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1.
Dokl Biol Sci ; 510(1): 167-171, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37582993

RESUMO

The study explored the potential of an animal opsin nonselectively expressed in various neuronal elements of the degenerative retina to restore the impaired visual function. A knockout murine model of inherited retinal dystrophy was used. Mice were injected intravitreally with either a virus carrying the gene of short-wavelength cone opsin associated with a reporter fluorescent protein or a control virus carrying the sequence of a modified fluorescent protein with enhanced membrane tropism. Viral transduction induced pronounced opsin expression in ganglion, bipolar, and horizontal retinal neurons. Behavioral testing included the visually guided task in the trapezoid Morris water maze and showed a partial recovery of the learning ability in the mice whose retinas had been transduced with cone opsin.


Assuntos
Opsinas dos Cones , Degeneração Retiniana , Camundongos , Animais , Opsinas dos Cones/genética , Opsinas dos Cones/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Retina , Opsinas/metabolismo , Camundongos Knockout
2.
Biophys Rev ; 14(4): 1051-1065, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36124271

RESUMO

Lipofuscin granules (LGs) are accumulated in the retinal pigment epithelium (RPE) cells. The progressive LG accumulation can somehow lead to pathology and accelerate the aging process. The review examines composition, spectral properties and photoactivity of LGs isolated from the human cadaver eyes. By use of atomic force microscopy and near-field microscopy, we have revealed the fluorescent heterogeneity of LGs. We have discovered the generation of reactive oxygen species by LGs, and found that LGs and melanolipofuscin granules are capable of photoinduced oxidation of lipids. It was shown that A2E, as the main fluorophore (bisretinoid) of LGs, is much less active as an oxidation photosensitizer than other fluorophores (bisretinoids) of LGs. Photooxidized products of bisretinoids pose a much greater danger to the cell than non-oxidized one. Our studies of the fluorescent properties of LGs and their fluorophores (bisretinoids) showed for the first time that their spectral characteristics change (shift to the short-wavelength region) in pathology and after exposure to ionizing radiation. By recording the fluorescence spectra and fluorescence decay kinetics of oxidized products of LG fluorophores, it is possible to improve the methods of early diagnosis of degenerative diseases. Lipofuscin ("aging pigment") is not an inert "slag". The photoactivity of LGs can pose a significant danger to the RPE cells. Fluorescence characteristics of LGs are a tool to detect early stages of degeneration in the retina and RPE.

3.
Biochemistry (Mosc) ; 85(6): 668-678, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32586230

RESUMO

The objective of this study was screening of ommochromes from the compound eyes of insects and comparison of their antioxidant properties. Ommochromes were isolated in preparative quantities from insects of five different families: Stratiomyidae, Sphingidae, Blaberidae, Acrididae, and Tenebrionidae. The yield of ommochromes (dry pigment weight) was 0.9-5.4% of tissue wet weight depending on the insect species. Isolated pigments were analyzed by high-performance liquid chromatography and represented a mixture of several ommochromes of the ommatin series. The isolated ommochromes displayed a pronounced fluorescence with the emission maxima at 435-450 nm and 520-535 nm; furthermore, the emission intensity increased significantly upon ommochrome oxidation with hydrogen peroxide. The ommochromes produced a stable EPR signal consisting of a singlet line with g = 2.0045-2.0048, width of 1.20-1.27 mT, and high concentration of paramagnetic centers (> 1017 spin/g dry weight). All the investigated ommochromes demonstrated high antiradical activity measured from the degree of chemiluminescence quenching in a model system containing luminol, hemoglobin, and hydrogen peroxide. The ommochromes strongly inhibited peroxidation of the photoreceptor cell outer segments induced by visible light in the presence of lipofuscin granules from the human retinal pigment epithelium, as well as suppressed iron/ascorbate-mediated lipid peroxidation. The obtained results are important for understanding the biological functions of ommochromes in invertebrates and identifying invertebrate species that could be used as efficient sources of ommochromes for pharmacological preparations to prevent and treat pathologies associated with the oxidative stress development.


Assuntos
Antioxidantes/farmacologia , Fenômenos Químicos , Olho Composto de Artrópodes/química , Insetos/metabolismo , Fenotiazinas/farmacologia , Epitélio Pigmentado da Retina/metabolismo , Animais , Olho Composto de Artrópodes/metabolismo , Peróxido de Hidrogênio , Insetos/efeitos dos fármacos , Luz , Peroxidação de Lipídeos , Oxirredução , Epitélio Pigmentado da Retina/efeitos dos fármacos
4.
Dokl Biol Sci ; 487(1): 95-97, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31571073

RESUMO

The visually driven instrumental conditioning of a single monkey (Macaca mulatta) was conducted after single-dose cranial irradiation with high-energy protons. The monkey executed saccades toward the visual stimuli and then responded by manually pressing right or left lever for stimuli in right or left half-field, respectively. The percentage of correct responses with dominant right hand exceeded the percentage of such responses with left hand and temporarily decreased two months after irradiation. A month later, the percentage of correct right-hand responses returned to the level before irradiation. No significant dynamic was found for changes in percentage of correct left-hand responses. The proton irradiation effect on right-hand responses suggests possible short-term disturbances in the eye-hand coordination for right handedness while the visual perception remains unaffected.


Assuntos
Irradiação Craniana/efeitos adversos , Prótons/efeitos adversos , Desempenho Psicomotor , Animais , Condicionamento Operante , Radiação Cósmica/efeitos adversos , Lateralidade Funcional , Macaca mulatta , Masculino
5.
Dokl Biochem Biophys ; 486(1): 184-186, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367817

RESUMO

To perform optogenetic prosthetics of the retinal ganglion cell receptive field, a bicistronic genetic construct carrying the genes encoding the excitatory (channelrhodopsin-2) and inhibitory (Guillardia theta anion channelrhodopsin GtACR2) rhodopsins was created. A characteristic feature of this construct was the combination of these two genes with a mutant IRES insertion between them, which ensures the exact ratio of expression levels of the first and second genes in each transfected cell. Illumination of the central part of the neuron with light with a wavelength of 470 nm induced the action potential generation in the cell. Stimulation of the peripheral neuronal region with light induced the inhibition of action potential generation. Thus, using optogenetics methods, we simulated the ON-OFF interaction in the retinal ganglion cell receptive field. Theoretically, this construct can be used for optogenetic prosthetics of degenerative retina in the case of its delivery to the ganglion cells with lentiviral vectors.


Assuntos
Channelrhodopsins/genética , Optogenética/métodos , Retina/patologia , Células Ganglionares da Retina/metabolismo , Animais , Luz , Neurônios/citologia , Neurônios/metabolismo , Neurônios/efeitos da radiação , Ratos , Retina/efeitos da radiação , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/efeitos da radiação , Transfecção
6.
Int J Biol Macromol ; 140: 736-748, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31445149

RESUMO

α-Crystallin maintains the transparency of the lens by preventing the aggregation of damaged proteins. The aim of our work was to study the chaperone-like activity of native α-crystallin in near physiological conditions (temperature, ionic power, pH) using UV-damaged ßL-crystallin as the target protein. α-Crystallin in concentration depended manner inhibits the aggregation of UV-damaged ßL-crystallin. DSC investigation has shown that refolding of denatured UV-damaged ßL-crystallin was not observed under incubation with α-crystallin. α-Crystallin and UV-damaged ßL-crystallin form dynamic complexes with masses from 75 to several thousand kDa. The content of UV-damaged ßL-crystallin in such complexes increases with the mass of the complex. Complexes containing >10% of UV-damaged ßL-crystallin are prone to precipitation whereas those containing <10% of the target protein are relatively stable. Formation of a stable 75 kDa complex is indicative of α-crystallin dissociation. We suppose that α-crystallin dissociation is the result of an interaction of comparable amounts of the chaperone-like protein and the target protein. In the lens simultaneous damage of such amounts of protein, mainly ß and gamma-crystallins, is impossible. The authors suggest that in the lens rare molecules of the damaged protein interact with undissociated oligomers of α-crystallin, and thus preventing aggregation.


Assuntos
Cristalino/metabolismo , alfa-Cristalinas/metabolismo , beta-Cristalinas/metabolismo , Chaperonas Moleculares/metabolismo , Agregados Proteicos/fisiologia , Temperatura , Raios Ultravioleta
7.
Biochemistry (Mosc) ; 84(5): 479-490, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31234763

RESUMO

The review discusses the prospects of using rhodopsin as an optogenetic tool for prosthetics of degenerative (blind) eye retina and the principles of optogenetic techniques. Retinal-containing proteins that depolarize/hyperpolarize the plasma membrane of nerve cells and, accordingly, excite/inhibit physiological activity of neurons, are described. The problem of what cells of the degenerative retina can be treated with what particular rhodopsins is discussed in detail. Viruses and promoters required for the rhodopsin gene delivery into the degenerative retina cells are described. In conclusion, main concepts and tasks associated with the optogenetic prosthetic treatment of degenerative retina employing rhodopsins are presented.


Assuntos
Optogenética/métodos , Degeneração Retiniana/patologia , Animais , Terapia Genética , Humanos , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Degeneração Retiniana/terapia , Células Ganglionares da Retina/metabolismo , Rodopsina/genética , Rodopsina/metabolismo
8.
Dokl Biochem Biophys ; 475(1): 283-286, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28864898

RESUMO

For the first time, it was found that the hormone melatonin exhibited antiglycation activity in vitro. It was shown that melatonin significantly slowed down the accumulation of fluorescent Schiff adducts formed as a result of BSA modification in the presence of high concentration of fructose. It was noted that, unlike the fructosylation reaction, melatonin did not affect the process of modification of BSA by methylglyoxal. We assume that melatonin is able to inhibit the development of the Maillard reaction but does not affect the process of BSA modification by reactive carbonyls.


Assuntos
Melatonina/farmacologia , Animais , Antioxidantes/farmacologia , Bovinos , Relação Dose-Resposta a Droga , Frutose/metabolismo , Glicosilação/efeitos dos fármacos , Soroalbumina Bovina/metabolismo
9.
Biochemistry (Mosc) ; 82(8): 916-924, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28941459

RESUMO

The effect of superoxide radicals on melanin destruction and degradation of melanosomes isolated from cells of retinal pigment epithelium (RPE) of the human eye was studied. We found that potassium superoxide causes destruction of melanin in melanosomes of human and bovine RPE, as well as destruction of melanin from the ink bag of squid, with the formation of fluorescent decay products having an emission maximum at 520-525 nm. The initial kinetics of the accumulation of the fluorescent decay products is linear. Superoxide radicals lead simultaneously to a decrease in the number of melanosomes and to a decrease in concentration of paramagnetic centers in them. Complete degradation of melanosomes leads to the formation of a transparent solution containing dissolved proteins and melanin degradation products that do not exhibit paramagnetic properties. To completely degrade one melanosome of human RPE, 650 ± 100 fmol of superoxide are sufficient. The concentration of paramagnetic centers in a melanolipofuscin granule of human RPE is on average 32.5 ± 10.4% (p < 0.05, 150 eyes) lower than in a melanosome, which indicates melanin undergoing a destruction process in these granules. RPE cells also contain intermediate granules that have an EPR signal with a lower intensity than that of melanolipofuscin granules, but higher than that of lipofuscin granules. This signal is due to the presence of residual melanin in these granules. Irradiation of a mixture of melanosomes with lipofuscin granules with blue light (450 nm), in contrast to irradiation of only melanosomes, results in the appearance of fluorescent melanin degradation products. We suggest that one of the main mechanisms of age-related decrease in melanin concentration in human RPE cells is its destruction in melanolipofuscin granules under the action of superoxide radicals formed during photoinduced oxygen reduction by lipofuscin fluorophores.


Assuntos
Melaninas/metabolismo , Melanossomas/metabolismo , Animais , Bovinos , Decapodiformes/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Cinética , Luz , Lipofuscina/química , Lipofuscina/metabolismo , Melaninas/química , Melanossomas/efeitos dos fármacos , Melanossomas/efeitos da radiação , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Superóxidos/química , Superóxidos/metabolismo , Superóxidos/farmacologia
10.
Dokl Biochem Biophys ; 474(1): 239-243, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28726093

RESUMO

A comparative analysis of fluorescence lifetime of lipofuscin granule fluorophores contained in the retinal pigment epithelium cells from human cadaver eyes in normal state and in the case of visualized pathology was carried out. Measurements of fluorescence lifetimes of bis-retinoids and their photooxidation and photodegradation products were carried out using the method of counting time-correlated photons. Comparative analysis showed that, in the case of visualized pathology, the contribution of photooxidation and photodegradation products of bis-retinoids to the total fluorescence of the retinal pigment epithelium cell suspension increases in comparison with the norm.


Assuntos
Oftalmopatias/patologia , Lipofuscina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Cadáver , Humanos , Cinética , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/patologia , Espectrometria de Fluorescência
11.
Dokl Biochem Biophys ; 472(1): 19-22, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28421434

RESUMO

The fluorescence lifetimes of lipofuscin fluorophores contained in chloroform extracts from retinal pigment epithelium (RPE) of human cadaver eyes without signs of pathology were evaluated by single photon counting. The comparison of fluorescence lifetimes of N-retinylidene-N-retinylethanolamine (A2E) and its photooxidation and photodegradation products has been carried out. It was shown that the contribution of A2E to the total fluorescence of chloroform extract from lipofuscin granules is not major. The results are important for the improvement of noninvasive diagnostic method of degenerative diseases of the retina and RPE-fundus autofluorescence (FAF).


Assuntos
Lipofuscina/química , Epitélio Pigmentado da Retina/metabolismo , Cadáver , Grânulos Citoplasmáticos/metabolismo , Fluorescência , Humanos , Lipofuscina/metabolismo , Oxirredução , Fótons , Retinoides/química
12.
Biochemistry (Mosc) ; 82(4): 490-500, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28371607

RESUMO

Photochemical reaction dynamics of the primary events in recombinant bacteriorhodopsin (bRrec) was studied by femtosecond laser absorption spectroscopy with 25-fs time resolution. bRrec was produced in an Escherichia coli expression system. Since bRrec was prepared in a DMPC-CHAPS micelle system in the monomeric form, its comparison with trimeric and monomeric forms of the native bacteriorhodopsin (bRtrim and bRmon, respectively) was carried out. We found that bRrec intermediate I (excited state of bR) was formed in the range of 100 fs, as in the case of bRtrim and bRmon. Further processes, namely the decay of the excited state I and the formation of intermediates J and K of bRrec, occurred more slowly compared to bRtrim, but similarly to bRmon. The lifetime of intermediate I, judging from the signal of ΔAESA(470-480 nm), was 0.68 ps (78%) and 4.4 ps (22%) for bRrec, 0.52 ps (73%) and 1.7 ps (27%) for bRmon, and 0.45 ps (90%) and 1.75 ps (10%) for bRtrim. The formation time of intermediate K, judging from the signal of ΔAGSA(625-635 nm), was 13.5 ps for bRrec, 9.8 ps for bRmon, and 4.3 ps for bRtrim. In addition, there was a decrease in the photoreaction efficiency of bRrec and bRmon as seen by a decrease in absorbance in the differential spectrum of the intermediate K by ~14%. Since photochemical properties of bRrec are similar to those of the monomeric form of the native protein, bRrec and its mutants can be considered as a basis for further studies of the mechanism of bacteriorhodopsin functioning.


Assuntos
Bacteriorodopsinas/química , Biopolímeros/química , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Proteínas Recombinantes/química , Espectrofotometria Ultravioleta
13.
Neurosci Lett ; 640: 76-80, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28093304

RESUMO

Optogenetics is a powerful technique in neuroscience that provided a great success in studying the brain functions during the last decade. Progress of optogenetics crucially depends on development of new molecular tools. Light-activated cation-conducting channelrhodopsin2 was widely used for excitation of cells since the emergence of optogenetics. In 2015 a family of natural light activated chloride channels GtACR was identified which appeared to be a very promising tool for using in optogenetics experiments as a cell silencer. Here we examined properties of GtACR2 channel expressed in the rat layer 2/3 pyramidal neurons by means of in utero electroporation. We have found that despite strong inhibition the light stimulation of GtACR2-positive neurons can surprisingly lead to generation of action potentials, presumably initiated in the axonal terminals. Thus, when using the GtACR2 in optogenetics experiments, its ability to induce action potentials should be taken into account. Our results also open an interesting possibility of using the GtACR2 both as cell silencer and cell activator in the same experiment varying the pattern of light stimulation.


Assuntos
Potenciais de Ação/efeitos da radiação , Córtex Cerebral/efeitos da radiação , Canais de Cloreto/fisiologia , Luz , Células Piramidais/efeitos da radiação , Potenciais de Ação/fisiologia , Animais , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Canais de Cloreto/genética , Feminino , Humanos , Proteínas Luminescentes/genética , Masculino , Células Piramidais/fisiologia , Ratos , Proteínas Recombinantes de Fusão/genética
14.
Dokl Biochem Biophys ; 471(1): 435-439, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28058680

RESUMO

A quantum-classical model of photoisomerization of the visual pigment rhodopsin chromophore is proposed. At certain (and more realistic) parameter value combinations, the model is shown to accurately reproduce a number of independent experimental data on the photoreaction dynamics: the quantum yield, the time to reach the point of conical intersection of potential energy surfaces, the termination time of the evolution of quantum subsystem, as well as the characteristic low frequencies of retinal molecular lattice fluctuations during photoisomerization. In addition, the model behavior is in good accordance with experimental data about coherence and local character of quantum transition.


Assuntos
Modelos Teóricos , Teoria Quântica , Rodopsina/química , Vitamina A/química , Algoritmos , Isomerismo , Estrutura Molecular , Processos Fotoquímicos , Retina/química
15.
Dokl Biochem Biophys ; 471(1): 440-442, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28058692

RESUMO

Anion-selective opsins slow ChloC and ACR2 were expressed in rat brain cortical neurons by electroporation in utero. It is shown that the light-activated channel ACR2 has pronounced advantages in terms of both the inactivation kinetics and the neuron inhibition intensity, which is associated with a more negative value of the light-activated current reversal potential compared to the slow ChloC channel. The identified properties of opsin ACR2 indicate that it can be used for strictly controlled suppression of neuronal activity in optogenetic experiments, including the expression in the retinal ganglionic cells for reconstituting the OFF-component of their receptive field, which is essential for optogenetic prosthetics of degenerative retina.


Assuntos
Optogenética , Rodopsina/metabolismo , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Animais , Córtex Cerebral/fisiologia , Córtex Cerebral/efeitos da radiação , Eletroporação , Luz , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Neurônios/fisiologia , Neurônios/efeitos da radiação , Optogenética/métodos , Técnicas de Patch-Clamp , Ratos , Rodopsina/genética , Técnicas de Cultura de Tecidos , Canal de Ânion 2 Dependente de Voltagem/genética
16.
Tsitologiia ; 57(2): 119-28, 2015.
Artigo em Russo | MEDLINE | ID: mdl-26035969

RESUMO

Emerging body of data indicate protecting effect of low level of stress (preconditioning) on retina. Our previous studies have revealed a non-linear dose-response relationship for cytotoxic effect of both ionizing radiation and N-methyl-N-nitrosourea (MNU) on mouse retina. Moreover, non-cytotoxic dose of MNU increased tolerance of retina to following challenge dose of MNU. This result displays protection of retina through mechanism of recovery. In the present study we used the mouse model for MNU-induced retinal degeneration to evaluate the adaptive response of the retina to proton irradiation and implication of glial Muller cells in this response. In this paper, we have shown that the recovery of the retina after exposure to genotoxic agents is associated with an increased efficiency of DNA damage repair and lowered death of retinal photoreceptors.


Assuntos
Metilnitrosoureia/toxicidade , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Retina/efeitos dos fármacos , Retina/efeitos da radiação , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Camundongos , Células Fotorreceptoras de Vertebrados/patologia , Radiação Ionizante , Retina/patologia , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/patologia , Estresse Fisiológico
17.
Dokl Biochem Biophys ; 465: 420-3, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26728739

RESUMO

Supramolecular organization of rhodopsin in the photoreceptor membrane was investigated by small-angle neutron scattering method. The experiments, which were performed with mixtures of heavy/light water as solvent (contrast variation method), were aimed at obtaining information about the lipid and protein components of the photoreceptor disc membrane separately. It was shown that the packaging density of the rhodopsin molecules in the photoreceptor membrane was unusually high: the distance between the centers of the molecules was approximately 56 Å. The probability of the monomeric state of rhodopsin molecules in the photoreceptor membrane, according to the data obtained, is rather high.


Assuntos
Membrana Celular/química , Rodopsina/química , Animais , Bovinos , Difração de Nêutrons , Células Fotorreceptoras de Vertebrados/química , Espalhamento a Baixo Ângulo
18.
Dokl Biochem Biophys ; 465: 424-7, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26728740

RESUMO

Anionic channelrhodopsin slow ChloC was expressed in the culture of nerve cells and in vivo in mouse brain. We demonstrated ability of slow ChloC to suppress effectively the activity of the neuron in response to the illumination with the visible light. It has been shown for a first time that slow ChloC works equally efficiently in both neuronal culture and in the whole brain being expressed in vivo. Thus, slow ChloC could be considered as an effective optogenetic tool capable in response to light stimulation to inhibit the generation of action potentials in the neuron.


Assuntos
Potenciais de Ação , Encéfalo/metabolismo , Neurônios/metabolismo , Rodopsina/metabolismo , Animais , Encéfalo/citologia , Encéfalo/fisiologia , Células Cultivadas , Luz , Camundongos , Neurônios/fisiologia , Optogenética/métodos , Rodopsina/genética , Rodopsina/efeitos da radiação
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