Role of active forms of oxygen in the induction of phytoalexin synthesis in Allium cepa cells

The results of studies on the role of plant superoxidesynthase signal system in elicitation of antimicrobial phytoalexin (PA) synthesis in cultured Allium cepa cells are presented. Exogenic application of O2- and H2O2 generators results in formation of PA--Tcibulins 1D and 2 [symbol: see text] in A. cepa cells. The mechanism of PA elicitation does not require peroxidase activity. However, the inhibition of one of the possible sources of the reactive oxygen, HADPH oxidase, suppresses elicitor-stimulated PA production. "Oxidative burst" modulation by different chemical compounds in A. cepa cells results in changes of PA synthesis elicitation. The results obtained suggest the tough correlation between "oxidative burst" and elicitation of defense responses, PA synthesis in particular.     

To the research of the gene pool of the Gagauz population of Moldavia

Population genetic data on Gagauzes from Moldavia are reported here for the first time. AB0 and Rhesus blood groups, serum protein group (HP, TF, GC) and the red cell enzyme polymorphism PGM1 were determined in 190 Gagauzes. In addition to this the ability to taste PTC was tested. The following allele frequencies were found: AB0*0 = 0.5241, AB0*A = 0.3279, AB0*B = 0.1480; RH*D = 0.6083, RH*d = 0.3917; HP*1 = 0.3544, HP*2 = 0.6456; TF*C1 = 0.7472, TF*C2 = 0.1770, TF*C3 = 0.0730, TF*B = 0.0028; GC*1F = 0.1025, GC*1S = 0.5932, GC*2 = 0.3043; PGM*1+ = 0.5932; PGM*1- = 0.1000, PGM*2+ = 0.2607, PGM*2- = 0.1107. The frequency of the PTC*T allele was found to be 0.5298. These frequencies and genetic distance analyses show that the gene pool of the Gagauzes is similar to that of neighbouring southeastern European populations.     

Seasonal dynamics of amino acids in two small Siberian reservoirs dominated by prokaryotic and eukaryotic phytoplankton

The comparison of the dynamics of phytoplankton biomass and total amino acid composition was made for two water bodies: in one the phytoplankton were dominated by prokaryotes (i.e., there was a bloom of cyanobacteria) and by eukaryotic microalgae in the other. The dynamics of phytoplankton biomass and of total amino acid composition of water were investigated during the vegetation season. It was found that the only factor that significantly changed the percentages of amino acids in water was the bloom of cyanobacteria in the blooming water body. During the bloom of cyanobacteria, the absolute and relative content of the Leu-Glu group increased, while the contents of other acids generally dropped. Before and after the bloom, no significant variations in the total amino acid composition were recorded. In the reservoir where eukaryotic microalgae dominated, no significant variations in amino acid composition were recorded during the season.     

Truncated aspartate aminotransferase from alkalophilic Bacillus circulans with deletion of N-terminal 32 amino acids is a non-functional monomer in a partially structured state

Aspartate aminotransferase (AspAT) from alkalophilic Bacillus circulans contains an additional N-terminal sequence of 32 amino acid residues that are absent in all other AspATs from different sources. Modeling suggested that this sequence forms two alpha-helical segments which establish a continuous network of interactions on the surface of the molecule. In the present study, we studied the role of the N-terminal sequence in folding and stability of AspAT by applying the scanning calorimetry, and CD and fluorescence spectroscopies to the native and truncated enzymes. Truncated AspAT (Delta2alpha mutant) devoid of N-terminal residues cannot provide sufficient potential of quaternary intersubunit and subunit-cofactor interactions, which results in a monomeric non-functional conformation. However, the residual tertiary interactions in the Delta2alpha mutant are sufficient to: i) provide stability of a residual structure over a wide pH range; ii) confer moderate cooperativity of the denaturant-induced transition while only low cooperativity of the thermal transition, and iii) maintain the hydrophobic core of a part of the structure which prevents aromatic fluorophores from quenching by water. Furthermore, the present study provides evidence that AspAT from the alkalophilic bacterium follows unfolding pathway comprising a stable non-functional intermediate, in contrast to a two-state mechanism of the thermophilic AspAT from Sulfolobus solfataricus.     

Expression, refolding, and ferritin-binding activity of the isolated VL-domain of monoclonal antibody F11

Expression of the VL-domain of mouse monoclonal antibody F11 to human spleen ferritin in Escherichia coli cells is associated with the formation of insoluble protein aggregates (inclusion bodies). The aggregates were solubilized in the presence of guanidine hydrochloride and the recombinant VL-domain was purified by immobilized metal affinity chromatography (IMAC). Subsequent renaturation results in approximately 99% pure preparation with high yield. The VL-domain forms dimers at concentrations from 1 to 10 mg/ml. Monomeric form is detected only at protein concentrations below 0.5 mg/ml. Functional activity of the VL-domain was verified by two variants of ELISA. The affinity of the VL-domain ((0.2-1.2). 108 M(-1)) is similar to the affinity of the full-length parental antibody F11 because when the immobilized VL-domain was used, the binding constant of ferritin to the VL-domain was only 4-6-fold lower than that in the case of F11 antibody. In another ELISA system with immobilized ferritin, affinity was decreased 30-fold. The VL-domain of antibody F11 is the first example of the recombinant variable domain of the immunoglobulin light chain that preserves the antigen-binding activity in the absence of the partner VH-domain. The data indicate that the recombinant VL-domain can be used in construction of chimeric immunotoxins and other antigen-binding proteins in immunotherapy and in studies of correlations between folding, stability, and activity of immunoglobulins.     

Antigen-binding activity of monoclonal antibodies after incubation with organic solvents

Effects of four organic solvents--methanol, trifluoroethanol, dimethylsulfoxide, and dimethylformamide (DMF)--on the ferritin-binding activity of three monoclonal mouse antibodies of IgG2a and IgG1 subclasses were studied. The ferritin-binding constants of monoclonal antibodies G10 and F11 (the IgG2a subclass) were increased 2-6-fold after incubation with DMF and removal of the organic solvent by gel filtration. The maximum effect on the F11 antibodies was found in the presence of 5-13% DMF and on the G10 antibodies at 11-40% DMF. The effect remained after the removal of DMF from the incubation medium, and this suggests that the incubation with DMF resulted in irreversible conformational changes of the antibodies and in production of active conformers of the G10 and F11 antibodies. These conformations occurred within 15-60 min. The long-term stability and the fluorescence of the antibodies exposed to DMF suggest that the conformational changes were not global, but involved small and relatively independent structural elements of the antibodies, either of hypervariable CDR loops in variable domains or of the hinge region of the antibodies. The affinity of the C5 antibodies of the mouse IgG1 subclass was decreased after incubation with DMF. The activation was a solvent-specific effect because incubation of the G10 antibodies with methanol and dimethylsulfoxide decreased the affinity for the antigen, and incubation with trifluoroethanol virtually did not affect it. Relatively small changes in the antigen-binding activity of the antibodies were found even after the incubation with 5% organic solvent.     

Antiferritin VL homodimer binds human spleen ferritin with high specificity

The antiferritin variable light domain (VL) dimer binds human spleen ferritin ( approximately 85% L subunits) but with approximately 50-fold lower affinity, K(a)=4 x 10(7) x M(-1), than the parent F11 antibody (K(a)=2.1 x 10(9) x M(-1)). The VL dimer does not recognize either rL (100% L subunits) or rH (100% H subunits) human ferritin, whereas the parent antibody recognizes rL-ferritin. To help explain the differences in ferritin binding affinities and specificities, the crystal structure of the VL domain (2.8A resolution) was determined by molecular replacement and models of the antiferritin VL-VH dimer were made on the basis of antilysozyme antibody D1.3. The domain interface is smaller in the VL dimer but a larger number of interdomain hydrogen bonds may prevent rearrangement on antigen binding. The antigen binding surface of the VL dimer is flatter, lacking a negatively charged pocket found in the VL-VH models, contributed by the CDR3 loop of the VH domain. Loop CDR2 (VL dimer) is located away from the antigen binding site, while the corresponding loop of the VH domain would be located within the antigen binding site. Together these differences lead to 50-fold lower binding affinity in the VL dimer and to more restricted specificity than is seen for the parent antibody.     

Effect of heavy water on the growth, glucose assimilation and stability of Escherichia coli to freezing-thawing]

Growth and resistance to freezing--thawing of Escherichia coli B-1640 were investigated during cultivation in synthetic media prepared with H2O and D2O. It is found that during cultivation in D2O the maximum specific growth rate decreases and the duration of the exponential growth phase increases. During the growth in D2O the glucose consumption rate drops in the exponential growth phase, the lactate content in the culture liquid is lower by two orders than that in H2O; the resistance to freezing--thawing is lower than that in H2O. After leaving the exponential phase the culture in D2O restores specific growth rate, glucose consumption rate and resistance to freezing--thawing up to the values obtained during the growth in H2O. The translation ability of ribosomes isolated from cells grown in D2O and H2O is the same. We conclude that the culture adapts to D2O during the exponential growth phase. It is suggested that during the adaptation the second carbon source is used which compensates the consequences of the disturbances of glucose metabolism and transport caused by deuteration of the cell content in the adaptation to D2O.