RolB gene-induced production of isoflavonoids in transformed Maackia amurensis cells.

Maackia amurensis Rupr. et Maxim is a valuable leguminous tree grown in the Russian Far East, in China, and in Korea. Polyphenols from the heartwood of this species (primarily stilbenes and isoflavonoids) possess strong hepatoprotective activity. Callus culture of M. amurensis produced isoflavonoids and their derivatives. In pharmacological experiments, the callus complex was at least as effective, as the plant complex. To increase the yield of isoflavonoids, calli were transformed with the rolB gene of Agrobacterium rhizogenes. Neomycin phosphotransferase (nptII) gene was used for transgenic cell selection. Three rolB transgenic callus lines with different levels of the rolB gene expression were established. Insertion of the rolB gene caused alterations in callus structure, growth, and isoflavonoid production, and stronger alterations were observed with higher expression levels. MB1, MB2, and MB4 cultures accumulated 1.4, 1.5, and 2.1 % of dry weight (DW) isoflavonoids, respectively. In contrast, the empty vector-transformed MV culture accumulated 1.22 % DW. Isoflavonoid productivity of the obtained MB1, MB2, and MB4 cultures was equal to 117, 112, and 199 mg/L of medium, respectively, comparing to 106 mg/L for the MV culture. High level of expression of the rolB gene in MB4 culture led to a 2-fold increase in the isoflavonoid content and productivity and reliably increased dry biomass accumulation. Lower expression levels of the rolB gene in MB1 and MB2 calli did not significantly enhance biomass accumulation and isoflavonoid content, although the rolB gene activated isoflavonoid biosynthesis during the early growth stages and caused the increased content of several distinct compounds.

[Calcium-dependent mechanism of somatic embryogenesis in oncogene rolC expressing cell cultures of Panax ginseng].

It was shown earlier, that ginseng embryogenic cell culture 2c3 was obtained as a result of callus cells transformation with the Agrobacterium rhizogenes rolC oncogene. In the present report we determine that inhibitors of Ca2+-channels (LaCl3, verapamil, niflumic acid) certainly lowered the quantity of somatic embryos in the 2c3 cell culture. This is the evidence of the influence of calcium-dependent signal system on plant embryogenesis. Protein kinases inhibitors W7 and H7 also caused the lowering of somatic embryos quantity in the 2c3 cell culture. We analysed changes of CDPK genes expression in embryogenic 2c3 cell culture. Total expression decreased 1.2-1.5 times comparing with the control callus culture. CDPK expression in the 2c3 embryogenic culture lowered by the inhibition of expression of the gene subfamilies PgCDPK1 (PgCDPK1a and PgCDPK1b) and PgCDPK3 (PgCDPK3a). At the same time, expression of PgCDPK2 gene subfamily (PgCDPK2b and PgCDPK2d) was increased. We suppose that genes of PgCDPK2 subfamily might be responsible for the embryogenesis initiation in the 2c3 ginseng cell culture. It was shown for the first time that the rolC gene and the process of embryogenesis could change expression of particular forms of CDPK genes.

Ion regulation of the kinetics of potential-dependent potassium channels.

We apply a theoretical approach developed earlier. The interaction ofions that permeate a channel with slowly relaxing charged channel-forminggroups (ion-conformational interaction - ICI) is addressed by thisapproach. One can describe the ion concentration influence (ion regulation)on channel functioning in this manner. A patch-clamp method in a'whole-cell' configuration is used to study the ICI. For this purpose theinfluence of an external concentration of potassium ions on thepotential-dependent potassium current (I(A)) in the externalmembrane of GH(3) cells was studied. The increase of[K(+) (out)] from 5 mM to 100 mM causes anon-monotonous shift of current-voltage dependencies. The dependence of bothan activation time constant tgr(n) and a steady-state activation(n(∞)) on [K(+)](out) have a minimum andmaximum respectively. The analysis of the results suggests that the observedeffects are caused by ICI. A physical model is developed to describe thedependence of the potassium channel kinetics on the external concentrationof the ions and the membrane potential. The 'deformation' of the closedstate of the gate and the corresponding energy shifts cause the observednon-monotonous dependencies due to ICI. Thus, the general theoreticalapproach has an experimental confirmation and is applied to concreteexamples. Formulas for concentrational dependencies of the channel kineticsare given for practical uses.

[Intensity of lipid peroxidation processes and activity of antioxidant enzymes in erythrocytes during anemia in pregnancy]. / Intensivnost' protsessov perekisnogo okisleniia lipidov i aktivnost' antioksidantnykh fermentov v éritrotsitakh pri anemiiakh beremennykh.

The given report deals with investigation of the level of spontaneous lipid peroxidation (LPO) (as judged by the accumulation of MDA) and activity of antioxidative enzymes (GSH-peroxidase, GSH-reductase) in red blood cells of pregnant women (24-26 weeks) suffering from minor (blood Hb is 95 g/l) and medium (blood Hb is 85-90 g/l) anemias. We have found that as the anemia becomes deeper, the red blood cells of pregnant women demonstrate a linear increase in the level of spontaneous LPO, which rises by 54% during anemia of moderate severity (p < 0.02) as compared with control. At the same time, simultaneously with LPO, the activity of the above glutathione-containing enzymes increases as the anemia progresses, which, apparently, is of the compensatory nature, aimed at the maintenance of the reduced glutathione pool, the latter being an important component of the cell antioxidative system during LPO activation. The authors think the increased activity of the physiological antioxidative system and intensification of the LPO processes to be a natural adaptive process, since lipid hydroperoxides are the activators of synthesis of prostaglandins, which are required in delivery.