypercholesterolemia symptoms in adulthood. It is suggested that enzymic imprinting is due to amplification of genes coding the inducible enzymes on account of preferential copying of the induced mRNA's by reverse transcription. It is shown that the enzymic induction was accompanied by activation of RNA dependent DNA synthesis and the activity of this enzymatic system was distinctly higher in newborn animals.

[High-polymeric xenogenic RNA as an antitumor immunity stimulator]. / Vysokopolimernaia ksenogennaia RNK kak stimuliator protivoopukholevogo immuniteta.

To immunize CC57BR mice a suspension of live cells of Krebs-2 ascites tumour was administered intradermally into the tail partially amputated afterwards. The growth of the tumour transplanted intraperitoneally was inhibited by 23% only after twofold immunization. Single immunization with tumour cell incubated with the cattle liver RNA preparation in conjunction with intraperitoneal administration of RNA following tumour transplantation inhibited its growth by 43--53%, while twofold administration by 84--88%. The high polymeric fraction of the preparation enhanced the immunization effect to the same measures the initial overall preparation. The treatment of the preparation with RNAase and partial depolymerization of RNA in the course of isolation resulted in the activity loss. It is concluded that the capacity of the RNA preparation for stimulating antitumour immunity is due to high polymeric fraction of RNA.

[Effect of exogenous RNA on the activity of poly(A).oligo(dT)-dependent DNA-polymerase in mouse tumors]. / Vliianie ekzogennoi RNK na aktivnost' poli (A). Oligo(dT)-zavisimoi DNK-polimerazy v opukholiakh myshei.

The effects of exogenous RNA on the poly(A).oligo(dT)-dependent DNA-polymerase activity of factor and factor-free carcinomas of the mammary gland of C3H mice and Ehrlich ascite carcinoma were studied. It was shown that after intraparenteral injection of the RNA the enzyme activity is increased in the oncornavirus-containing carcinomas and remains unchanged in the virus-free tumours. The effect of RNA is not due to the glucocorticoid release into the blood and is suppressed by actinomycin D one hour prior to the addition of RNA. The increase of the poly(A).oligo(dT)-dependent DNA-polymerase activity of the cytoplasmic fraction of tumour cells is correlated with the rise of cytoplasmic RNA-polymerase activity and the increased incorporation of the precursors into the cytoplasmic RNA. Thus, in virus-containing tumours exogenous RNA induces the synthesis of cytoplasmic RNAs responsible for the synthesis of RNA-dependent DNA-polymerase.

Stimulation of reparative processes in the damaged myocardium by the RNA preparations.

RNA preparations were used as stimulants of reparative processes in rat myocardium damaged by diathermocoagulation. Administration of RNA accelerated the proteosynthesis in the zone of the lesion, as indicated by the intensity of incorporation of 14C-labelled amino-acids. Morphological examinations of the myocardium revealed increased synthetic activity in fibroblasts, proliferation of fibroblasts, and accelerated scar healing. The results prove the stimulating effect of RNA preparations on reparative processes in the damaged myocardium.