Water-Soluble Products of Photooxidative Destruction of the Bisretinoid A2E Cause Proteins Modification in the Dark.

Aging of the retina is accompanied by a sharp increase in the content of lipofuscin granules and bisretinoid A2E in the cells of the retinal pigment epithelium (RPE) of the human eye. It is known that A2E can have a toxic effect on RPE cells. However, the specific mechanisms of the toxic effect of A2E are poorly understood. We investigated the effect of the products of photooxidative destruction of A2E on the modification of bovine serum albumin (BSA) and hemoglobin from bovine erythrocytes. A2E was irradiated with a blue light-emitting diode (LED) source (450 nm) or full visible light (400-700 nm) of a halogen lamp, and the resulting water-soluble products of photooxidative destruction were investigated for the content of carbonyl compounds by mass spectrometry and reaction with thiobarbituric acid. It has been shown that water-soluble products formed during A2E photooxidation and containing carbonyl compounds cause modification of serum albumin and hemoglobin, measured by an increase in fluorescence intensity at 440-455 nm. The antiglycation agent aminoguanidine inhibited the process of modification of proteins. It is assumed that water-soluble carbonyl products formed as a result of A2E photodestruction led to the formation of modified proteins, activation of the inflammation process, and, as a consequence, to the progression of various senile eye pathologies.

Lipofuscin Granule Bisretinoid Oxidation in the Human Retinal Pigment Epithelium forms Cytotoxic Carbonyls.

Age-related macular degeneration (AMD) is the primary cause of central blindness among the elderly. AMD is associated with progressive accumulation of lipofuscin granules in retinal pigment epithelium (RPE) cells. Lipofuscin contains bisretinoid fluorophores, which are photosensitizers and are phototoxic to RPE and neuroretinal cells. In the presence of oxygen, bisretinoids are also oxidized, forming various products, consisting primarily of aldehydes and ketones, which are also potentially cytotoxic. In a prior study, we identified that in AMD, bisretinoid oxidation products are increased in RPE lipofuscin granules. The purpose of the present study was to determine if these products were toxic to cellular structures. The physicochemical characteristics of bisretinoid oxidation products in lipofuscin, which were obtained from healthy donor eyes, were studied. Raman spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis identified the presence of free-state aldehydes and ketones within the lipofuscin granules. Together, fluorescence spectroscopy, high-performance liquid chromatography, and mass spectrometry revealed that bisretinoid oxidation products have both hydrophilic and amphiphilic properties, allowing their diffusion through lipofuscin granule membrane into the RPE cell cytoplasm. These products contain cytotoxic carbonyls, which can modify cellular proteins and lipids. Therefore, bisretinoid oxidation products are a likely aggravating factor in the pathogenesis of AMD.

Antioxidative Properties of Melanins and Ommochromes from Black Soldier Fly Hermetia illucens.

A comparative study of melanin and ommochrome-containing samples, isolated from the black soldier fly (BSF) by enzymatic hydrolysis, alkaline and acid alcohol extraction or by acid hydrolysis, was carried out. Melanin was isolated both as a melanin-chitin complex and as a water-soluble melanin. Acid hydrolysis followed by delipidization yielded a more concentrated melanin sample, the electron spin resonance (ESR) signal of which was 2.6 × 1018 spin/g. The ommochromes were extracted from the BSF eyes with acid methanol. The antiradical activity of BSF melanins and ommochromes was determined by the method of quenching of luminol chemiluminescence. It has been shown that delipidization of water-soluble melanin increases its antioxidant properties. A comparison of the antioxidant activity of BSF melanins and ommochromes in relation to photoinduced lipid peroxidation was carried out. The ESR characteristics of native and oxidized melanins and ommochromes were studied. It is assumed that H. illucens adult flies can be a useful source of natural pigments with antioxidant properties.

Lipofuscins prepared by modification of photoreceptor cells via glycation or lipid peroxidation show the similar phototoxicity.

AIM: To investigate the effect of two ways of lipofuscin production (lipid peroxidation and glycation) on lipofuscin fluorescence characteristics and phototoxicity and to compare them with the properties of natural lipofuscin. METHODS: Model lipofuscins were prepared on the basis of bovine photoreceptor outer segments (POS) with bisretinoid A2E addition. One set of samples was prepared from POS modified by lipid peroxidation, while another set from POS modified by glycation with fructose. Fluorescent properties and kinetics of photoinduced superoxide generation of model lipofuscins and human retinal pigment epithelium (RPE) lipofuscin were compared. The fluorescence spectra of samples were measured at 365 nm excitation wavelength and 380-650 emission wavelength. RESULTS: The fluorescence spectra of model lipofuscins are almost the same as the spectrum of natural lipofuscin. Visible light irradiation of both model lipofuscins and natural lipofuscin isolated from RPE cells leads to decrease of a fluorescence maximum at 550 nm and to appearance of a distinct, new maximum at 445-460 nm. The rate of photogeneration of reactive oxygen forms by both model lipofuscins was almost the same and approximately two times less than that of RPE lipofuscin granules. CONCLUSION: These data suggest that fluorescent characteristics and phototoxicity of lipofuscin granules depend only to an insignificant degree on the oxidative modification of POS proteins and lipids, and generally are defined by the bisretinoid fluorophores contained in them.

3D molecular modeling, free radical modulating and immune cells signaling activities of the novel peptidomimetic L-glutamyl-histamine: possible immunostimulating role.

An original representative of the patented by author family of histamine-containing peptidomimetics L-glutamyl-histamine (L-Glu-Hist) was synthesized and characterized as a biologically active compound with a role of cytokine mimic leading to cellular responses of improved specificity. The study assesses the ability of L-Glu-Hist to affect molecular modeling, modulate free radical activity and influence immune cell signaling. The energy-minimized 3D conformations of L-Glu-Hist derived from its chemical structure resulted in stabilization for Fe2+ chelating complexes. L-Glu-Hist accelerated the decrease of ferrous iron in the ferrous sulfate solution in a concentration-dependent mode and showed the ferroxidase-like activity at concentrations less than 3 mM in the phenanthroline assay, whereas in the concentration range 3-20 mM L-Glu-Hist restricted the availability of Fe2+ to phenanthroline due to binding of ferrous ions in chelating complexes. L-Glu-Hist showed stimulatory effect on phosphatidylcholine liposomal peroxidation (LPO) catalyzed by the superoxide anion radical (O2*-)-generating system (Fe2+ + ascorbate) at low (less or about 1 mM) L-Glu-Hist concentrations and both revealed the inhibitory effect on LPO in this system of high (approximately 10 mM) L-Glu-Hist concentration. The stimulation of LPO by L-Glu-Hist was related to the ability of peptidomimetic in small (approximately 0.05 mM) concentrations to release O2*- free radicals as determined by the superoxide dismutase-inhibitable cytochrome c reduction assay. O2*- release by L-Glu-Hist might result from its ferroxidase-like activity, while inhibition of LPO by L-Glu-Hist was caused by its chelating activity to Fe2+ ions, prevention of free radical generation and lipid hydroperoxide-degrading ability of 5-20 mM L-Glu-Hist. L-Glu-Hist released O2*- in concentrations which stimulated [3H]-thymidine incorporation into DNA and proliferation of mouse spleen lymphocytes and mononuclear cells from human blood. L-Glu-Hist modulates the ability of oxygen free radicals to act as signaling agents at low concentrations, influencing gene expression. The structural peptide-like analogues of L-Glu-Hist such as L-Glu-Trp, carcinine (beta-alanylhistamine), but not L-Pro-Glu-Trp were active in stimulating thymidine incorporation and in inducing proliferation of mononuclear cells as compared to mitogen concanavalin A at doses 2.5-25.0 microg/ml. Our data provide evidence that L-Glu-Hist may act as a very fast, specific and sensitive trigger for lymphocyte proliferation and immunoregulation. The cited abilities and further obtained in vivo results make Immudilin ((INCI: glutamylamidoethyl imidazole, aqueous solution), L-Glu-Hist) a useful immunoregulatory agent.