卷 52, 编号 2 (2016)

Cytokines are a family of signaling polypeptides involved in intercellular interactions in the process of the immune response, as well as in the regulation of a number of normal physiological functions. Cytokines are used in medicine for the treatment of cancer, immune disorders, viral infections, and other socially significant diseases, but the extent of their use is limited by the high production cost of the active agent. The development of this area of pharmacology is associated with the success of genetic engineering, which allows the production of significant amounts of protein by transgenic organisms. The review discusses the latest advances in the production of various cytokines with the use of genetically modified plants.

In a basic investigation, the molecular interactions between 2 different fused products of Arabidopsis cysteine proteinase (CP) and cysteine proteinase inhibitor (CPI) pair “namely R1: H₂N-maltose-binding protein- CPI-CP-COOH and R2: H₂N-maltose binding protein-CP-CPI-COOH” and 4 different L-amino acids including L-Ala, L-Ser, L-Asp, and L-Phe were analyzed using experimental methods and computational tools. The activity of CP relatively increased in purified R2 product in which test L-amino acids tend to interact with CP/CPI pair. On the other hand, the functionality of R1 product (having no tendency to interact with CP/CPI pair) was not influenced by L-amino acids. Detection of the effect of L-enantiomers of amino acids on the fusion forms of 2 functionally related proteins such as CP and CPI is the first ever time work on this research area. As a research recommendation, manufacturing the switchable biological systems expressing the fused forms of CP/CPI pair was proposed to control the relative activities of proteolytic compounds in different environments in the future.

No eukaryotic species has a system for homologous DNA recombination of the mitochondrial genome. We report on an integrative genetic system based on the pQ-SRUS construct that allows the expression of the RecA recombinase from Bacillus subtilis and its transportation to mitochondria of Yarrowia lipolytica. The targeting of recombinant RecA to mitochondria is provided by leader sequences (5'-UTR and 3'-UTR) derived from the SOD2 gene mRNA, which exhibit affinity to the outer mitochondrial membrane and provides cotranslational import of RecA to the inner space of mitochondria. The accumulation of RecA in mitochondria of the Y. lipolytica recombinant strain bearing the pQ-SRUS construct has been shown by immunoblotting of purified mitochondrial preparations.

A system for the production of mutant recombinant human alpha-fetoprotein (rhAFP0) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS115/pPICZαA/rhAFP0, which produces unglycosylated rhAFP0 and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

The capacity of fungus Aspergillus niger to degrade anionic surfactants (AS) and coproduce the detergent compatible enzymes in a liquid Czapek-Dox medium supplemented with 0.3% powder detergent Merix (Henkel, Serbia) was examined in this study. The fungus was isolated from wastewater of Lepenica River (Kragujevac, Serbia), at a place where municipal wastewater discharged into the river. The concentration of AS in tested detergent and detergent-supplemented culture media was determined using a methylene blue active substances assay. The physico-chemical and biochemical changes of pH, redox potential, biomass dry weight, activities of alkaline protease and α-amylase were evaluated during fungal growth from 3-rd to 16- th day. The detergent caused an inhibitory effect on fungal growth and biomass dry weight (about 51.4%). Nevertheless, A. niger decomposed approximately 180.2 μg/mL or 30% of AS after 16 days. The results also showed that activities of alkaline protease and α-amylase were enhanced in presence of tested detergent for 372.0 and 12.7%, respectively. Overall, the obtained results indicate the potential application of fungus in wastewater treatment and detergent industry.

The influence of colonization of the pea (Pisum sativum L.) by aerobic methylobacteria of five different species (Methylophilus flavus Ship, Methylobacterium extorquens G10, Methylobacillus arboreus Iva, Methylopila musalis MUSA, Methylopila turkiensis Side1) on plant resistance to paraquat-induced stresses has been studied. The normal conditions of pea colonization by methylobacteria were characterized by a decrease in the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidases) and in the concentrations of endogenous H₂O₂, proline, and malonic dialdehyde, which is a product of lipid peroxidation and indicator of damage to plant cell membranes, and an increase in the activity of the photosynthetic apparatus (the content of chlorophylls а, b and carotenoids). In the presence of paraquat, the colonized plants had higher activities of antioxidant enzymes, stable photosynthetic indices, and a less intensive accumulation of the products of lipid peroxidation as compared to noncolonized plants. Thus, colonization by methylobacteria considerably increased the adaptive protection of pea plants to the paraquat-induced oxidative stress.

The effects of the salt stress (200 mM NaCl) and exogenous jasmonic acid (JA) on levels of osmolytes and flavonoids in leaves of four-week-old Arabidopsis thaliana L. plants of the wild-type (WT) Columbia-0 (Col-0) and the mutant jin1 (jasmonate insensitive 1) with impaired jasmonate signaling were studied. The increase in proline content caused by the salt stress was higher in the Col-0 plants than in the mutant jin1. This difference was especially marked if the plants had been pretreated with exogenous 0.1 μM JA. The sugar content increased in response to the salt stress in the JA-treated WT plants but decreased in the jin1 mutant. Treatment with JA of the WT plants but not mutant defective in jasmonate signaling also enhanced the levels of anthocyanins and flavonoids absorbed in UV-B range in leaves. The presence of JA increased salinity resistance of the Col-0 plants, since the accumulation of lipid peroxidation products and growth inhibition caused by NaCl were less pronounced. Under salt stress, JA almost did not render a positive effect on the jin1 plants. It is concluded that the protein JIN1/MYC2 is involved in control of protective systems under salt stress.

A number of alkylated (quaternized) and acylated derivatives of low–molecular weight chitosan were obtained. The structure and composition of the compounds were confirmed by the results of IR and PMR spectroscopy, as well as conductometric titration. The effect of the acyl substituent and the degree of substitution of N-(2-hydroxy-3-trimethylammonium) propyl fragment appended to amino groups of the C₂ atom of polymer chains on antibacterial activity against typical representatives of gram-positive and gramnegative microorganisms (Staphylococcus epidermidis and Escherichia coli) was studied. The highest activity was in the case of N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC) with the maximal substitution (98%). The minimal inhibitory concentration of the derivative was 0.48 μg/mL and 3.90 μg/mL for S. epidermis and E. coli, respectively.

Fluorescent and optical spectroscopy were used to study the interaction of alcohol dehydrogenase (ADH) with negatively charged polystyrene sulfonate (PSS) and dextran sulfate (DS), as well as positively charged poly(diallyldimethylammonium) (PDADMA). As found, DS and PDADMA did not affect the structural and catalytic enzyme properties. In contrast, PSS slightly decreased the protein self-fluorescence over 1 h of incubation, which is associated with partial destruction of its quaternary (globular) structure. Investigation of the ADH activity with and without PSS showed its dependency on the incubation time and the PSS presence. Sodium chloride (2.0 and 0.2 M) or ammonium sulfate (0.1 M) added to the reaction mixture did not completely protect the enzyme quaternary structure from the PSS action. However ammonium sulfate or 0.2 M sodium chloride stabilized the enzyme and partially inhibited the negative PSS effect.