[Expression of foreign gene by cysteine proteinase null recombinant baculovirus].

The baculovirus expression vector systems (BEVS) are broadly used for producing foreign proteins in lepidopteran larvae. Most commercial BEVS are engineered to insert foreign genes into the polyhedrin (polh) locus and lack the polh gene. These viruses cannot produce occlusion bodies and are inconvenient for per os inoculation of larvae. Current knowledge in baculovirus genomics makes it possible to engineer BEVS into other parts of the virus genome. In our work, we have expressed recombinant M-HBsAg (middle surface antigen of human hepatitis B) in the baculovirus construct, rBmNPV-Deltav-cath-M-HBsAg, inserting foreign gene into the v-cath locus of the Bombyx mori nucleopolyhedrovirus (BmNPV) such that the v-cath gene is deleted and the native polh gene is retained. Silkworm larvae were infected per os and M-HBsAg was observed to be abundantly produced at a very late stage of infection.

The Homingbac baculovirus cloning system: An alternative way to introduce foreign DNA into baculovirus genomes.

An in vitro baculovirus cloning system has been developed for direct cloning of foreign DNA into baculovirus genomes. This system is called the "Homingbac system" because it uses homing endonucleases. The Homingbac system was engineered into the baculoviruses AcMNPV, BmNPV, PxMNPV, RoMNPV, HaSNPV and HzSNPV. All Homingbac viruses were designed to retain the polyhedra phenotype so that they could be inoculated per os to insects. This is the first time a common in vitro baculovirus cloning system has been made for multiple baculovirus species that include both groups I and II nucleopolyhedroviruses (NPVs). In this study, the Homingbac system was demonstrated by directly cloning a PCR-amplified beta-glucuronidase gene cassette into a parent Homingbac virus. This new collection of groups I and II NPV Homingbac viruses are a significant expansion of in vitro cloning technology and are new tools for making recombinant baculoviruses.

[Biosynthesis of recombinant human hepatitis B M-HBsAg in silkworm larvae]. / Biosintez rekombinantnogo M-HBsAg virusa gepatita B cheloveka v lichinkakh tutovogo shelkopriada.

The middle surface antigen (M-HBsAg) of human hepatitis B virus is virus envelope protein. It's used as a basis for development of vaccine and test-system for detecting of hepatitis B virus. The cDNA of M-HBsAg was inserted into transfer vector pBK273 under the polyhedron promoter with obtaining of recombinant plasmid DNA pBHep-2. As a result of cotransfection pBHep-2 with wild type BmNPV the recombinant baculovirus rBmNPVHep which included the cDNA of M-HBsAg under the polyhedron promoter was obtained. Infection of silkworm larvae Bombyx mori with recombinant virus resulted in expression of foreign gene and accumulation of middle surface antigen of human hepatitis B virus mostly (>90%) in fat bodies of silkworm larvae.

[The translocation and specific binding of the pesticide fluometuron (cotoran) with the proteins of rat liver nuclei]. / Translokatsiia i spetsificheskoe sviazyvanie pestitsida fluometurona (kotorana) s belkami iader pecheni krys.

Intracellular distribution of labeled cotoran was studied. 3H-cotoran was shown to penetrate through the nuclear membrane to accumulate uneventfully in the intranuclear components. An insignificant amount of 3H-cotoran was associated with the nucleoplasm and the outer nuclear membrane. At the same time, essential radioactivity was observed in the proteins of the nuclear matrix (up to 30%) and in non-histone proteins of chromatin (up to 60%). Acception of 3H-cotoran on metaphase chromosomes of cultured cells as well as specificity of cotoran binding with non-histone proteins of chromatin in vivo and in vitro was studied by radioautography. It was revealed that cotoran was translocated into the interphase nuclei to be accepted by metaphase chromosomes of the HeLa line cells and fibroblasts in human embryo, and specifically, in receptor-like manner, bound to chromatin proteins.

Transfection of insect cell lines using polyethylenimine.

Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been frequently done using labor intensive and cytotoxic liposome-based transfection reagents. In the current study we have optimized a new kind of polyethylenimine-based DNA transfection reagent on the Spodoptera frugiperda Sf9 insect cell line. A plasmid vector that transiently expresses green fluorescent protein (GFP) was effectively delivered into Sf9 cells. A transfection efficiency of 54% and cell viability of 85-90% were obtained for Sf9 cells. The developed transfection protocol has now been successfully used to transfect eight insect cell lines derived from Bombyx mori, Trichoplusia ni, Helicoverpa zea, Heliothis virescens and S. frugiperda with GFP and GUS with transfection efficiencies of at least 45%. This method provides high heterologous protein expression levels, transfection efficacy and cell viability, and could be used for transient gene expression in other lepidopteran cell lines.

[Induction of Ca2+ ion transport in human thrombocytes as affected by thyroid hormone receptors from malignant transformed cells]. / Induktsiia transporta ionov Ca2+ v trombotsitakh cheloveka pod deistvie retseptora tireoidnykh gormonov zlokachestvenno transformirovannykh kletok.

The effect of thyroid hormone receptors isolated from normal and malignant cells on the induction of Ca2+ transport to platelets was studied. It is established that the receptor isolated from malignant cells by thyroxin (T4) or triiodothyronine (T3) stimulates (three-fold) the induction of Ca2+ transport to platelets. The effect is blocked by verapamil, which is the inhibitor of Ca2+ channels in plasma membranes. It is shown that neither the receptor of cancer cells, nor hormones (T3 or T4) influence the transport of Ca2+ to platelets without preliminary complexing. The hormone-receptor complex failed to affect the concentration of "cytoplasmic" Ca2+ in platelets analogous experimental conditions. It is suggested that the increased Ca2+ content in cancer cells can be partially attributed to the ability of thyroid hormone receptor of cancer cells to induce Ca2+ transport across the plasma membrane.

[The nature of thyroid hormone receptors. The role of serum thyroxine binding prealbumin in the realization of the hormonal effect]. / O prirode retseptorov tireoidnykh gormonov. Rol' tiroksinsviazyvaiushchego preal'bumina syvorotki krovi v realizatsii gormonal'nogo éffekta.

Data from determination of molecular weight and competitive displacement suggest that T3 and T4 are bound to the same protein in chromatin. It was shown that the antigenic determinants of T3 and T4 for the chromatin-binding protein coincide with those for blood serum thyroxine-binding prealbumin (TBPA). It was found also that the binding either to T3 and T4 decreases proportionally to the amount of the TBPA removed from the subcellular fractions. It may thus be concluded that blood serum TBPA is responsible for the binding to T3 and T4 as well as for the realization of the hormonal response.

[Nature of the intracellular thyroid hormone receptor]. / O prirode vnutrikletochnogo retseptora tireoidynkh gormonov.

Thyroxine-binding proteins were isolated and purified by means of affine chromatography from the blood serum, 105 000 g of cytosol supernatant, non-histone protein fractions of chromatin, extracted with 0.4 M KCl. Identity of their molecular weights and of end aminoacids was found. A comparative study of proteins above, using chromatoelectrophoresis, has shown identity of their primary structure. The data obtained indicate, that chromatin and cytosol contain universal, thyroxine-binding protein, which structure is identical with that of prealbumin thyroxine-binding protein. This protein is considered to be a receptor, realizing the control of genome expression by thyroid hormones.

[Effect of thyroid hormone receptors in normal and cancerous cells on the ionic conductivity of bilayer phospholipid membranes]. / Vliianie retseptorov tireoidnykh gormonov normal'nykh i rakovykh kletok na ionnuiu provodimost' bisloinykh fosfolipidnykh membran.

The effect of thyroid hormones receptors isolated from normal and cancer cells on bilayer phospholipid membranes (BPhLM) conductivity, has been studied. The receptor isolated from normal cells in complex X with the hormone selectively induces H+-conductivity of BPhLM generating transmembrane potential equal to 42 mV on the membrane at pH gradient equal to 1. In the presence of K+, Na+, Ca+, Mn2+, Sr2+, Mg2+ the changes of BPhLM are not observed. Neither hormones (T3, T4) nor receptor in free position affect the BPhLM conductivity. Thyroid hormone receptor isolated from mamalignantly transformed cells in a complex with T3 or T4 increases the BPhLM permeability for Ca2+. The transmembrane potential measured at 10fold Ca2+ ion concentration is equal to 16 mV. In the presence of H+, K+, Na+, Mn2+, Sr2+, Mg2+, Ba2+, the resistance of BPhLM doesn't change.

[The nature of thyroid hormone receptors. Intracellular functions of thyroxine-binding prealbumin]. / O prirode retseptorov tireoidnykh gormonov. Vnutrikletochnye funktsii tiroksinsviazyvaiushchego preal'bumina.

The effect of tyroxin-binding prealbumin (TBPA) of blood serum on the template activity of chromatin was studied. It was found that the values of binding constants of TBPA for T3 and T4 are 2 X 10(-11) M and 5 X 10(-10) M, respectively. The receptors isolated from 0.4 M KCl extract of chromatin and mitochondria as well as hormone-bound TBPA cause similar effects on the template activity of chromatin. Based on experimental results and the previously published comparative data on the structure of TBPA, nuclear, cytoplasmic and mitochondrial receptors of thyroid hormones as well as on translocation across the plasma membrane and intracellular transport of TBPA, a conclusion was drawn, which suggested that TBPA is the "core" of the true thyroid hormone receptor. It was shown that T3-bound TBPA caused histone H1-dependent conformational changes in chromatin. Based on the studies with the interaction of the TBPA-T3 complex with spin-labeled chromatin, a scheme of functioning of the thyroid hormone nuclear receptor was proposed.

[Effects of pesticide fluometuron (cotoran) on template synthesis of RNA]. / Vliianie pestitsida fluometurona (kotorana) na matrichnyi sintez RNK.

The experiments on the investigation of pesticide fluometuron (cotoran) influence on nuclease sensitivity and template activity of rat liver chromatin were carried out. Cotoran was found to bind specifically with non-histone proteins of chromatin. It was shown that this pesticide considerably decreases template activity of chromatin and its sensitivity to the action of nucleases. It suggests, that certain conformation changes occur in chromatin upon the action of cotoran.

[RNA synthesis and transport in the rat liver under the effects of pesticide cotoran (fluometuron)]. / Sintez i transport RNK v pecheni krys pri deistvii pestitsida kotorana (fluometurona).

The experiments on the investigation of pesticide cotoran-effect on RNA synthesis and transport were carried out. Cotoran was shown to destroy considerably the processes of RNA biosynthesis in rat liver, that results in the decrease of RNA transport from nuclei into cytoplasm. By special experiments it was established that functional activity and the integrity of nuclear membrane (according to the alteration in the activity of nuclear membrane enzyme Mg2-dependent ATP-ase) was not destroyed.

[The nature of thyroid hormone receptors. Translocation of thyroid hormones through plasma membranes]. / O prirode retseptorov tireoidnykh gormonov. Translokatsiia tireoidnykh gormonov cherez plazmaticheskuiu membranu.

The in vivo translocation of thyroxine-binding blood serum prealbumin (TBPA) was studied. It was found that the TBPA-hormone complex penetrates-through the plasma membrane into the cytoplasm of target cells. Electron microscopic autoradiography revealed that blood serum TBPA is localized in ribosomes of target cells as well as in mitochondria, lipid droplets and Golgi complex. Negligible amounts of the translocated TBPA is localized in lysosomes of the cells insensitive to thyroid hormones (spleen macrophages). Study of T4- and T3-binding proteins from rat liver cytoplasm demonstrated that one of them has the antigenic determinants common with those of TBPA. It was shown autoimmunoradiographically that the structure of TBPA is not altered during its translocation.

[The nature of thyroid hormone receptors. Thyroxine- and triiodothyronine-binding proteins of mitochondria]. / O prirode retseptorov tireoidnykh gormonov. Tiroksin- i triiodtironinsviazyvaiushchie belki mitokhondrii.

T4- and T3-binding proteins of rat liver were studied. It was found that the external mitochondrial membranes and matrix contain a protein whose electrophoretic mobility is similar to that of thyroxine-binding blood serum prealbumin (TBPA) and which binds either T4 or T3. This protein is precipitated by monospecific antibodies against TBPA. The internal mitochondrial membrane has two proteins able to bind thyroid hormones, one of which is localized in the cathode part of the gel and binds only T3, while the second one capable of binding T4 rather than T3 and possessing the electrophoretic mobility similar to that of TBPA. Radioimmunoprecipitation with monospecific antibodies against TBPA revealed that this protein also the antigenic determinants common with those of TBPA. The in vivo translocation of 125I-TBPA into submitochondrial fractions was studied. The analysis of densitograms of submitochondrial protein fraction showed that both TBPA and hormones are localized in the same protein fractions. Electron microscopic autoradiography demonstrated that 125I-TBPA enters the cytoplasm through the external membrane and is localized on the internal mitochondrial membrane and matrix.

[The nature of thyroid hormone receptors. Intracellular transport and compartmentalization of chromatin receptors and thyroid hormones]. / O prirode retseptorov tireoidnykh gormonov. Vnutrikletochnyi transport i kompartmentalizatsiia khromatinovogo retseptora tireoidnykh gormonov.

It is well established that in vivo administered labelled TBPA penetrates into liver, brain and lung cells, is translocated from cytosol into the nucleus and is accepted by chromatin without being affected by modifications touching upon the antigenic determinants of this protein. Electron microscopic autoradiography demonstrated that 125I-TBPA translocated from cytosol into the nucleus is localized on the border between hetero- and euchromatin. The data obtained may serve as an additional proof of the universal structure of intracellular thyroid hormone receptors and suggest that TBPA participate in manifestation of genetic effects of thyroid hormones.