PERSPECTIVES OF THE DEVELOPMENT OF MUCOSAL VACCINES AGAINST DANGEROUS INFECTIONS ON THE BASE OF TRANSGENIC PLANTS

Mucosal vaccines created on the base of transgenic plants reacting with mucosal layers of the intestines and other organs are considered to be the perspective method of the vaccination. These vaccines induce both mucosal and general humoral immunogenicity after the peroral administration. The folding of antigenic proteins synthesizing in plants occurs via eukaryotic type and has advantages before yeast and prokaryotic platforms. This feature results to more adequate synthesis of antibodies against pathogens and to the interaction with effector molecules of complement. Earlier we together with The State Scientific Center “Vector”, Institute of chemical biology and fundamental medicine SB RAS and Dr R.Hammond from Laboratory of Plant Pathology (Maryland, USA) created two candidate vaccines : one of them against AIDS (HIV-1) and hepatitis B on the base of the chimeric gene TBI-HBS, encoding simultaneously 9 antigenic determinants of HIV-1 and the main surface antigen of hepatitis B (HBsAg). The second candidate vaccine was created against hepatitis B on the base of the genetic construct with the gene preS2-S encoding the synthesis of two subunits of the main surface antigen of hepatitis B and the signal peptide HDEL which directed antigens for the accumulation on ER. Both vaccines were tested on mice and confirmed their immunogenicity as the pronounced antibodies response. Twice vaccinated mice maintained the antibodies response during 11 months after there was little tendency to lowering. It was established that transgenic plants – vaccines (tomato) kept the capability to the synthesis of antigenic determinants in seven seed generations during 7 years. The results of the development of the mucosal vaccine against cervical carcinoma (carcinoma of uterine cervix) evoked by human papillomaviruses of high oncogenic risks were presented in this report. We created the genetic construct consisting of 35S CaMV promoter, Ώ (omega) leader of TMV, the target gene HPV16 L1 and the nos terminator. The target gene HPV16 L1 of the most oncogenic type 16 of human papillomavirus was choosen as the object. Different procedures of the plant transformation were elaborated and the transgenic plants synthesizing the antigenic protein L1 of human papillomavirus of type 16 were obtained. The insertion and the expression of the target gene were controlled by northern blotting, the synthesis of antigenic protein HPV16 L1 was determined by ELISA and western blot. The antigenic protein of HPV16 L1 was synthesized in amount of 20 – 50 ng/mg of total soluble proteins in tomato transgenic plants. The results of the examination of the immunogenicity of the vaccine obtained by means of the peroral immunization of mice were showed in the report. Therefore it was demonstrated the principal opportunity of the creation of mucosal vaccines on the base of transgenic plants against several dangerous diseases

The influence of osmotic stress on the content of calcium ions in the red beet vacuoles and on the transport activity of tonoplast proton pumps

The contents of calcium ions in the isolated vacuoles and in intact red beets under the conditions of dormancy and osmotic stress was determined. It is demonstrated that the content of calcium ions in the red beet vacuoles not exposed to osmotic stress makes 13.3% of the total content these ions in intact red beets. Under the conditions of osmotic stress, this indicator increases substantially. Furthermore, under the conditions of hyperosmotic stress, the content of calcium ions in the vacuoles was 30%, while under hypoosmotic stress it was 49% of the total content of these ions in the intact red beet. The transition of calcium ions from the cytoplasm and other compartments into the vacuole under the conditions of osmotic stress is, probably, one of forms of participation of the vacuole in adaptation processes of the plant cell under this kind of abiotic stress. It has been demonstrated for the first time that tonoplast proton pumps, which actively participate in provision of calcium homeostasis in cytoplasm, substantially activate their transport activity under osmotic stress, what allows one to speak about their important role in the cell’s protective programs. Under normal (no stress) conditions, artificial elevation of the content of calcium ions led to inhibition of activity of the tonoplast proton pumps, while under gipoosmotic stress the activity of tonoplast proton pumps increased, what might aid to restoring homeoctasis with respect to calcium ions in cytoplasm

Glutathione Reductase of Vacuole. Comparison of Glutathione Reductase Activity of Vacuole and Tissue Extract of Red Beet Root (Beta vulgaris L.)

Glutathione reductase (GR, EC 1.8.1.7) is the enzyme that reduces oxidized glutathione (GSSG) and thus regulates the redox state of glutathione (GSH/GSSG). GR has been studied in most plants. This enzyme has been identified in chloroplasts and cytosol, so these cellular compartments are considered to be the main place of the enzyme localization. In the same time, just a little is known about GR vacuoles. There are no conclusive evidences to prove the presence or absence of this enzyme in the vacuoles. GR activity was found in the vacuoles of red beet root cells (Beta vulgaris L.). The level of activity, the optimum pH and isoenzyme composition of GR were compared in the vacuoles and tissue extract of beet root. Vacuolar GR activity was quite high, it was 1.5-2 times higher than the activity of the tissue extract. Enzyme pH optimum of all the objects were identical. pH-optimum depend on the pyridine nucleotide nature: pH 7.0-8.0 was an optimal range with NADPH; pH 5.0 – with NADH. GR activity of the vacuoles and tissue extracts decreased in the presence of a noncompetitive inhibitor 1-chloro-2.4-dinitrobenzene (CDNB), indicating the specificity of this enzymatic reaction. Two bands with glutathione reductase activity have been identified in the vacuoles and tissue extracts using zymography method to determine the enzymatic activity in PAAG after electrophoresis of proteins. Belonging to the GR isoforms of these bands was confirmed by enzyme immunoassay (Western blotting). The electric mobility of isoforms of the study objects did not differ significantly. It is concluded that the biochemical characteristics of vacuolar glutathione reductase were substantially identical to the biochemical characteristics of other localization GR

Redox Enzymes of Red Beetroot Vacuoles (Beta vulgaris L.)

Years of research have shown that some of the redox elements (enzymes, coenzymes, and co-substrate) are isolated from each other kinetic and spatial manner (compartmentalization) in the eukaryotic cells. The redox elements forming the "highly" and "widely" specialized redox system are found in all cell structures: mitochondria, plastids, peroxisomes, apoplast, nucleus etc. In recent years the active involvement of the central vacuole in the maintenance of the plant cell redox homeostasis is discussed, actually the information about the vacuolar redox system is very small. The high-priority redox processes and "redox-specialization" of the vacuolar compartment are not known. We have begun a study of red beet-root vacuole redox systems (Beta vulgaris L.) and have identified redox enzymes such as: phenol peroxidase (EC 1.11.1.7), superoxide dismutase (EC 1.15.1.1) and glutathione reductase (EC 1.8.1.7). This paper presents some of the characteristics of these enzymes and considers the probable ways of their functioning in vacuolar redox chains

The Effect of Herbicides on Hydrogen Peroxide Generation in Isolated Vacuoles of Red Beet Root (Beta vulgaris L.)

Influence of herbicides on the hydrogen peroxide generation in vacuolar extracts of red beet root (Beta vulgaris L.) was investigated. Belonging to different chemical classes of herbicide compounds have been used. Herbicides differ from each other in the mechanism of effects on plants. Clopyralid (aromatic acid herbicide, derivative of picolinic acid) and 2.4-D (phenoxyacetic herbicide), characterized by hormone-like effects, contributed to the formation of H2O2 in vacuolar extracts. Fluorodifen (nitrophenyl ether herbicide) and diuron (urea herbicide) also have increased contents H2O2. These compounds inhibit the electron transport, photosynthesis, and photorespiration in sensitive plants. Herbicidal effect of glyphosate (organophosphorus herbicide) is due to the inhibition of amino acid synthesis in plant cells. Glyphosate did not affect the content of H2O2 in vacuolar extracts. Herbicide dependent H2O2-generation did not occur with oxidoreductase inhibitors, potassium cyanide and sodium azide. The results suggest that the formation of ROS in the vacuoles due to activity of oxidoreductases, which could interact with herbicides

Determination of Glutathione and Its Redox Status in Isolated Vacuoles of Red Beetroot Cells

The glutathione of the red beetroot vacuoles (Beta vulgaris L.) was measured using three well-known methods: the spectrofluorimetric method with orthophthalic aldehyde (OPT); the spectrophotometric method with 5.5'-dithiobis-2-nitrobenzoic acid (DTNB); the high-performance liquid chromatography (HPLC). The content of reduced (GSH) and oxidized glutathione (GSSG) differed depending on the research method. With OPT the concentration of glutathione was: GSH – 0.059 µmol /mg protein; GSSG – 0.019 µmol/mg protein and total glutathione (GSHtotal) – 0.097 µmol/mg protein. In the case of determining with DTNB the concentration of glutathione was: GSH – 0.091 µmol/mg protein; GSSG – 0.031 µmol/mg protein; GSHtotal – 0.153 µmol/mg protein. HPLC-defined concentration of glutathione was lower: GSH – 0.039 µmol/mg protein; GSSG – 0.007 µmol/mg protein; GSHtotal – 0.053 µmol/mg protein. Redox ratio of GSH/GSSG was also dependent on the method of determination: with OPT – 3.11; with DTNB – 2.96 and HPLC – 5.57. Redox ratio of glutathione in vacuoles was much lower than the tissue extracts of red beetroot, which, depending on the method of determination, was: 7.23, 7.16 and 9.22. The results showed the vacuoles of red beetroot parenchyma cells contain glutathione. Despite the low value of the redox ratio GSH/GSSG, in vacuoles the pool of reduced glutathione prevailed over the pool of oxidized glutathione