Stopped-flow spectrofluorimetry and a theoretical method for predicting protonation equilibria in... more Stopped-flow spectrofluorimetry and a theoretical method for predicting protonation equilibria in polyelectrolytes were combined in an analysis of the pH dependence of the kinetics of binding of analogues of the 5'-mRNA cap to the cap binding protein eIF4E. The computer simulations and available experimental data indicate that there are two titratable groups in the binding site of the protein and two titratable groups on the ligands directly involved in the binding, in addition to stacking interactions described by other groups. The observed pH dependencies of the rate constants obtained from the stopped-flow experiments are consistent with this finding. In particular, it is concluded that binding of both forms of the cap analogs regarding protonation at the N1 position of the guanine ring is efficient, and the shift to a predominantly protonated form of the ring takes place after formation of the complex.
In order to initiate hairy root culture initiation cotyledons and hypocotyls of Calendula officin... more In order to initiate hairy root culture initiation cotyledons and hypocotyls of Calendula officinalis L. were infected with Agrobacterium rhizogenes strain ATCC 15834 or the same strain containing pCAMBIA 1381Z vector with β-glucuronidase reporter gene under control of promoter of NIK (Nematode Induced Kinase) gene. The efficiency of induction of hairy roots reached 33.8% for cotyledons and 66.6% for hypocotyls together for both transformation experiments. Finally, eight control and nine modified lines were established as a long-term culture. The hairy root cultures showed the ability to synthesize oleanolic acid mainly (97%) as glycosides; control lines contained it at the average 8.42 mg · g(-1) dry weight in tissue and 0.23 mg · dm(-3) in medium; modified lines: 4.59 mg · g(-1) for the tissue, and 0.48 mg · dm(-3) for the medium. Additionally lines showed high positive correlation between dry/fresh weight and oleanolic acid concentration in tissue. Using the Killiani mixture in a...
ABSTRACT Substantial qualitative and quantitative differences in the triterpenoid profiles of mar... more ABSTRACT Substantial qualitative and quantitative differences in the triterpenoid profiles of marigold Calendula officinalis L. cv. Persimmon Beauty native plant organs (whole capitular inflorescences, separated ligulate flowers, leaves, and roots) and of various types of marigold in vitro cultures (a suspension culture initiated from seedlings, a hairy root culture, a suspension culture initiated from hairy roots) were demonstrated through GC–MS/FID analysis. The ligulate flowers and whole inflorescences were the richest sources of pentacyclic triterpenoids occurring in free and ester forms, whereas the in vitro cultures synthesized primarily sterols, although the profile of these compounds was considerably different than that of the native plant. The synthesis and accumulation of triterpenoids of taraxastane-type skeletons were determined to occur only in flowers, and D:A-friedooleanane (friedelane) triterpenoids were detected only in the roots, whereas oleanane, ursane and lupane skeletons were determined to be synthesized in all parts of the plant. Certain metabolic features of the parent plant were retained in the in vitro cultures (e.g., the capacity to synthesize oleanane, ursane and lupane skeletons), whereas others were lost. The obtained results confirm the existence of a strong correlation between morphological differentiation and the biosynthesis of plant secondary metabolites, which influences the potential productivity of in vitro cultures.
Journal of Biochemical and Biophysical Methods, 2002
A method for extracting kinetic and optical parameters from progress curves for protein-ligand as... more A method for extracting kinetic and optical parameters from progress curves for protein-ligand association, obtained by stopped-flow experiments, is described. The method is limited to one-step and two-step association kinetics, but it allows concentration of protein and offset of the signals to be adjustable parameters during an interactive non-linear least-squares fitting procedure. The method is tested on simulated pseudo-experimental data and applied to progress curves obtained in a stopped-flow spectrofluorimeter, for association of the translation initiation factor eIF4E with 7-methyl-GDP, an analog of 5'-end of mRNA.
ABSTRACT The effects of elicitors on cell growth and oleanolic acid (OA) accumulation in shaken c... more ABSTRACT The effects of elicitors on cell growth and oleanolic acid (OA) accumulation in shaken cell suspension cultures of Calendula officinalis were investigated. Elicitors were added individually at various concentrations to 5-day-old cell cultures and their effects monitored at 24 h intervals for 4 days. Different effects on OA accumulation were observed depending on the day of treatment. Jasmonic acid was the most efficient elicitor. After 72 h of treatment with 100 μM JA, the intracellular content of OA reached its maximum value (0.84 mg g−1 DW), which was 9.4-fold greater than that recorded in an untreated control cultures. The addition of chitosan at 50 mg l−1 produced a 5-fold enhancement of OA accumulation (0.37 mg g−1 DW) after 48 h of treatment. Treatment with yeast extract at 200 mg l−1 for 96 h or with pectin at 2 mg l−1 for 48 h produced identical cellular levels of OA (0.22 mg g−1 DW). Lastly, 48 h elicitation with homogenate of the fungus Trichoderma viride produced a 1.8-fold increase in oleanolic acid content (0.12 mg g−1 DW). In addition to significantly stimulating OA accumulation and its secretion into the culture medium, the elicitors also caused slight inhibition of cell growth.
The paper presents a method of identification of DC motor parameters using computer. The problem ... more The paper presents a method of identification of DC motor parameters using computer. The problem of identification of unknown parameters has been reformulated to the problem of optimization of given cost function. The cost function should be matched so that it has only one global minimum for optimal values of wanted model parameters. The minimum of cost function was searched numerically by using MATLAB software. Application of computers to identify model parameters allows a quick and effective solving this kind of tasks.
Stopped-flow spectrofluorimetry and a theoretical method for predicting protonation equilibria in... more Stopped-flow spectrofluorimetry and a theoretical method for predicting protonation equilibria in polyelectrolytes were combined in an analysis of the pH dependence of the kinetics of binding of analogues of the 5'-mRNA cap to the cap binding protein eIF4E. The computer simulations and available experimental data indicate that there are two titratable groups in the binding site of the protein and two titratable groups on the ligands directly involved in the binding, in addition to stacking interactions described by other groups. The observed pH dependencies of the rate constants obtained from the stopped-flow experiments are consistent with this finding. In particular, it is concluded that binding of both forms of the cap analogs regarding protonation at the N1 position of the guanine ring is efficient, and the shift to a predominantly protonated form of the ring takes place after formation of the complex.
In order to initiate hairy root culture initiation cotyledons and hypocotyls of Calendula officin... more In order to initiate hairy root culture initiation cotyledons and hypocotyls of Calendula officinalis L. were infected with Agrobacterium rhizogenes strain ATCC 15834 or the same strain containing pCAMBIA 1381Z vector with β-glucuronidase reporter gene under control of promoter of NIK (Nematode Induced Kinase) gene. The efficiency of induction of hairy roots reached 33.8% for cotyledons and 66.6% for hypocotyls together for both transformation experiments. Finally, eight control and nine modified lines were established as a long-term culture. The hairy root cultures showed the ability to synthesize oleanolic acid mainly (97%) as glycosides; control lines contained it at the average 8.42 mg · g(-1) dry weight in tissue and 0.23 mg · dm(-3) in medium; modified lines: 4.59 mg · g(-1) for the tissue, and 0.48 mg · dm(-3) for the medium. Additionally lines showed high positive correlation between dry/fresh weight and oleanolic acid concentration in tissue. Using the Killiani mixture in a...
ABSTRACT Substantial qualitative and quantitative differences in the triterpenoid profiles of mar... more ABSTRACT Substantial qualitative and quantitative differences in the triterpenoid profiles of marigold Calendula officinalis L. cv. Persimmon Beauty native plant organs (whole capitular inflorescences, separated ligulate flowers, leaves, and roots) and of various types of marigold in vitro cultures (a suspension culture initiated from seedlings, a hairy root culture, a suspension culture initiated from hairy roots) were demonstrated through GC–MS/FID analysis. The ligulate flowers and whole inflorescences were the richest sources of pentacyclic triterpenoids occurring in free and ester forms, whereas the in vitro cultures synthesized primarily sterols, although the profile of these compounds was considerably different than that of the native plant. The synthesis and accumulation of triterpenoids of taraxastane-type skeletons were determined to occur only in flowers, and D:A-friedooleanane (friedelane) triterpenoids were detected only in the roots, whereas oleanane, ursane and lupane skeletons were determined to be synthesized in all parts of the plant. Certain metabolic features of the parent plant were retained in the in vitro cultures (e.g., the capacity to synthesize oleanane, ursane and lupane skeletons), whereas others were lost. The obtained results confirm the existence of a strong correlation between morphological differentiation and the biosynthesis of plant secondary metabolites, which influences the potential productivity of in vitro cultures.
Journal of Biochemical and Biophysical Methods, 2002
A method for extracting kinetic and optical parameters from progress curves for protein-ligand as... more A method for extracting kinetic and optical parameters from progress curves for protein-ligand association, obtained by stopped-flow experiments, is described. The method is limited to one-step and two-step association kinetics, but it allows concentration of protein and offset of the signals to be adjustable parameters during an interactive non-linear least-squares fitting procedure. The method is tested on simulated pseudo-experimental data and applied to progress curves obtained in a stopped-flow spectrofluorimeter, for association of the translation initiation factor eIF4E with 7-methyl-GDP, an analog of 5'-end of mRNA.
ABSTRACT The effects of elicitors on cell growth and oleanolic acid (OA) accumulation in shaken c... more ABSTRACT The effects of elicitors on cell growth and oleanolic acid (OA) accumulation in shaken cell suspension cultures of Calendula officinalis were investigated. Elicitors were added individually at various concentrations to 5-day-old cell cultures and their effects monitored at 24 h intervals for 4 days. Different effects on OA accumulation were observed depending on the day of treatment. Jasmonic acid was the most efficient elicitor. After 72 h of treatment with 100 μM JA, the intracellular content of OA reached its maximum value (0.84 mg g−1 DW), which was 9.4-fold greater than that recorded in an untreated control cultures. The addition of chitosan at 50 mg l−1 produced a 5-fold enhancement of OA accumulation (0.37 mg g−1 DW) after 48 h of treatment. Treatment with yeast extract at 200 mg l−1 for 96 h or with pectin at 2 mg l−1 for 48 h produced identical cellular levels of OA (0.22 mg g−1 DW). Lastly, 48 h elicitation with homogenate of the fungus Trichoderma viride produced a 1.8-fold increase in oleanolic acid content (0.12 mg g−1 DW). In addition to significantly stimulating OA accumulation and its secretion into the culture medium, the elicitors also caused slight inhibition of cell growth.
The paper presents a method of identification of DC motor parameters using computer. The problem ... more The paper presents a method of identification of DC motor parameters using computer. The problem of identification of unknown parameters has been reformulated to the problem of optimization of given cost function. The cost function should be matched so that it has only one global minimum for optimal values of wanted model parameters. The minimum of cost function was searched numerically by using MATLAB software. Application of computers to identify model parameters allows a quick and effective solving this kind of tasks.
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