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1.
Photodiagnosis Photodyn Ther ; 44: 103853, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37863377

ABSTRACT

BACKGROUND: The development of multidrug resistance (MDR) in infectious agents is one of the most serious global problems facing humanity. Antimicrobial photodynamic therapy (APDT) shows encouraging results in the fight against MDR pathogens, including those in biofilms. METHODS: Photosensitizers (PS), monocationic methylene blue, polycationic and polyanionic derivatives of phthalocyanines, electroneutral and polycationic derivatives of bacteriochlorin were used to study photodynamic inactivation of Gram-positive and Gram-negative planktonic bacteria and biofilms under LED irradiation. Zeta potential measurements, confocal fluorescence imaging, and coarse-grained modeling were used to evaluate the interactions of PS with bacteria. PS aggregation and photobleaching were studied using absorption and fluorescence spectroscopy. RESULTS: The main approaches to ensure high efficiency of bacteria photosensitization are analyzed. CONCLUSIONS: PS must maintain a delicate balance between binding to exocellular and external structures of bacterial cells and penetration through the cell wall so as not to get stuck on the way to photooxidation-sensitive structures of the bacterial cell.


Subject(s)
Anti-Infective Agents , Photochemotherapy , Photosensitizing Agents/pharmacology , Photosensitizing Agents/chemistry , Photochemotherapy/methods , Gram-Negative Bacteria , Biofilms/radiation effects
2.
Photosynth Res ; 147(2): 197-209, 2021 Feb.
Article in English | MEDLINE | ID: mdl-33389445

ABSTRACT

Chromatophores of purple non-sulfur bacteria (PNSB) are invaginations of the cytoplasmic membrane that contain a relatively simple system of light-harvesting protein-pigment complexes, a photosynthetic reaction center (RC), a cytochrome complex, and ATP synthase, which transform light energy into the energy of synthesized ATP. The high content of negatively charged phosphatidylglycerol (PG) and cardiolipin (CL) in PNSB chromatophore membranes makes these structures potential targets that bind cationic antiseptics. We used the methods of stationary and kinetic fluorescence spectroscopy to study the effect of some cationic antiseptics (chlorhexidine, picloxydine, miramistin, and octenidine at concentrations up to 100 µM) on the spectral and kinetic characteristics of the components of the photosynthetic apparatus of Rhodobacter sphaeroides chromatophores. Here we present the experimental data on the reduced efficiency of light energy conversion in the chromatophore membranes isolated from the photosynthetic bacterium Rb. sphaeroides in the presence of cationic antiseptics. The addition of antiseptics did not affect the energy transfer between the light-harvesting LH1 complex and reaction center (RC). However, it significantly reduced the efficiency of the interaction between the LH2 and LH1 complexes. The effect was maximal with 100 µM octenidine. It has been proved that molecules of cationic antiseptics, which apparently bind to the heads of negatively charged cardiolipin molecules located in the rings of light-harvesting pigments on the cytoplasmic surface of the chromatophores, can disturb the optimal conditions for efficient energy migration in chromatophore membranes.


Subject(s)
Anti-Infective Agents, Local/pharmacology , Bacterial Chromatophores/drug effects , Energy Transfer/drug effects , Photosynthetic Reaction Center Complex Proteins/drug effects , Rhodobacter sphaeroides/physiology , Cardiolipins/chemistry , Cell Membrane/drug effects , Kinetics , Light , Light-Harvesting Protein Complexes/drug effects , Phosphatidylglycerols/chemistry , Photosynthesis/drug effects , Rhodobacter sphaeroides/chemistry , Spectrometry, Fluorescence
3.
Oxid Med Cell Longev ; 2019: 9710208, 2019.
Article in English | MEDLINE | ID: mdl-31827716

ABSTRACT

Peroxidation of cardiolipin (CL) in the inner mitochondrial membrane plays a key role in the development of various pathologies and, probably, aging. The four fatty acid tails of CL are usually polyunsaturated, which makes CL particularly sensitive to peroxidation. Peroxidation of CL is involved in the initiation of apoptosis, as well as in some other important cellular signaling chains. However, the studies of CL peroxidation are strongly limited by the lack of methods for its tracing in living cells. We have synthesized a new mitochondria-targeted fluorescent probe sensitive to lipid peroxidation (dubbed MitoCLox), where the BODIPY fluorophore, carrying a diene-containing moiety (as in the C11-BODIPY (581/591) probe), is conjugated with a triphenylphosphonium cation (TPP+) via a long flexible linker that contains two amide bonds. The oxidation of MitoCLox could be measured either as a decrease of absorbance at 588 nm or as an increase of fluorescence in the ratiometric mode at 520/590 nm (emission). In CL-containing liposomes, MitoCLox oxidation was induced by cytochrome c and developed in parallel with cardiolipin oxidation. TPP+-based mitochondria-targeted antioxidant SkQ1, in its reduced form, inhibited oxidation of MitoCLox concurrently with the peroxidation of cardiolipin. Molecular dynamic simulations of MitoCLox in a cardiolipin-containing membrane showed affinity of positively charged MitoCLox to negatively charged CL molecules; the oxidizable diene moiety of MitoCLox resided on the same depth as the cardiolipin lipid peroxides. We suggest that MitoCLox could be used for monitoring CL oxidation in vivo and, owing to its flexible linker, also serve as a platform for producing peroxidation sensors with affinity to particular lipids.


Subject(s)
Cardiolipins/chemistry , Cytochromes c/metabolism , Fluorescent Dyes/chemistry , Lipid Peroxidation , Mitochondria/metabolism , Apoptosis , Cardiolipins/metabolism , Fluorescence , Fluorescent Dyes/metabolism , Humans , Liposomes/metabolism , Oxidation-Reduction , Reactive Oxygen Species/metabolism
4.
Anal Biochem ; 587: 113445, 2019 12 15.
Article in English | MEDLINE | ID: mdl-31542342

ABSTRACT

Lipopolysaccharides (LPS) are the Gram-negative bacteria cell wall components capable to induce the system inflammatory response even at picomolar concentrations. LPS detection at these concentrations is necessary to develop new sorbents for the efficient purification of the biological fluids. LAL-test widely used for LPS concentration estimation is based on the LPS biological activity measurement and thus may depend on the LPS concentration in a non-linear way. Here we propose a new explicit method for the LPS concentration measurement based on fluorescently labeled LPS and direct photon counting and develop the new protocol for LPS adsorption efficiency measurement. Following the suggested protocol in the experiments on novel sorbents, we demonstrate that LPS adsorption at small biologically relevant concentrations is non-Langmuir.


Subject(s)
Lipopolysaccharides/analysis , Adsorption , Molecular Structure
5.
PLoS Comput Biol ; 15(8): e1007327, 2019 08.
Article in English | MEDLINE | ID: mdl-31469822

ABSTRACT

Thirteen tubulin protofilaments, made of αß-tubulin heterodimers, interact laterally to produce cytoskeletal microtubules. Microtubules exhibit the striking property of dynamic instability, manifested in their intermittent growth and shrinkage at both ends. This behavior is key to many cellular processes, such as cell division, migration, maintenance of cell shape, etc. Although assembly and disassembly of microtubules is known to be linked to hydrolysis of a guanosine triphosphate molecule in the pocket of ß-tubulin, detailed mechanistic understanding of corresponding conformational changes is still lacking. Here we take advantage of the recent generation of in-microtubule structures of tubulin to examine the properties of protofilaments, which serve as important microtubule assembly and disassembly intermediates. We find that initially straight tubulin protofilaments, relax to similar non-radially curved and slightly twisted conformations. Our analysis further suggests that guanosine triphosphate hydrolysis primarily affects the flexibility and conformation of the inter-dimer interface, without a strong impact on the shape or flexibility of αß-heterodimer. Inter-dimer interfaces are significantly more flexible compared to intra-dimer interfaces. We argue that such a difference in flexibility could be key for distinct stability of the plus and minus microtubule ends. The higher flexibility of the inter-dimer interface may have implications for development of pulling force by curving tubulin protofilaments during microtubule disassembly, a process of major importance for chromosome motions in mitosis.


Subject(s)
Tubulin/chemistry , Biomechanical Phenomena , Computational Biology , Cryoelectron Microscopy , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Hydrolysis , Microtubules/chemistry , Microtubules/metabolism , Microtubules/ultrastructure , Models, Molecular , Molecular Dynamics Simulation , Principal Component Analysis , Protein Conformation , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Stability , Protein Structure, Quaternary , Tubulin/metabolism , Tubulin/ultrastructure
6.
J Phys Chem B ; 122(14): 3711-3722, 2018 04 12.
Article in English | MEDLINE | ID: mdl-29553736

ABSTRACT

Phthalocyanines are aromatic macrocyclic compounds, which are structurally related to porphyrins. In clinical practice, phthalocyanines are used in fluorescence imaging and photodynamic therapy of cancer and noncancer lesions. Certain forms of the substituted polycationic metallophthalocyanines have been previously shown to be active in photodynamic inactivation of both Gram-negative and Gram-positive bacteria; one of them is zinc octakis(cholinyl)phthalocyanine (ZnPcChol8+). However, the molecular details of how these compounds translocate across bacterial membranes still remain unclear. In the present work, we have developed a coarse-grained (CG) molecular model of ZnPcChol8+ within the framework of the popular MARTINI CG force field. The obtained model was used to probe the solvation behavior of phthalocyanine molecules, which agreed with experimental results. Subsequently, it was used to investigate the molecular details of interactions between phthalocyanines and membranes of various compositions. The results demonstrate that ZnPcChol8+ has high affinity to both the inner and the outer model membranes of Gram-negative bacteria, although this species does not show noticeable affinity to the 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylcholine membrane. Furthermore, we found out that the process of ZnPcChol8+ penetration toward the center of the outer bacterial membrane is energetically favorable and leads to its overall disturbance and formation of the aqueous pore. Such intramembrane localization of ZnPcChol8+ suggests their twofold cytotoxic effect on bacterial cells: (1) via induction of lipid peroxidation by enhanced production of reactive oxygen species (i.e., photodynamic toxicity); (2) via rendering the bacterial membrane more permeable for additional Pc molecules as well as other compounds. We also found that the kinetics of penetration depends on the presence of phospholipid defects in the lipopolysaccharide leaflet of the outer membrane and the type of counterions, which stabilize it. Thus, the results of our simulations provide a detailed molecular view of ZnPcChol8+ "self-promoted uptake", the pathway previously proposed for some small molecules crossing the outer bacterial membrane.

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