Antioxidant properties of melanin in retinal pigment epithelial cells

The retinal pigment epithelium (RPE) is a monolayer of highly pigmented cells lining the inner aspect of Bruch's membrane. This pigmentation is due to eumelanin and a possible antioxidant role of melanin is reported here. The photo-oxidation of A2E, a constituent of RPE lipofuscin, leads to the sequential addition of up to nine oxygen atoms and/or the addition or loss of two hydrogen atoms. These photo-oxidations were investigated in the presence and absence of either calf or human RPE melanin in A2E-laden RPE cells. It was found that calf melanin was protective against the photo-oxidation of A2E, with an inhibition of oxidation of up to 50% in the case of the addition of two oxygen atoms. Calf melanin was also protective against blue light-induced damage to RPE cells. In addition this ability appears to decrease in humans as they grow older. With aging, a melanin-lipofuscin complex called melanolipofuscin forms. It is suggested that the oxidation or photo-oxidation of A2E in vivo may contribute to the age-related deterioration of the anti-oxidant role of RPE melanin and lead to various retinal disorders, such as age-related macular degeneration.     

Blue light irradiation inhibits the production of HGF by human retinal pigment epithelium cells in vitro

Blue visible light damage to retinal pigment epithelial cells occurs through a photooxidative mechanism and the resultant damage is hypothesized to induce or exacerbate age-related macular degeneration. The purpose of the present study was to identify changes in the cell growth and the expression of hepatocyte growth factor (HGF) in cultured human retinal pigment epithelium (RPE) cells as a result of both blue and red light irradiation. HGF is a growth factor and neurotrophic factor that stimulates growth of various ocular cells and promotes the survival of RPE and retinal neurons. Early passages of human RPE cells were exposed to blue light (460 nm) and red light (640 nm). Nonirradiated cells were used as controls. After 24 and 48 h, conditioned medium was collected and the amount of HGF was measured by ELISA. Cells were detached from the well and counted. Cell viability was evaluated by trypan-blue exclusion study. Blue light at dosage of 63 J/cm(2) significantly inhibited the growth of RPE cells without affecting of cell viability. Amounts of HGF in the culture medium were significantly inhibited by blue-light irradiation at the dosage from 32 to 63 J/cm(2). Red light at a dose of 174 J/cm(2) causes a nonsignificant inhibition of growth of RPE cells and a slight decrease of secretion of HGF. As HGF promotes survival of RPE cells and retinal neurons, the inhibition of production of HGF by visible light, especially by blue light, may enhance the phototoxic effects of visible light on the RPE and retinal neurons.     

In vitro assays for evaluating the ultraviolet B-induced damage in cultured human retinal pigment epithelial cells

The present study demonstrates broadband UV-B-induced damage of cultured human retinal pigment epithelial cells as an effort to develop an in vitro model that can be used, along with in vivo research and other in vitro efforts, to evaluate the need for retinal UV protection in humans after cataract removal. The human retinal pigment epithelial cell line, ARPE-19, was cultured in two groups: control and treated. Treated cells were irradiated with three broadband UVB radiations at energy levels of 0.05, 0.1 and 0.2J/cm(2). After irradiation, cells were incubated for 48h while cellular viability, morphology, and phagocytotic activity were analyzed using the Alamar blue assay, confocal microscopy, and fluorescent microspheres. Confocal analysis concentrated on the study of the cell nuclei and mitochondria. The Alamar blue assay of UV-B-exposed cells showed dose and time-dependent decreases in cellular viability in comparison to control cells. Loss of cell viability was measured at the two higher energy levels (0.2 and 0.1J/cm(2)), but the cell group exposed to 0.05J/cm(2) showed no significant viability change at 1-h time point. Morphological evaluation also showed dose and time-dependent degradation of mitochondria and nucleic acids. Cells exposed with 0.05J/cm(2) UVB did not show significant degradation of mitochondria and nucleic acids during the entire culture period. Phagocytotic activity assay data for UVB-exposed cells showed dose-dependent decreases in phagocytotic activity in comparison with the control cells. The control cells have significantly greater capacities for uptake than the 0.1 and 0.2J/cm(2) UV-B-exposed cells, while the 0.05J/cm(2) UV-B-exposed cell group showed no significant difference from the control cell group. The findings suggest that UVB radiation-induced cultured RPE cell damage can be evaluated by assays that probe cellular viability, morphological change, and phagocytotic activity, and that these assay methods together provide a valuable in vitro model for ultraviolet radiation-induced retinal toxicology research.     

Optimization of bulk cell electrofusion in vitro for production of human-mouse heterohybridoma cells

Cell electrofusion is a phenomenon that occurs, when cells are in close contact and exposed to short high-voltage electric pulses. The consequence of exposure to pulses is transient and nonselective permeabilization of cell membranes. Cell electrofusion and permeabilization depend on the values of electric field parameters including amplitude, duration and number of electric pulses and direction of the electric field. In our study, we first investigated the influence of the direction of the electric field on cell fusion in two cell lines. In both cell lines, applications of pulses in two directions perpendicular to each other were the most successful. Cell electrofusion was finally used for production of human-mouse heterohybridoma cells with modified Koehler and Milstein hybridoma technology, which was not done previously. The results, obtained by cell electrofusion, are comparable to usually used polyethylene glycol mediated fusion on the same type of cells.     

Synthetic melanin is a model for soluble natural eumelanin in UVA-photosensitised superoxide production

Studies to UV-irradiate natural eumelanins in vitro have used insoluble pigment obtained by acid hydrolysis, which lacks melanoprotein. Eumelanin synthesised in the presence of a protein is not insoluble, and the insoluble form of melanin from acid hydrolysis may not have the same physicochemical properties as the natural pigment synthesised in vivo in the melanosome. Here we investigated radical production by three natural eumelanins exposed to solar levels of UVA; sepia melanin from Sepia officinalis, and eumelanins isolated from Oriental human and domestic cat hair. UVA irradiation of sepia melanin in solution at pH 4.5 in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) gave hydroperoxyl and hydroxyl radical-adducts, maximal at 0.6-2.5 mg/ml melanin concentrations. Hydroperoxyl radical production was relatively low in acetate buffer, but detected in aqueous suspensions of sepia melanin. Hair eumelanins were photoreactive with hydroperoxyl radical-adduct production at low concentrations (0.1-0.4 mg/ml melanin). Synthetic pigment after synthesis undergoes photo-oxidation (producing superoxide) at low concentrations (0.3 mg/ml) and its oxidation increases the photoreactivity at higher melanin concentrations. These findings may be physiologically relevant to the properties and function of eumelanin in vivo when it is at low concentration (found in a small proportion of Caucasian melanocytes), and suggest that synthetic melanin has the potential for the basis of a model for natural eumelanin.     

N-Alkylamides from Piper longum L. and their stimulative effects on the melanin content and tyrosinase activity in B16 melanoma cells

Abstract

Piper longum L., known as long pepper, is an edible and medicinal plant used as spice and for the treatment of stomach disease and analgesia in traditional Chinese medicine. N-Alkylamides are the major secondary metabolites in this plant. Sixteen known N-alkylamides were isolated from P. longum. Their structures were established on the basis of spectroscopic data and comparison to reported literatures. Among them, five compounds were isolated from this plant for the first time. Ethanol extract, compounds 1, 2, 3, 7 and 11 exhibited potent ability to increase the melanin content and weak stimulative effect on the tyrosinase activity in a concentration-dependent manner. Moreover, compound 2 also presented strong capacity to increase the tyrosinase activity in a concentration-dependent manner. These results indicated that P. longum might be a good natural source of lead compound for skin disorder diseases.     

Percutaneous penetration, melanin activation and toxicity evaluation of a phytotherapic formulation for vitiligo therapeutic

The aim of this work was to apply photoacoustic spectroscopy for the ex vivo determination of the penetration rate of a phytotherapic formulation for vitiligo therapeutic, with or without salicylic acid as the promoter agent. In addition, the compound toxicity and morphophysiology effects were evaluated for different concentrations of salicylic acid. The experiments were performed as a function of the period of time of treatment in a well-controlled group of rabbits. Toxic effects were not observed with any of the tested products. All formulations containing salicylic acid induced cutaneous reaction which was dose dependent. The histological analysis showed that the use of the medication was associated with an increased comedogenic effect in relation to the control group, regardless of salicylic acid concentration. Inflammatory reactions and acanthosis were observed only in the animals treated with formulations containing higher concentrations of salicylic acid, while none of these effects were detected with the use of the formulation containing 2.5% (wt/vol) of salicylic acid. Photoacoustic depth monitoring showed that both formulations, with or without salicylic acid, propagated through the skin up to the melanocytes region, suggesting that the transport of the active agent may occur through the epithelial structure without the need of using queratinolitic substances, which are known to induce side effects in the animals.     

Inhibition effect of phenyl compounds from the Oryza sativa roots on melanin production in murine B16-F10 melanoma cells

Five phenyl compounds, vanillin (1), methyl trans-ferulate (2), trans-p-coumaric acid methyl ester (3), N-benzoyltryptamine (4), and N-(trans-cinnamoyl)tryptamine (5), were isolated from the roots of Oryza sativa L. and identified on the basis of spectroscopic data. Compounds 3 and 5 showed strong inhibition effect on melanin production in murine B16-F10 melanoma cells and tyrosinase activity. Also, the quantitative analysis of the compounds was carried out using LC/MS/MS experiment. Compounds 3 and 5 could be used as skin-whitening agents.     

In vitro accelerated mass propagation and ex vitro evaluation of Aloe vera L. with aloin content and superoxide dismutase activity

An innovative protocol on accelerated in vitro propagation and acclimatisation was developed in Aloe vera L. Culture was initiated with rhizomatous stem where Murashige and Skoog (MS) medium fortified with 0.5?mg?L(-1) α-naphthalene acetic acid and 1.5?mg?L(-1) N(6)-benzylaminopurine (BAP) promoted earliest shoot induction. Maximum shoot multiplication was achieved in MS medium supplemented with 2.5?mg?L(-1)BAP. The best in vitro rooting was observed in the MS medium with 0.5?mg?L(-1) indole-3-acetic acid plus 2?g?L(-1) activated charcoal. The simple acclimatisation process, primarily with a combination of sand and soil (1?:?1 v/v) and finally with a blend of sand, soil and farm yard manure (2?:?1?:?1 v/v), ensured a 98% survival rate. Overall, 192 true-to-type plantlets were achieved from a single explant within 85 days. Morphologically, in vitro generated plants performed better than conventionally propagated plants; nevertheless the similarity in aloin content, gel content and superoxide dismutase activity was corroborated.     

In vitro production of Clostridium difficile spores for use in the efficacy evaluation of disinfectants: a precollaborative investigation

Clostridium difficile is a strict anaerobic spore-forming bacterium, and an increasingly common nosocomial pathogen. The U.S. Environmental Protection Agency (EPA) is responsible for the registration of disinfectants, including products designed to treat environmental surfaces contaminated with spores of C. difficile. Product efficacy data are required for registration; however, there is a lack of methodology for generating high-quality spore suspensions for evaluating product performance. As such, a study was carried out to select a suitable C. difficile strain and to develop a stand-alone method to prepare a spore suspension that meets specific criteria necessary for quantitative testing of disinfectants. The criteria are: (1) a spore titer of > 8 log10/mL, (2) > or = 90% spores to vegetative cells, and (3) resistance of spores (determined by viability) to 2.5 M hydrochloric acid (HCl). Several strains of C. difficile (toxigenic and nontoxigenic) were grown on various media (solid and liquid) for varying lengths of time to determine the best combination of incubation conditions and media to optimize spore production and quality. Once the spore production procedure was optimized, a toxigenic strain of C. difficile [American Type Culture Collection (ATCC) 43598] was selected for use in trials to verify repeatability from one production run to the next. The spore suspension was initiated by spreading vegetative cells of C. difficile (ATCC 43598) on CDC anaerobic 5% sheep blood agar plates and incubating for 7-10 days at 36 +/- 1 degrees C under anaerobic conditions. Spores were harvested when > or = 90% of the cells converted to spores as determined by observation using phase-contrast microscopy. The spores were washed three times with saline-Tween-80, resuspended in cold deionized water, heated to 70 degrees C for 10 min, evaluated microscopically for quality, and enumerated on cycloserine-cefoxitin-fructose agar containing horse blood and taurocholate. The spore suspension was used to inoculate brushed stainless steel carriers (1 cm in diameter) with and without a soil load in accordance with the Standard Quantitative Carrier Disk Test Method (ASTM E-2197-02) to determine carrier load. Once it was determined that > 6 log10 spores/carrier could be recovered, spores were evaluated for resistance to HCI. The sporulation method presented in this report is simple and repeatable and results in spore suspension of high titer (> 8 log10/mL) and quality (> or = 90% spores to vegetative cells) that met acid resistance criteria (spores were resistant to 2.5 M HCI for 10 min). In addition, recovery from brushed stainless steel carriers with and without soil load was > 6 log10 spores/carrier. A 6 log10 performance standard was set forth in the EPA's interim guidance for generating data to support a label claim for effectiveness against C. difficile spores on hard, nonporous surfaces. This precollaborative investigation successfully demonstrated the use of a methodology for in vitro production of C. difficile spores (ATCC 43598) necessary for conducting efficacy tests. A proposal will be submitted to the AOAC INTERNATIONAL Methods Committee on Antimicrobial Efficacy Testing for a collaborative study; see Appendix.     

Synthesis and evaluation of in vitro bioactivity for polysubstituted N-arylpyrazole derivatives

New polysubstituted N-arylpyrazole derivatives were synthesized from N1-arylsydnone with acetylene and boronic acid, including 2-thiophenyl, 3-thiophenyl, 2-benzo[b]thiophenyl, or dibenzothiophenyl-4-boronic acid, via 1,3-dipolar cycloaddition and Suzuki coupling reaction. Based on the growth inhibitory activity results against lung carcinoma (NCI-H226), nasopharyngeal (NPC-TW01), and T-cell leukemia (Jurkat) cancer cells, compounds 5d and 7d with dibenzothiophenyl bioisostere possessed the significant inhibitory activity for NPC-TW01 (32 μM and 16 μM) and NCI-H226 (16 μM and 8.9 μM), respectively.     

Mn loaded apoferritin as an MRI sensor of melanin formation in melanoma cells

Mn(III)-loaded apoferritin is promptly reduced to Mn(II)-apoferritin by the oxidation of L-DOPA to melanin. The process is nicely witnessed by a marked relaxation enhancement of water proton relaxation rate that has been detected both in cultured melanoma cells and in tumor animal models.     

Gamma radiation interacts with melanin to alter its oxidation-reduction potential and results in electric current production

The presence of melanin pigments in organisms is implicated in radioprotection and in some cases, enhanced growth in the presence of high levels of ionizing radiation. An understanding of this phenomenon will be useful in the design of radioprotective materials. However, the protective mechanism of microbial melanin in ionizing radiation fields has not yet been elucidated. Here we demonstrate through the electrochemical techniques of chronoamperometry, chronopotentiometry and cyclic voltammetry that microbial melanin is continuously oxidized in the presence of gamma radiation. Our findings establish that ionizing radiation interacts with melanin to alter its oxidation-reduction potential. Sustained oxidation resulted in electric current production and was most pronounced in the presence of a reductant, which extended the redox cycling capacity of melanin. This work is the first to establish that gamma radiation alters the oxidation-reduction behavior of melanin, resulting in electric current production. The significance of the work is that it provides the first step in understanding the initial interactions between melanin and ionizing radiation taking place and offers some insight for production of biomimetic radioprotective materials.     

Changes in the energy distribution between chlorophyll-protein complexes of thylakoid membranes from pea mutants with modified pigment content. I. Changes due to the modified pigment content

The low-temperature (77 K) emission and excitation chlorophyll fluorescence spectra in thylakoid membranes isolated from pea mutants were investigated. The mutants have modified pigment content, structural organization, different surface electric properties and functions [Dobrikova et al., Photosynth. Res. 65 (2000) 165]. The emission spectra of thylakoid membranes were decomposed into bands belonging to the main pigment protein complexes. By an integration of the areas under them, the changes in the energy distribution between the two photosystems as well as within each one of them were estimated. It was shown that the excitation energy flow to the light harvesting, core antenna and RC complexes of photosystem II increases with the total amount of pigments in the mutants, relative to the that to photosystem I complexes. A reduction of the fluorescence ratio between aggregated trimers of LHC II and its trimeric and monomeric forms with the increase of the pigment content (chlorophyll a, chlorophyll b, and lutein) was observed. This implies that the closer packing in the complexes with a higher extent of aggregation regulates the energy distribution to the PS II core antenna and reaction centers complexes. Based on the reduced energy flow to PS II, i.e., the relative increased energy flow to PS I, we hypothesize that aggregation of LHC II switches the energy flow toward LHC I. These results suggest an additive regulatory mechanism, which redistributes the excitation energy between the two photosystems and operates at non-excess light intensities but at reduced pigment content.     

ASV determination of cadmium complexation in seawater. Methodology evaluation

The methodology for using DPASV to study cadmium complexation in seawater is evaluated using EDTA as a model ligand and by analysing natural samples. The results show that the methodology gives an accurate evaluation of metal complexation when inert complexes are studied, both as regards the ligand concentration and the conditional stability constant; the error for both the parameters is lower than 10% at a ligand concentration of about 10(-8) M and a conditional stability constant of 10(9) M-1. Cadmium complexes with ligands present in natural seawater show an evident kinetic lability that may lead to underestimation of the conditional stability constant when a working electrode characterised by a very thick diffusion layer is used. The conditional stability constant in one water sample of the Adriatic coast ranged between 0.14 and 1.4 l/nmol using a rotating disk electrode at rotation rates of 300 and 6000 rpm. The results of cadmium complexation obtained for samples collected in coastal seawater show that the ligands present low specificity for the metal.     

Comparison of the accuracy of approximate methods TrESP and TrCAMM for evaluation of pigment coupling in light-harvesting complexes

Due to the large size of light-harvesting complexes, approximate methods are commonly used for calculating the coupling energies between their constituents. Two approximate methods are studied in this work: TrESP and the recently suggested TrCAMM method, based on the expansion of the one-particle density matrix. The quality of approximation of the electrostatic potential in the framework of these two approaches has been compared for two biological pigments as an example—chlorophyll a and bacteriochlorophyll a. It has been shown that both approaches provide high accuracy of approximation of the matrix elements of the electrostatic potential operator. In addition, it has been demonstrated that symmetrization of the one-particle density matrix in the framework of the TrCAMM method significantly improves the calculation accuracy and makes the computational procedure unambiguous.     

Nitric oxide-dependent pigment migration induced by ultraviolet radiation in retinal pigment cells of the crab Neohelice granulata

The purpose of this study was to verify the occurrence of pigment dispersion in retinal pigment cells exposed to UVA and UVB radiation, and to investigate the possible participation of a nitric oxide (NO) pathway. Retinal pigment cells from Neohelice granulata were obtained by cellular dissociation. Cells were analyzed for 30 min in the dark (control) and then exposed to 1.1 and 3.3 J cm⁻² UVA, 0.07 and 0.9 J cm⁻² UVB, 20 n m β-PDH (pigment dispersing hormone) or 10 μ m SIN-1 (NO donor). Histological analyses were performed to verify the UV effect in vivo . Cultured cells were exposed to 250 μ m L-NAME (NO synthase blocker) and afterwards were treated with UVA, UVB or β-PDH. The retinal cells in culture displayed significant pigment dispersion in response to UVA, UVB and β-PDH. The same responses to UVA and UVB were observed in vivo . SIN-1 did not induce pigment dispersion in the cell cultures. l-NAME significantly decreased the pigment dispersion induced by UVA and UVB but not by β-PDH. All retinal cells showed an immunopositive reaction against neuronal nitric oxide synthases. Therefore, UVA and UVB radiation are capable of inducing pigment dispersion in retinal pigment cells of Neohelice granulata and this dispersion may be nitric oxide synthase dependent.     

Viscometric determination of dialdehyde content in periodate oxycellulose. Part I. Methodology

Periodate oxycellulose was subjected to alkaline hydrolysis at room temperature in both homogeneous (cupriethylenediamine) and heterogeneous (sodium hydroxide) medium, and the degradation kinetics was followed for nearly three months. By comparing the obtained results with the degradation kinetics of both hydrocellulose and periodate oxycellulose reduced with tert-butylamine borane, it was demonstrated that the -alkoxy fragmentation of oxidised sites is a very fast reaction, which reaches completeness during the preparation of samples for viscometric analyses. The subsequent degradation is due to other mechanisms, such as autoxidation and peeling. A comparison between the degrees of polymerisation of periodate oxycellulose before and after its reduction allows the quantitative determination of dialdehyde groups, without the interference of reducing end groups. Although this technique might not be valid for other kinds of oxycellulose, it supplies a simple and fast method for the analysis of mildly oxidised cellulose.     

Modifications of in vitro skin penetration under solar irradiation: evaluation on flow-through diffusion cells

The effect of solar irradiation on ex vivo dermatomed hairless rat skin samples maintained in culture on flow-through diffusion cells for at least 24 h was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and by histological observations. Transepidermal water loss (TEWL) measurements and kinetic analysis of the permeation of both tritiated water and 14C caffeine through the skin were performed after full-spectrum solar exposure involving the use of a xenon arc solar simulator. After a UV exposure of less than 420 mJ/cm2, skin integrity and permeation of both water and caffeine did not change significantly. In contrast, after a 420 mJ/cm2 UV exposure, the epidermis appeared more contracted, associated with an increase of 55% of TEWL and 220% of the skin permeation of tritiated water after 6 h. The data suggested a dramatic alteration of the skin barrier integrity. Moreover, the flux of 14C caffeine increased rapidly by 338% of the absorption of water 12 h after irradiation. These results reveal the presence of a threshold UV exposure that would not modify skin penetration.