Dimethyl- and monomethyloxyluciferins as analogs of the product of the bioluminescence reaction catalyzed by firefly luciferase.

The absorption and fluorescence spectra of dimethyloxyluciferin (DMOL) and monomethyloxyluciferin (MMOL) were studied at pH 3.0-12.0. In the range of pH 3.0-8.0, the fluorescence spectrum of DMOL exhibits a maximum at lambda(em) = 639 nm. At higher pH values an additional emission maximum appears at lambda(em) = 500 nm (wavelength of excitation maximum lambda(ex) = 350 nm), which intensity increases with time. It is shown that this peak corresponds to the product of DMOL decomposition at pH > 8.0. The absorption spectra of MMOL were studied in the range of pH 6.0-9.0. At pH 8.0-9.0, the absorption spectrum of MMOL exhibits one peak at lambda(abs) = 440 nm. At pH 7.3-7.7, an additional band appears with maximum at lambda(abs) = 390 nm. At pH 6.0-7.0 two maxima are observed, at lambda(abs) = 375 and 440 nm. The fluorescence spectra of MMOL (pH 6.0-9.7, lambda(ex) = 440 or 375 nm) exhibit one maximum. It is shown that decomposition of DMOL and MMOL in aqueous solutions results in products of similar structure. DMOL and MMOL are rather stable at the pH optimum of luciferase. It is suggested that they can be used as fluorescent markers for investigation of the active site of the enzyme.

[The cytotoxicity of mevalonate, lovastatin and carminomycin with respect to MOLT-4 human malignant T-lymphoblasts in vitro]. / Tsitotoksichnost' mevalonata, lovastatina i karminomitsina v otnoshenii chelovecheskikh zlokachestvennykh T-limfoblastov MOLT-4 in vitro.

It was shown in vitro that high concentrations of lovastatin, a competitive inhibitor of hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibited human malignant cells MOLT-4. The activity of lovastatin in doses of 50-250 microM was dose-dependent. Addition of mevalonate in a concentration of 3 mM to the growth medium completely prevented the cytotoxic effect of 100 microM of lovastatin. At the same time, exogenous mevalonate did not decrease the cytotoxicity of the anthracycline antibiotic carminomycin. Moreover, in a high concentration (7 mM) mevalonate slowly but significantly inhibited the growth of the malignant target cells and the effect was added to the cytotoxic effect of carminomycin low concentrations (0.08 to 0.175 microgram/ml). The results and the literature data suggested that combination of mevalonate, HMG-CoA reductase inhibitors and anthracyclines could be useful in tumor chemotherapy. The suggestion needs further investigation.

[Participation of DnaK chaperone and ATP in in vivo folding of firefly luciferase, synthesized by E. coli cells]. / Uchastie shaperona DnaK i ATP v svorachivanii in vivo liutsiferazy svetliako, sinteziruemoi kletkami E. coli.

The time-courses of protein accumulation and luciferase activity of Luciola mingrelica firefly luciferase synthesized on plasmid pJG lambda in E. coli strains omega 238 and B178groE7 (DnaK- and GroEL-deficient, correspondingly) and omega 237 and W3110 that are control to them, have been studied. It was shown that the amounts of the luciferase protein synthesized by all strains was approximately equal to 3.0-3.9% of the total cell protein. Luciferase was synthesized in a catalytically inactive form and transformed into an active enzyme during incubation at 21 degrees C. In the DnaK-deficient E. coli strain omega 238, the enzyme transformation into a catalytically active form did not occur which provides evidence for participation of the DnaK chaperone in transformation of newly synthesized luciferase into catalytically active form. Luciferase folding was accelerated with an increase in ATP concentration inside the cell. It is possible to increase the ATP concentration inside the cell by treatment with polymyxin and addition of ATP to the culture medium, which significantly accelerates the luciferase folding.

[14-O-hemiesters and 13-hydrazones of anthracyline antibiotics of the daunorubicin series. Synthesis and cytostatic activity with respect to tumor cells sensitive or resistant to doxorubicin]. / 14-O-gemiéfiry i 13-gidrazony antratsiklinovykh antibiotikov riada daunorubitsina. Sintez i tsitostaticheskaia aktivnost' v otnoshenii opukholevykh kletok, chuvstvitel'nykh ili ustoichivykh k doksorubitsinu.

Doxorubicin and 14-hydroxycarminomycin 14-O-hemiadipates and 14-O-hemipimelates, synthesized from 14-bromo derivatives of daunorubicin and carminomycin and monosodium adipate and pimelate, were converted to the corresponding N-trifluoroacetylated compounds. 13-(4-Methylpiperazine-1-yl)imino derivatives of the anthracycline antibiotics were also obtained. The cytostatic activity of the compounds synthesized was studied using a panel of human and animal tumor cell lines sensitive or resistant to doxorubicin. N-Trifluoroacetylation of the antibiotics resulted in a decrease in the cytostatic activity. The activity of the water-soluble 13-(4-methylpiperazine-l-yl)imino derivatives is close to that of the corresponding parent antibiotics.

[Kinetics of luciferase gene expression from fireflies Photinus pyralis and Luciola mingrelica in Escherichia coli cells. I. Kinetics of the transformation of recombinant luciferase into enzymatically-active conformers]. / Kinetika ékspressii genov liutsiferaz svetliakov Photinus pyralis i Luciola mingrelica v kletkakh Escherichia coli. I. Kinetika transformatsii rekombinantnykh liutsiferaz v fermentativno-aktivnye konfermery.

The Photinus pyralis and Luciola mingrelica luciferases genes expression has been studied on the pME61 or pJG lambda plasmids with a thermoinducible lambda Pr promoter in the E. coli strain CA using three independent methods: SDS gel electrophoresis to quantify the synthesized luciferase protein, EIA to quantify the enzyme native conformer and activity measurements. The cultures were incubated by the temperature schemes 28 degrees-42 degrees-21 degrees C and 28 degrees-21 degrees C. The maximal amount of the Ph. pyralis protein reached 4.5%, while that of L. mingrelica luciferase was 4.1% of the total amount of the cellular proteins after 10-hr incubation. The amount of the native conformer and the luciferase activity began to grow after a long induction period, reaching the maximal level by hour 50-60 after the thermoinduction. The increase in the enzyme activity correlated well with the increase in the ATP content in the cell. The observed "low-temperature induction" of the enzyme activity is interpreted as a protein transition to an active conformation. This transformation is considerably behind the protein biosynthesis process. Intracellular metabolic reactions have been shown to play an active part in these conformational changes.

[Kinetics of luciferase gene expression from fireflies Photinus pyralis and Luciola mingrelica in Escherichia coli cells. II. The role of pH in the biosynthesis of proteins and their transformation into active conformers]. / Kinetika ékspressii genov liutsiferaz svetliakov Photinus pyralis i Luciola mingrelica v kletkakh Escherichia coli. II. Rol' pH v biosinteze belkov i ikh transformatsii v aktivnye konfermery.

The Photinus pyralis and Luciola mingrelica luciferases genes expression kinetics has been studied, repectively, on the pME61 and pJG lambda plasmids with a thermoinducible lambda PR promoter in the E. coli strain CA under conditions of a pH shift. An increase in the enzyme activity after a pH shift has been revealed. The maximal amount of the firefly Ph. pyralis luciferase reaches 5.3%, while the maximal amount of the L. mingrelica enzyme reaches 4.9% of the total amount of cellular proteins in case of pHout shift to 9.0 according to the SDS gel electrophoresis data; that in case of a pHout shift to 8.5 is 4.9% and 4.5%, respectively. The enzyme activation correlates well with the dynamics of pHout and pHin.

[Synthesis and properties of new rubomycin derivatives]. / Sintez i svoistva novykh proizvodnykh rubomitsina.

Condensation of rubomycin (daunorubicin) with respective hydrazides yielded novel substituted hydrazones: 13-cyanoacetyl hydrazone rubomycin, 13-L-phenylalanyl hydrazone rubomycin, 13-BOC-3-(uracilyl-1)-DL-alanyl hydrazone rubomycin and 13-BOC-3-(adenylyl-9)-DL-alanyl hydrazone rubomycin. With successive treatment of rubomycin with hydrazine hydrate and respective ketones novel asymmetric azines were prepared: 13-cyclopentylidene hydrazone rubomycin, 13-alpha,alpha'-dimethyl-cyclopentylidene hydrazone rubomycin and 13-(1-phenylethylidene-1) hydrazone rubomycin. 14-Adenylyl-N9-rubomycin was synthesized by interaction of 14-bromorubomycin with adenine and hydrogenation of its analog, 14-N-imidazolyl rubomycin by sodium borhydrite yielded 13-dihydro-14-N-imidazolyl rubomycin. There was observed correlation between the antimicrobial activity of the derivatives against B. mycoides and their cytostatic effect on the cells of murine leukemia NK/LI. The high in vitro activity of 13-cyclopentylidene hydrazone rubomycin showed satisfactory correlation with the results of the study on the antitumor effect in animals.