Eosin interaction of alpha-synuclein leading to protein self-oligomerization.
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Among various dyes including congo red, thioflavin S, thioflavin T, eosin, rhodamine 6G, and phenol red, the eosin was the only dye that induced self-oligomerization of alpha-synuclein in the presence of a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline. To analyze chemical nature of the eosin interaction with alpha-synuclein, the phenomenon of self-oligomerization was further examined with eosin congeners such as ethyl eosin, eosin B, phloxine B, erythrosin B, and rose bengal. The followings are the conclusions we have reached. First of all, intactness of the benzoate moiety of eosin and the negative charge on the carboxylic group of the dye are important factors leading to the specific interaction with alpha-synuclein. Secondly, the localized negative charge on the xanthene moiety of eosin is another critical factor for the interaction. As far as substituting halides are concerned, bromides and iodides on the xanthene moiety of the dyes do not make any difference on the alpha-synuclein interaction because both eosin and erythrosin B have induced the common phenomenon of self-oligomerization. The binding curve between eosin and alpha-synuclein was sigmoidal as the dye concentrations were increased. A double reciprocal plot of the saturation curve showed that the maximum number of eosin binding sites on alpha-synuclein was 1.85 with a dissociation constant of 390 microM. The dye binding to the protein appeared to occur via a positive cooperativity. The eosin binding site(s) was suggested to be located predominantly on the NAC region and partly related to the acidic C-terminus of alpha-synuclein. It has been, therefore, expected that this information might be useful to develop alpha-synuclein interactive molecules, which could provide eventual preventive or possible therapeutic means against various alpha-synuclein related disorders including Parkinson's disease. (+info)
Photochemical keratodesmos for repair of lamellar corneal incisions.
(10/243)
PURPOSE: To determine the efficacy of photochemical keratodesmos (PKD) for closing surgical incisions in the cornea of enucleated rabbit eyes compared with that achieved using sutures and self-sealing incisions. METHODS: A 3.5-mm incision, at an angle parallel to the iris, was made in the cornea of enucleated New Zealand White rabbit eyes. The intraocular pressure required to cause leakage (IOP(L)) from the untreated incision was then recorded. Photochemical keratodesmos treatment was then performed by application of a dye, Rose Bengal (RB), in saline solution to the surfaces of the incision wound, followed by laser irradiation at 514 nm from an argon ion laser. Immediately after treatment, the IOP(L) was measured. Both dose and laser irradiance dependencies were studied in five or more eyes for each condition and appropriate control eyes. The IOP(L)s were compared with those obtained using conventional interrupted 10-0 nylon sutures. Other dyes were tested in a similar fashion. RESULTS: The IOP(L) of 300 mm Hg was obtained using a fluence of 1270 J/cm(2) with an irradiance of 1.27 W/cm(2) (laser exposure time, 16 minutes 40 seconds). No sealing was observed using dye or light alone where control pressures of approximately 30 mm Hg were found. At higher dose (1524 J/cm(2)) and irradiance (3.82 W/cm(2); 6 minutes 35 seconds), PKD was less effective, which may be attributable to thermal effects. PKD produced IOP(L)s similar to those in closure by sutures. Other dyes such as riboflavin-5-phosphate and N:-hydroxy-pyridine thione also produced efficient bonding after PKD. Nonphotochemically active dyes did not produce significant increases in the IOP(L) at which leakage occurred. CONCLUSIONS: The increase in IOP(L) after PKD treatment, comparable with that with sutures, in enucleated rabbit eyes demonstrates the feasibility of this technique ex vivo. (+info)
Use of prawn blood agar hemolysis to screen for bacteria pathogenic to cultured tiger prawns Penaeus monodon.
(11/243)
A newly developed prawn blood agar consisting of 1 ml of tiger prawn hemolymph in medium containing 200 ppm Rose Bengal was used to determine the hemolytic activity of 35 isolates of bacteria obtained from cultured tiger prawns Penaeus monodon and their rearing water. For comparison, the hemolytic activity of these isolates was also determined in sheep blood agar. Nine isolates (25.7% of total) showed different hemolytic reactions on prawn blood agar and sheep blood agar. From the 35 isolates, 8 with various hemolytic characteristics were selected and the relationship between the type of hemolytic activity and pathogenicity was determined and compared. Four isolates that showed hemolytic activity in prawn blood agar caused high mortality to cultured tiger prawns. By contrast, a significantly lower mortality rate was observed for tiger prawns injected with 4 isolates that did not exhibit hemolytic activity on prawn blood agar. Results further showed that mortality did not correlate with hemolytic activity determined using sheep blood agar. Prawn blood agar containing P. monodon hemocytes was faster and more accurate for determining prawn hemolytic activity of bacterial isolates. (+info)
Laser-induced noninvasive vascular injury models in mice generate platelet- and coagulation-dependent thrombi.
(12/243)
A minimally invasive laser-induced injury model is described to study thrombus development in mice in vivo. The protocol involves focusing the beam of an argon-ion laser through a compound microscope on the vasculature of a mouse ear that is sufficiently thin such that blood flow can be visualized by intravital microscopy. Two distinct injury models have been established. The first involves direct laser illumination with a short, high-intensity pulse. In this case, thrombus formation is inhibited by the GPIIb/IIIa antagonist, G4120. However, the anticoagulants, hirulog, PPACK, and NapC2 have minimal effect. This indicates that thrombus development induced by this model mainly involves platelet interactions. The second model involves low-intensity laser illumination of mice injected with Rose Bengal dye to induce photochemical injury in the region of laser illumination. Thrombi generated by this latter procedure have a slower development and are inhibited by both anticoagulant and anti-platelet compounds. (+info)
Even after UVA-exposure will nitric oxide protect cells from reactive oxygen intermediate-mediated apoptosis and necrosis.
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Reactive oxygen species (ROS) play a pivotal role in UVA-induced cell damage. As expression of the inducible nitric oxide synthase (iNOS) is a normal response of human skin to UV radiation we examined the role of nitric oxide (NO) as a protective agent during or even after UVA1- or ROS-exposure against apoptosis or necrosis of rat endothelial cells. When added during or up to 2 h subsequent to UVA1 or ROS exposure the NO-donor S-nitroso-cysteine (SNOC) at concentrations from 100-1000 microM significantly protects from both apoptosis as well as necrosis. The NO-mediated protection strongly correlates with complete inhibition of lipid peroxidation (sixfold increase of malonedialdehyde formation in untreated versus 1.2-fold with 1 mM SNOC). NO-mediated protection of membrane function was also shown by the inhibition of cytochrome c leakage in UVA1 treated cells, a process not accompanied by alterations in Bax and Bcl-2 protein levels. Thus, the experiments presented demonstrate that NO exposure during or even after a ROS-mediated toxic insult fully protects from apoptosis or necrosis by maintaining membrane integrity and function. (+info)
Iodine-123-Rose bengal: An improved hepatobiliary imaging agent.
(14/243)
A practical method for preparing )23-I-rose bengal that allows for its rapid and efficient incorporation into the molecule is reported. Administration of 123-I-rose bengal to normal healthy patients showing the normal uptake and excretory pattern visualized with this radio pharmaceutical is also presented. The overall reduction in imaging time and radiation exposure together with the improved images possible should greatly improve our diagnostic capabilities in evaluating the jaundiced patient. (+info)
Interaction of rabbit hemopexin with rose bengal and photooxidation of the rose bengal-hemopexin complex.
(15/243)
Rabbit hemopexin associates with rose bengal producing a hypochromic shift in the absorption spectrum of the dye; the extinction coefficient of the dye bound to heme-saturated hemopexin is approximately 20% lower than that of the dye bound to the apoprotein. The interaction of apo- and heme-saturated hemopexin with rose bengal was studied in detail by difference spectroscopy. Apo-hemopexin has one tight binding site for the dye with a dissociation constant in the micromolar range and a set of several weaker binding sites. In contrast, heme-saturated hemopexin has a very low affinity for the dye. Evidence that histidine residues of hemopexin participate in the binding of heme was obtained by photooxidation of hemopexin sensitized by rose bengal. Progressive modification of the 16 histidine residues of hemopexin is effected by illumination of the dye-hemopexin complexes. The midpoint of this pH-dependent reaction is at pH 6.8 +/- 0.1. In 15 min of irradiation, apo-hemopexin loses 50% of its ability to form a low spin hemichrome complex with deuteroheme while only 10% of the ligand coordination to heme iron of the deuteroheme-hemopexin is lost. At that time, approximately 2 more histidine residues are modified in apo-hemopexin than in deuteroheme-hemopexin, and no change is found in other potentially photolabile amino acid residues. The characteristic circular dichroism positive extremum at 231 nm of hemopexin also was decreased by photooxidation, and the loss was slower in the deuteroheme-hemopexin complex than in the apoprotein. When deuteroporphyrin IX was used as the photosensitizing agent, similar results were obtained. (+info)
Evidence for essential histidine residues in bovine-liver mitochondrial monoamine oxidase.
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Ethoxyformic acid anhydride, amino-1H-tetrazole, and photooxidation in the presence of rose bengal, which are reagents known to react with histidine residues of proteins, were shown to inactivate monoamine oxidase. Ethoxyformic acid anhydride reacted with about 6 histidine residues per 100 000 g of protein under the experimental conditions adopted and completely inactivated the enzyme. However, NH2OH reactivated the ethaxyformic acid derivative t only. Since NH2OH specifically deacylates N-ethoxyformylimidazole, it was shown that at least some of the histidine residues are essential for activity. In addition, photooxidation experiments in the presence of 0.01% rose bengal confirmed that only histidine residues of bovine hepatic monoamine oxidase are destroyed under the designated experimental conditions. About 9 histidine residues per 100 000 g of protein were destroyed during the photooxidation experiments. In the presence of substrate, kynuramine or benzylamine, only 7 histidine residues were destroyed, which indicates that 2 histidine residues per 100 000 g of protein are essential for activity. (+info)