BODIPY Dye Derivative for Irreversible Fluorescent Labeling of Eukaryotic Cells and Their Simultaneous Cytometric Analysis.

In this work, we synthesized a green fluorescent dye derivative, 1,3,5,7-tetramethyl-BODIPY, with a heptyl substituent at the 8-position. The obtained highly hydrophobic compound was able to rapidly and irreversibly bind to eukaryotic cells. Incubation of cells with the dye over different periods of time or at different concentrations allowed us to control the degree of cell labeling and the level of fluorescence. This made it possible to modulate the fluorescence level of different eukaryotic cell cultures and then distinguish them by their level of fluorescence signal in the green channel in cytometric experiments. The labeled cells can be combined and further analyzed in the same test tube under identical conditions using the channels in which the dye does not fluoresce. This approach has been tested on a number of tumor cell cultures containing the HER2 receptor on their surface. The representation of the receptor in these cells was analyzed in one test tube in one run using a HER2-specific ligand based on the hybrid protein DARPin9_29-mCherry, which fluoresces in the red region of the spectrum.

Synthetic Fluorophores for Visualizing Biomolecules in Living Systems.

The last decade has witnessed significant advance in the imaging of living systems using fluorescent markers. This progress has been primarily associated with the discovery of different spectral variants of fluorescent proteins. However, the fluorescent protein technology has its own limitations and, in some cases, the use of low-molecular-weight fluorophores is preferable. In this review, we describe the arsenal of synthetic fluorescent tools that are currently in researchers' hands and span virtually the entire spectrum, from the UV to visible and, further, to the near-infrared region. An overview of recent advances in site-directed introduction of synthetic fluorophores into target cellular objects is provided. Application of these fluorescent probes to the solution of a wide range of biological problems, in particular, to the determination of local ion concentrations and pH in living systems, is discussed.

[The Development and Application of BODIPY-Labeled Oligonucleotides in Real-Time Polymerase Chain Reaction].

A BODIPY-based (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene, TMB) green fluorescent probe for quantitative real-time polymerase chain reaction (qPCR) was synthesized by azide-alkyne cycloaddition reaction. Comparative studies with analogous fluorescein-based probe were carryed out. We demonstrate that fluorescent probes with TMB fluorophore can be used in qPCR experiments as well.

[Ph-Sensor Properties of a Fluorescent Protein from Dendronephthya sp].

Genetically encoded biosensors based on fluorescent proteins are now widely applicable for monitoring pH changes in live cells. Here, we have shown that a fluorescent protein from Dendronephthya sp. (DendFP) exhibits a pronounced pH-sensitivity. Unlike most of known genetically encoded pH-sensors, fluorescence of the protein is not quenched upon medium acidification, but is shifting from the red to green spectral range. Therefore, quantitative measurements of intracellular pH are feasible by ratiometric comparison of emission intensities in the red and green spectral ranges, which makes DendFP advantageous compared with other genetically encoded pH-sensors.

[Age-dependent occupational stress in doctors of various specialties].

A comparative assessment of the extent and structure of the various forms of professional burnout among doctors-organizers as well as therapeutists and surgeons depending on age was given. It is shown that the professional burnout in doctors-organizers conjugates with a high level of emotional tension manifested in avoidance of over-saturated emotional and professional communication outside professional activities, increased irritability and temper, reluctance to exercise empathy towards colleagues and compassion for patients. The comparison of three age groups of physicians to each other in terms of the level and features of the burnout was adduced; the results demonstrate the significant differences between the age periods of 30 and 40 years and over 41 years old. Physicians in the age group of 30-40 years old are inclined to depersonalization at a relatively low level of reduction of professional achievement. It was found that age specificity of formation of the syndrome of professional burnout among surgeons was caused by the increased tendency to development of professional burnout syndrome of young and middle-aged surgeons at low levels of professional burnout among older people.

[3D-structure determination of fluorescent proteins by homology modeling combined with mass spectrometry].

A method for the 3D-structure generation of GFP-like fluorescent proteins is presented. The method is based on a combination of homology modeling for the overall spatial structure determination and mass spectrometry for the chromophore structure identification. The proposed approach can be applied to the spatial structure determination ofnoncrystalizable GFP homologs.

[Posttranslational reactions resulting in a long-wavelength shift in the spectra of asFP595 protein from Anemonia sulcata].

In most fluorescent proteins characterized by light absorption and emission in the red and the far-red spectral region (550-650 nm), the chromophore pi system is extended at the expense of the additional oxidation of the GFP-like structure and the formation of an acylimine substituent. As distinct from these proteins, the photoactivateable protein asFP595 contains a chromophore with the keto group substituted for an acylimine substituent. In this work, we studied the reactions that result in a bathochromic shift in the spectrum of asFP595. Maturation kinetics analysis has shown the generation of the immature form containing a protonated chromophore (absorption at 420 nm) at the intermediate step, as in the case of other red fluorescent proteins, which then is isobestically converted into the final mature form (568 nm). Mass spectrometric analysis of the chromopeptide isolated from immature asFP595 has demonstrated that the intermediate form contains a GFP-type chromophore. It has also been found that the oxidation of the GFP chromophore is accompanied by the generation of an equimolar amount of hydro gen peroxide. The intermediate products of oxidation have been analyzed by the mutagenesis of the first chromophore-generating amino acid residue. It has been demonstrated that in the case of all mutants studied, chromophore synthesis does not terminate at the stage of the acylimine derivative, but immediately results in the fragmentation of the main chain of the protein and in the formation of the keto form.

Posttranslational chemistry of proteins of the GFP family.

This review focuses on the current knowledge about posttranslational chemistry underlying the diverse optical properties of GFP-like proteins.

Photoconversion of the chromophore of a fluorescent protein from Dendronephthya sp.

A green fluorescent protein from the coral Dendronephthya sp. (Dend FP) is characterized by an irreversible light-dependent conversion to a red-emitting form. The molecular basis of this phenomenon was studied in the present work. Upon UV-irradiation at 366 nm, the absorption maximum of the protein shifted from 494 nm (the green form) to 557 nm (the red form). Concurrently, in the fluorescence spectra the emission maximum shifted from 508 to 575 nm. The green form of native Dend FP was shown to be a dimer, and the oligomerization state of the protein did not change during its conversion to the red form. By contrast, UV-irradiation caused significant intramolecular changes. Unlike the green form, which migrates in SDS-polyacrylamide gels as a single band corresponding to a full-length 28-kD protein, the red form of Dend FP migrated as two fragments of 18- and 10-kD. To determine the chemical basis of these events, the denatured red form of Dend FP was subjected to proteolysis with trypsin. From the resulting hydrolyzate, a chromophore-containing peptide was isolated by HPLC. The structure of the chromophore from the Dend FP red form was established by methods of ESI, tandem mass spectrometry (ESI/MS/MS), and NMR-spectroscopy. The findings suggest that the light-dependent conversion of Dend FP is caused by generation of an additional double bond in the side chain of His65 and a resulting extension of the conjugated system of the green form chromophore. Thus, classified by the chromophore structure, Dend FP should be referred to the Kaede subfamily of GFP-like proteins.