5 research outputs found
Consideration of the contribution of chemical (non-enzymatic) conversion of substrate in the general mechanism of enzyme reaction
When enzyme-catalyzed reactions are studied, it is necessary to take into account the contribution of the chemical (non-enzymatic) conversion of the substrate to the product, which is carried out together with the enzyme-catalyzed conversion of the substrate. It is generally believed that the difference of the product concentration that was formed in the presence of the enzyme and in its absence (during the same time interval) is the concentration of the product that was formed directly in the enzyme-catalyzed reaction, i.e. that there is additivity of the product concentrations at each time point. In this paper, we have analyzed when there is additivity and how to correctly take into account the contribution of chemical (non-catalytic) substrate conversion when the enzyme-catalyzed reactions are investigated. We have shown that the additivity of productΒ concentrations and initial rates is observed only for a period when the product concentration increases linearΒly with time. The longer the reaction proceeds the more the deviation from the additivity. Under equilibrium condition, there is no additivity of equilibrium product concentrations but under conditions of detailed balance the equilibrium product concentration of the overall reaction, including the enzyme-catalyzed and chemical (non-enzymatic) conversion of the substrate, is also at the same time the equilibrium concentration of the product of the enzyme-catalyzed conversion of the substrate
Examining c-di-GMP and possible quorum sensing regulation in Pseudomonas fluorescens SBW25:links between intra and inter-cellular regulation benefits community cooperative activities such as biofilm formation
Bacterial success in colonizing complex environments requires individual response to micro-scale conditions as well as community-level cooperation to produce large-scale structures such as biofilms. Connecting individual and community responses could be achieved by linking the intracellular sensory and regulatory systems mediated by bis-(3β²-5β²)-cyclic dimeric guanosine monophosphate (c-di-GMP) and other compounds of individuals with intercellular quorum sensing (QS) regulation controlling populations. There is growing evidence to suggest that biofilm formation by many pseudomonads is regulated by both intra and intercellular systems, though in the case of the model Pseudomonas fluorescens SBW25 Wrinkly Spreader in which mutations increasing c-di-GMP levels result in the production of a robust cellulose-based air-liquid interface biofilm, no evidence for the involvement of QS regulation has been reported. However, our recent review of the P. fluorescens SBW25 genome has identified a potential QS regulatory pathway and other QSβassociated genes linked to c-di-GMP homeostasis, and QS signal molecules have also been identified in culture supernatants. These findings suggest a possible link between c-di-GMP and QS regulation in P. fluorescens SBW25 which might allow a more sophisticated and responsive control of cellulose production and biofilm formation when colonising the soil and plant-associated environments P. fluorescens SBW25 normally inhabits.ΠΠ½Π°Π»ΠΈΠ· Ρ-Π΄ΠΈ-ΠΠΠ€ ΠΈ Π²ΠΎΠ·ΠΌΠΎΠΆΠ½ΠΎΠ³ΠΎ ΡΡΠ²ΡΡΠ²Π° ΠΊΠ²ΠΎΡΡΠΌΠ° Ρ Pseudomonas fluorescens SBW 25: ΡΠ²ΡΠ·Ρ ΠΌΠ΅ΠΆΠ΄Ρ Π²Π½ΡΡΡΠΈ ΠΈ ΠΌΠ΅ΠΆΠΊΠ»Π΅ΡΠΎΡΠ½ΠΎΠΉ ΡΠ΅Π³ΡΠ»ΡΡΠΈΠ΅ΠΉ ΡΠΏΠΎΡΠΎΠ±ΡΡΠ²ΡΠ΅Ρ ΠΊΠΎΠΎΠΏΠ΅ΡΠ°ΡΠΈΠ²Π½ΠΎΠΌΡ ΠΏΠΎΠ²Π΅Π΄Π΅Π½ΠΈΡ Π² ΡΠΎΠΎΠ±ΡΠ΅ΡΡΠ²Π΅ ΠΈ ΡΠΎΡΠΌΠΈΡΠΎΠ²Π°Π½ΠΈΡ Π±ΠΈΠΎΠΏΠ»ΡΠ½ΠΊΠΈΠ£ΡΠΏΠ΅ΡΠ½ΠΎΡΡΡ Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΠΎΠΉ ΠΊΠΎΠ»ΠΎΠ½ΠΈΠ·Π°ΡΠΈΠΈ ΡΠ»ΠΎΠΆΠ½ΡΡ
ΡΠΊΠΎΠ½ΠΈΡ ΡΡΠ΅Π±ΡΠ΅Ρ ΠΈΠ½Π΄ΠΈΠ²ΠΈΠ΄ΡΠ°Π»ΡΠ½ΠΎΠ³ΠΎ ΠΎΡΠ²Π΅ΡΠ° Π½Π° ΠΈΠ·ΠΌΠ΅Π½Π΅Π½ΠΈΡ ΡΡΠ»ΠΎΠ²ΠΈΠΉ Π½Π° ΠΌΠΈΠΊΡΠΎΡΡΠΎΠ²Π½Π΅ ΡΠ°Π²Π½ΠΎ ΠΊΠ°ΠΊ ΠΈ ΠΊΠΎΠΎΠΏΠ΅ΡΠ°ΡΠΈΠΈ Π½Π° ΡΡΠΎΠ²Π½Π΅ ΡΠΎΠΎΠ±ΡΠ΅ΡΡΠ²Π° Π΄Π»Ρ ΠΏΡΠΎΠ΄ΡΠΊΡΠΈΠΈ ΡΠ°ΠΊΠΈΡ
ΠΊΡΡΠΏΠ½ΠΎ ΠΌΠ°ΡΡΡΠ°Π±Π½ΡΡ
ΡΡΡΡΠΊΡΡΡ ΠΊΠ°ΠΊ Π±ΠΈΠΎΠΏΠ»ΡΠ½ΠΊΠΈ. ΠΠΎΠΎΡΠ΄ΠΈΠ½Π°ΡΠΈΡ ΠΈΠ½Π΄ΠΈΠ²ΠΈΠ΄ΡΠ°Π»ΡΠ½ΡΡ
ΠΎΡΠ²Π΅Ρ ΠΎΠ² ΠΈ ΠΎΡΠ²Π΅ΡΠΎΠ² ΡΠΎΠΎΠ±ΡΠ΅ΡΡΠ²Π° ΠΌΠΎΠΆΠ΅Ρ Π±ΡΡΡ Π΄ΠΎΡΡΠΈΠ³Π½ΡΡΠ° ΠΏΡΡΠ΅ΠΌ ΡΠ²ΡΠ·ΡΠ²Π°Π½ΠΈΡ Π²Π½ΡΡΡΠΈΠΊΠ»Π΅ΡΠΎΡΠ½ΡΡ
ΡΠ΅Π½ΡΠΎΡΠ½ΡΡ
ΠΈ ΡΠ΅Π³ΡΠ»ΡΡΠΎΡΠ½ΡΡ
ΡΠΈΡΡΠ΅ΠΌ, ΠΎΠΏΠΎΡΡΠ΅Π΄ΡΠ΅ΠΌΡΡ
Π±ΠΈΡ-(3',5')-ΡΠΈΠΊΠ»ΠΈΡΠ΅ΡΠΊΠΈΠΌ Π΄ΠΈΠΌΠ΅ΡΠ½ΡΠΌ Π³ΡΠ°Π½ΠΎΠ·ΠΈΠ½ΠΌΠΎΠ½ΠΎΡΠΎΡΡΠ°ΡΠΎΠΌ (Ρ-Π΄ΠΈ-ΠΠΠ€) ΠΈ Π΄ΡΡΠ³ΠΈΠΌΠΈ ΡΠΎΠ΅Π΄ΠΈΠ½Π΅Π½ΠΈΡΠΌΠΈ ΠΈΠ½Π΄ΠΈΠ²ΠΈΠ΄ΡΡΠΌΠΎΠ² Ρ ΠΌΠ΅ΠΆΠΊΠ»Π΅ΡΠΎΡΠ½ΠΎΠΉ ΡΠ΅Π³ΡΠ»ΡΡΠΈΠ΅ΠΉ - ΡΡΠ²ΡΡΠ²ΠΎΠΌ ΠΊΠ²ΠΎΡΡΠΌΠ° (Π§Π), ΠΊΠΎΠ½ΡΡΠΎΠ»ΠΈΡΡΡΡΠ΅ΠΌ ΠΏΠΎΠΏΡΠ»ΡΡΠΈ Ρ. ΠΠ°ΠΊΠ°ΠΏΠ»ΠΈΠ²Π°Π΅ΡΡΡ Π²ΡΡ Π±ΠΎΠ»ΡΡΠ΅ Π΄ΠΎΠΊΠ°Π·Π°ΡΠ΅Π»ΡΡΡΠ² ΡΠΎΠ³ΠΎ, ΡΡΠΎ ΡΠΎΡΠΌΠΈΡΠΎΠ²Π°Π½ΠΈΠ΅ Π±ΠΈΠΎΠΏΠ»Π΅Π½ΠΊΠΈ ΠΌΠ½ΠΎΠ³ΠΈΠΌΠΈ ΠΏΡΠ΅Π²Π΄ΠΎΠΌΠΎΠ½Π°Π΄Π°ΠΌΠΈ ΡΠ΅Π³ΡΠ»ΠΈΡΡΠ΅ΡΡΡ ΠΊΠ°ΠΊ Π²Π½ΡΡΡΠΈ ΠΊΠ»Π΅ΡΠΎΡΠ½ΡΠΌΠΈ, ΡΠ°ΠΊ ΠΈ ΠΌΠ΅ΠΆ ΠΊΠ»Π΅ΡΠΎΡΠ½ΡΠΌΠΈ ΡΠ΅Π³ΡΠ»ΡΡΠΎΡΠ½ΡΠΌΠΈ ΡΠΈΡΡΠ΅ΠΌΠ°ΠΌΠΈ, Ρ
ΠΎΡΡ Π² ΡΠ»ΡΡΠ°Π΅ ΠΌΠΎΠ΄Π΅Π»ΡΠ½ΠΎΠΉ Pseudomonas fluorescens SBW25 Wrinkly Spreader, Ρ ΠΊΠΎΡΠΎΡΠΎΠΉ ΠΌΡΡΠ°ΡΠΈΠΈ, ΠΏΠΎΠ²ΡΡΠ°ΡΡ ΠΈΠ΅ ΡΡΠΎΠ²Π½ΠΈ Ρ-Π΄ΠΈ-ΠΠΠ€, ΠΏΡΠΈΠ²ΠΎΠ΄ΡΡ ΠΊ ΡΠΎΠ·Π΄Π°Π½ΠΈΡ ΠΏΡΠΎΡΠ½ΠΎΠΉ ΡΠ΅Π»Π»ΡΠ»ΠΎΠ·Π½ΠΎΠΉ Π±ΠΈΠΎΠΏΠ»ΡΠ½ΠΊΠΈ Π½Π° Π³ΡΠ°Π½ΠΈΡΠ΅ ΡΠ°Π·Π΄Π΅Π»Π° ΡΠ°Π· Π²ΠΎΠ·Π΄ΡΡ
-ΠΆΠΈΠ΄ΠΊΠΎΡΡΡ, Π½Π΅ Π±ΡΠ»ΠΎ ΠΎΠ±Π½Π°ΡΡΠΆΠ΅Π½ΠΎ Π½ΠΈ ΠΊΠ° ΠΊΠΎΠ³ΠΎ ΡΠ²ΠΈΠ΄Π΅ΡΠ΅Π»ΡΡΡΠ²Π° Π²ΠΎΠ²Π»Π΅ΡΠ΅Π½ΠΈΡ ΠΊΠ²ΠΎΡΡΠΌ-Π·Π°Π²ΠΈΡΠΈΠΌΠΎΠΉ ΡΠ΅Π³ΡΠ»ΡΡΠΈΠΈ. ΠΠ΄Π½Π°ΠΊΠΎ Π½Π°Ρ Π½Π΅Π΄Π°Π²Π½ΠΈΠΉ ΠΎΠ±Π·ΠΎΡ Π³Π΅Π½ΠΎΠΌΠ° P. fluorescens SBW25 Π²ΡΡΠ²ΠΈΠ» ΠΏΠΎΡΠ΅Π½ΡΠΈΠ°Π»ΡΠ½ΡΠΉ Π§Π-Π·Π°Π²ΠΈΡΠΈΠΌΡΠΉ ΡΠ΅Π³ΡΠ»ΡΡΠΎΡΠ½ΡΠΉ ΠΏΡ ΡΡ ΠΈ Π΄ΡΡΠ³ΠΈΠ΅ Π§Π-Π·Π°Π²ΠΈΡΠΈΠΌΡΠ΅ Π³Π΅Π½Ρ, ΡΠ²ΡΠ·Π°Π½Π½ΡΠ΅ Ρ Π³ΠΎΠΌΠ΅ΠΎΡΡΠ°Π·ΠΎΠΌ Ρ-Π΄ΠΈ-ΠΠΠ€, Π° ΠΌΠΎΠ»Π΅ΠΊΡΠ»Ρ Π§Π-ΡΠΈΠ³Π½Π°Π»ΠΈΠ½Π³Π° Π±ΡΠ»ΠΈ ΠΈΠ΄Π΅Π½ΡΠΈΡΠΈΡΠΈΡΠΎΠ²Π°Π½Ρ Π² ΠΊΡΠ»ΡΡΡΡΠ΅. ΠΡΠΈ Π΄Π°Π½Π½ΡΠ΅ ΡΠ²ΠΈΠ΄Π΅ΡΠ΅Π»ΡΡΡΠ²ΡΡΡ ΠΎ Π²ΠΎΠ·ΠΌΠΎΠΆΠ½ΠΎΠΉ ΡΠ²ΡΠ·ΠΈ ΠΌΠ΅ΠΆΠ΄Ρ Ρ-Π΄ΠΈ-ΠΠΠ€-ΡΠ΅Π³ΡΠ»ΡΡΠΈΠ΅ΠΉ ΠΈ Π§Π Ρ P. fluorescens SBW25, ΡΡΠΎ ΠΏΠΎΠ·Π²ΠΎΠ»ΡΠ΅Ρ Π±ΠΎΠ»Π΅Π΅ ΡΠ»ΠΎΠΆΠ½ΡΠΉ ΠΈ Π³ΠΈΠ±ΠΊΠΈΠΉ ΠΊΠΎΠ½ΡΡΠΎΠ»Ρ Π½Π°Π΄ ΠΏΡΠΎΠ΄ΡΠΊΡΠΈΠ΅ΠΉ ΡΠ΅Π»Π»ΡΠ»ΠΎΠ·Ρ ΠΈ ΠΎΠ±ΡΠ°Π·ΠΎΠ²Π°Π½ΠΈ Π΅ΠΌ Π±ΠΈΠΎΠΏΠ»Π΅Π½ΠΊΠΈ ΠΏΡΠΈ ΠΊΠΎΠ»ΠΎΠ½ΠΈΠ·Π°ΡΠΈΠΈ ΠΏΠΎΡΠ² ΠΈ ΡΠΊΠΎΠ½ΠΈΡ, aΡΡΠΎΡΠΈΠΈΡΠΎΠ²Π°Π½Π½ΡΡ
Ρ ΡΠ°ΡΡΠ΅Π½ΠΈΡΠΌ ΠΈ, - Π΅ΡΡΠ΅ΡΡΠ²Π΅Π½Π½ΡΠΌΠΈ ΡΡΠ΅Π΄Π°ΠΌΠΈ ΠΎΠ±ΠΈΡΠ°Π½ΠΈΡ P. fluorescens SBW25
Electrochemical potential of the inner mitochondrial membrane and Ca(2+) homeostasis of myometrium cells
We demonstrated using Ca2+-sensitive fluorescent probe, mitochondria binding dyes, and confocal laser scanning microscopy, that elimination of electrochemical potential of uterus myocytesβ inner mitochondrial membrane by a protonophore carbonyl cyanide m-chlorophenyl hΡdrazone (10 ΞΌM), and by a respiratory chain complex IV inhibitor sodium azide (1 mM) is associated with substantial increase of Ca2+ concentration in myoplasm in the case of the protonophore effect only, but not in the case of the azide effect. In particular, with the use of nonyl acridine orange, a mitochondria-specific dye, and 9-aminoacridine, an agent that binds to membrane compartments in the presence of proton gradient, we showed that both the protonophore and the respiratory chain inhibitor cause the proton gradient on mitochondrial inner membrane to dissipate when introduced into incubation medium. We also proved with the help of 3,3β²-dihexyloxacarbocyanine, a potential-sensitive carbocyanine-derived fluorescent probe, that the application of these substances results in dissipation of the membraneβs electrical potential. The elimination of mitochondrial electrochemical potential by carbonyl cyanide m-chlorophenyl hΡdrazone causes substantial increase in fluorescence of Ca2+-sensitive Fluo-4 AM dye in myoplasm of smooth muscle cells. The results obtained were qualitatively confirmed with flow cytometry of mitochondria isolated through differential centrifugation and loaded with Fluo-4 AM. Particularly, Ca2+ matrix influx induced by addition of the exogenous cation is totally inhibited by carbonyl cyanide m-chlorophenyl hydrazone. Therefore, using two independent fluorometric methods, namely confocal laser scanning microscopy and flow cytometry, with Ca2+-sensitive Fluo-4 AM fluorescent probe, we proved on the models of freshly isolated myocytes and uterus smooth muscle mitochondria isolated by differential centrifugation sedimentation that the electrochemical gradient of inner membrane is an important component of mechanisms that regulate Ca2+ homeostasis in myometrium cells
2D-BN nanoparticles as a spectroscopic marker and drug delivery system with protection properties
An application of 2D-BN nanoparticles as a spectroscopic marker, weak luminescent marker and anticancer drug (doxorubicin, DOX) delivery system with protection properties was studied for the LNCaP strains of cancer cells using FTIR and Raman spectroscopy for analysing the cancer cells, cells with BN, the cancer cells with DOX, and the cancer cells with BN nanoparticles loaded by DOX. Study of IR absorption and Raman spectra of the LNCaP strains of cancer cells incubated with 2D-BN nanoparticles for 1 hour showed that the 2D-BN nanoparticles could pass through the cell membrane and localize inside the membrane or close to the membrane in the cytoplasm of the cells. We registered the spectra of the disturbed lipids during the DOX-2D-BN passing through the membrane. After incubation for 2 hours and more, spectral changes in other structural components of the cell (nuclei, cytoplasm, mitochondria) can be registered. Confocal microscopy showed that a gold nanostructured support enhances the fluorescence of the cancer cells with 2D-BN as well as that with DOX, however the double action of 2D-BN and DOX on the cancer cells aggravates the emission property of the studied system. An MTT test showed that the toxicity of DOX on the 2D-BN nanoparticles is less than that on the reference cells, and at the same time the efficiency of the DOX action on the cancer cells does not change
Identification of nitric oxide in mitochondria of myometrium cell
Aim. To demonstrate the possibility of NO synthesis in intact myocytes of uterus. Methods. Confocal scanning microscopy method, NO-sensitive fluorescent probe DAF-FM, MitoTracker Orange CM-H2TMRos. Results. The basal production of NO in intact myocytes was shown using DAF-FM. Incubation of myocytes with NO donor β sodium nitroprusside (SNP) β led to an increase of the DAF-FM-T fluorescent signal. On the contrary, the addition of NO-synthase inhibitor β N-nitro-L-arginine (NA) β results in the reduction of fluorescent intensity. It was demonstrated colocalizition of specific probe for mitochondria MitoTracker Orange CM-H2TMRos and NO-sensitive dye DAF-FM. Conclusions. For the first time it has been demonstrated the presence of NO in smooth muscle cell mitochondria using laser confocal microscopy, NO-sensitive probe DAF-FM and specific marker of the functionally active mitochondria MitoTracker Orange CM-H2TMRos