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Chemical modification, characterization and structure-anticoagulant activity relationships of Chinese lacquer polysaccharides.

A natural lacquer polysaccharide with complex branches was separated into two fractions, LPH (MW 16.9x10(4)) and LPL (MW 6.85x10(4)). Results of 13C NMR and FT-IR indicated they had the same structure. The treatment of LPL with sodium periodate led to a partial cut-off of side chains with 4-O-methyl-D-glucuronic acid in the terminal. These polysaccharides were sulfated in the presence of Py*SO3/DMSO. Depending on the reaction conditions, the products showed a different degree of sulfation (DS) ranging from 0.57 to 1.57 and different molecular weights ranging from 1.71x10(4) to 3.49x10(4). FT-IR analysis showed the equatorial primary OH at O-6 and the axial secondary OH at O-4 were sulfated. Activated partial thromboplastin time (APTT), prothrombin time and thrombin time (TT) assays showed the sulfated polysaccharides could prolong APTT and TT, but not TP. These activities strongly depended on the DS, the molecular weights (MW) and the branching structure of polysaccharides. DS of above 0.8 was essential for anticoagulant activity. The anticoagulant activity increased with the DS and the molecular weights. The molecular weights played a more important role. The branching structure of polysaccharides increased the activities. In our studies, the sulfated polysaccharides with the DS of 1.15 and the highest MW of 3.49x10(4) had the best blood anticoagulant activities.

Anticoagulants↗

Assay for Laccase activity by microcalorimetry: laccase was extracted from china lacquer of Rhus vernicifera.

The reactions between Laccase (extracted from China lacquer of Rhus vernicifera) and various substrates (3,4-Dihydroxybenzaldehyde, Guaiacol, Pyrogallol, Gallic acid) have been studied using LKB-2107 batch microcalorimetry system. Based on calorimetry, a new method has been proposed. Laccase activity and the Michaelis constant K(m) have been determined simultaneously by this method. The method is simple, sample-saving, and valid for a wider range of substrate concentrations. Furthermore, it can be extended for assaying other enzymes catalyzing reactions using this method.

Calorimetry↗

Toxicodendron radicans dermatitis with black lacquer deposit on the skin.

Four patients with clinical Toxicodendron dermatitis (poison ivy) presented with dramatic black lacquer-like deposits on several lesions. This black deposit was also observed at sites of injury on poison ivy plants and was reproduced on volunteers by the application of plant sap on the skin. Histologically, the observed material was identified in the stratum corneum. This little-recognized phenomenon has been mentioned in past dermatologic literature.

Adolescent↗

Optical properties of japanese-lacquer-tree (Rhus vernicifera) laccase depleted of type 2 copper(II). Involvement of type-2 copper(II) in the 330nm chromophore.

1. Spectroscopic and functional properties of Japanese-lacquer-tree (Rhus vernicifera) laccase were re-investigated, with special emphasis on the relationships between the different types of copper centres (Types 1, 2, and 3). 2. On removal of the Type 2 Cu(II), a decrease of absorbance occurred in the wavelength region above 650 nm (delta epsilon 750 = 300 M-1 . cm-1) and around 330 nm (delta episom 330 up to 2200 M-1 . cm-1). 3. Reductive titrations with ascorbic acid or ferrocyanide showed that the electron-accepting capacity of the partial apoprotein is one electron-equivalent lower than that of the native protein, i.e. the protein two-electron acceptor is present in the oxidized state in spite of absorbance loss at 330 nm. 4. The 330 nm chromophore apparently depends on the presence of both the Type 2 and the Type 3 copper in the oxidized state. 5. This finding may have implications in the relative location of Type 2 and 3 copper centres and on the redox behaviour of laccase.

Circular Dichroism↗

Titrations with ferrocyanide of japanese-lacquer-tree (Rhus vernicifera) laccase and of the type 2 copper-depleted enzyme. Interrelation of the copper sites.

1. Redox titrations are reported of the metal centres in Japanese-lacquer-tree (Rhus vernicifera) laccase with ferrocyanide. 2. The redox potential of Type 1 Cu was found to increase with ferrocyanide concentration up to a limiting value similar to that for the Type 1 Cu in Type 2 Cu-depleted enzyme (which is independent of ferrocyanide concentration). 3. The redox potential of the two-electron acceptor (Type 3 Cu) is also independent of ferrocyanide concentration in Type 2 Cu-depleted enzyme and lower than values reported for the native enzyme. 4. The two-electron acceptor is present in the oxidized state in the Type 2 Cu-depleted enzyme, though the latter lacks the 330 nm absorption band. 5. The redox potential of Type 2 Cu also depends on ferrocyanide concentration, at least in the presence of azide. 6. The redox potentials are affected by freezing the solutions and/or addition of azide, the latter binding to Type 2 Cu with affinity dependent on the redox state of the two-electron acceptor.

Binding Sites↗

Dependence on freezing of the geometry and redox potential of type 1 and type 2 copper sites of Japanese-lacquer-tree (Rhus vernicifera) laccase.

The room-temperature e.p.r. spectrum of the Japanese-lacquer-tree (Rhus vernicifera) laccase shows A parallel (the hyperfine splitting constant) and g parallel values of both the Type 1 and Type 2 Cu appreciably different from those measured at liquid-N2 temperature. The geometry of the sites, as inferred from the room-temperature e.p.r. parameters, is more consistent with their redox properties. A rough correlation is found between A parallel and g parallel values and redox potential of the blue copper in several enzymes.

Chemical Phenomena↗

Heterogeneity of the Type 3 copper in Japanese-lacquer-tree (Rhus vernicifera) laccase.

The two steps of the titration of the Japanese-lacquer-tree (Rhus vernicifera) laccase with N3- [Morpurgo, Rotilio, Finazzi-Agrò & Mondovi (1974) Biochim. Biophys. Acta 336, 324-328; LuBien, Winkler, Thamann, Scott, Co, Hodgson & Solomon (1981) J. Am. Chem. Soc. 103, 7014-7016] were shown to be two distinct reactions, each involving one different portion of the native enzyme molecules. The difference consists in the oxidation state of the Type 3 Cu, which is reduced in the portion with higher affinity for N3- and oxidized in the portion with lower affinity for N3-. The difference is eliminated by treatment with oxidizing (H2O2) or reducing agents, and a single N3- adduct is then formed. The e.p.r. spectra of the H2O2-treated enzyme and of its F- derivatives support this interpretation of the results. The similarity of the spectroscopic properties of the high-affinity N3- adduct to those of the N3- adducts of half-met-haemocyanins and half-met-tyrosinase is discussed.

Azides↗

Yeast copper-thionein can reconstitute the Japanese-lacquer-tree (Rhus vernicifera) laccase from the Type 2-copper-depleted enzyme via a direct copper(I)-transfer mechanism.

The Type 2-Cu-depleted laccase from the Japanese lacquer tree (Rhus vernicifera) can be reconstituted with CuSO4 aerobically and much more rapidly and efficiently under anaerobic reducing conditions. This is to be related to a more favourable conformation of a laccase in the reduced state, rather than to reduction of the metal ion. In fact, reconstitution with Cu(I)-thionein from baker's yeast (Saccharomyces cerevisiae) only proceeds under anaerobic reducing conditions, via a direct transfer of Cu(I).

Binding Sites↗

Low-temperature resonance-Raman spectra of Japanese-lacquer-tree (Rhus vernicifera) laccase, type-2-copper-depleted laccase and H2O2-treated type-2-copper-depleted laccase.

Resonance-Raman spectra of Japanese-lacquer-tree (Rhus vernicifera) laccase, type-2-copper-depleted laccase and the latter form treated with H2O2 were measured in liquid and frozen solution, on excitation into the 600 nm absorption band. Significant changes in intensity and/or frequency of the bands lying in the 370-430 cm-1 region were observed on freezing, indicating local structural rearrangements taking place at the blue copper site. These findings corroborate previous suggestions based on e.p.r. measurements and redox data [Morpurgo, Calabrese, Desideri & Rotilio (1981) Biochem. J. 193, 639-642]. They show the strong dependence of the physical properties of blue copper centres on local symmetry. Some conclusions on the origin of the Raman bands are also drawn.

Cold Temperature↗

Stability of Japanese-lacquer-tree (Rhus vernicifera) laccase to thermal and chemical denaturation: comparison with ascorbate oxidase.

The thermal denaturation of laccase from the Japanese lacquer tree (Rhus vernicifera) was studied by differential scanning calorimetry. The endotherms of holo-laccase, type 2-Cu-depleted laccase and apo-laccase were deconvoluted into two independent two-state transitions, providing evidence for a domain structure of the protein. The correlation of the two transitions with the bleaching of copper optical bands and the decrease of the transitions' enthalpy on Cu removal show that the process involves the denaturation of Cu sites. No detectable unfolding of secondary structure was observed, since the thermal transitions, characterized by low overall specific enthalpy, did not modify either the laccase c.d. spectra in the beta-fold region or the maximum wavelength of the fluorescence emission. On chemical denaturation, however, the emission was red-shifted by about 20 nm. The laccase behaviour is substantially different from that of stellacyanin, a protein containing a single blue Cu ion, in which the thermal transition had higher specific enthalpy and induced a large change of the c.d. spectrum in the beta-fold region. The laccase denaturation behaviour is similar to that of ascorbate oxidase from zucchini (courgette; Cucurbita pepo) [Savini, D'Alessio, Giartosio, Morpurgo and Avigliano (1990) Eur. J. Biochem. 190, 491-495], suggesting a structural analogy. In both proteins heating may cause a change of tertiary structure through modifications of Cu co-ordination with loosening of the bonds between the structural domains at the interface of which the trinuclear Cu cluster is located.

Ascorbate Oxidase↗

A randomized trial of amorolfine 5% solution nail lacquer in association with itraconazole pulse therapy compared with itraconazole alone in the treatment of Candida fingernail onychomycosis.

BACKGROUND: Treatment failures and relapses are not uncommon in onychomycosis. Therefore, it is worthwhile to consider the combination of systemic and topical antifungals to improve the cure rates further and to reduce the duration of systemic treatment. OBJECTIVES: To evaluate and compare itraconazole pulse therapy combined with amorolfine with itraconazole alone in the treatment of Candida fingernail onychomycosis. METHODS: Ninety patients with moderate to severe Candida fingernail onychomycosis were randomized into two treatment groups of 45 subjects each. Group 1 received itraconazole pulse therapy for 2 months and applied amorolfine 5% solution nail lacquer for 6 months, while group 2 received monotherapy with three pulses of itraconazole. The primary efficacy criterion was the result of mycological examination at 3 months. The secondary criterion was the combined mycological and clinical response at 9 months. A pharmacoeconomic analysis was also performed to compare the cost-effectiveness of combined therapy vs. monotherapy. RESULTS: Eighty-five patients were analysed (73 women and 12 men, mean +/- SD age 44.2 +/- 12.9 years). Patients had a mean +/- SD of 3.64 +/- 2.0 nails involved and 228.6 +/- 148.0 mm2 of their nail surface diseased. The mean duration of onychomycosis was 11 months. Paronychial involvement was evident in 71 patients. C. albicans was isolated in 85 cases, C. parapsilosis in three and other Candida species in two cases. Side-effects were uncommon and in only one case led to withdrawal. At the 3-month visit, mycological cure was seen in 32 (74%) of 43 patients in group 1 and in 25 (60%) of 42 patients in group 2. At the 9-month visit, a global cure was seen in 40 patients (93%) in group 1 and in 34 patients (81%) in group 2. Statistical analysis showed no statistically significant difference (P > 0.1) between the two treatment groups. The cost per cure ratio was 1.63 and 1.70euro for groups 1 and 2, respectively. CONCLUSIONS: The combination of amorolfine and oral itraconazole, which interfere with different steps of ergosterol synthesis, exhibited substantial synergy. Compared with oral itraconazole alone, the combination achieved greater mycological cure and increased total cure rate. However, no statistically significant difference was documented for this number of observations. Combination treatment with amorolfine and two pulses of itraconazole is at least as safe and effective as three pulses of itraconazole, with a lower cost per patient. In our opinion, the addition of amorolfine to oral itraconazole pulse therapy is of value in the treatment of moderate to severe Candida fingernail onychomycosis.

Administration, Cutaneous↗

Successful treatment of onychomycosis with ciclopirox nail lacquer: a case report.

Onychomycosis commonly causes patients to seek medical care. Its unpleasant cosmetic appearance and negative impact on a patient's quality of life often prompt these visits. Onychomycosis rarely resolves spontaneously and has a high rate of recurrence. Although a variety of treatment options are available, inconsistent results and poor patient compliance have often hindered their success. We report the case of a 75-year-old man with moderate-to-severe onychomycosis who was treated successfully with ciclopirox nail lacquer solution 8%, the first topical antifungal agent approved by the Food and Drug Administration for the treatment of this disease.

Administration, Topical↗

[Periodontal disease in the workers of paints and lacquer industry (author's transl)].

Examinations were performed by the author upon the influence of organic solvents and polycyclic aromatic hydrocarbons on parodontium in the workers of paints and lacquer industry. 195 persons employed in industry were examined. 100 persons whose conditions of work had no influence on their parodontium served as controls.--It was found that both, the frequency of incidents and the degree of intesity of changes in periodontium were significantly higher than those in the control group not exposed to the noxius action of work conditions.--This was analysed depending on sex, age, total time of employment and development of calculus dentis and plaques. The frequency, the intensivity of PI and the kind of parodontopathy was examined depending on various toxic condition at different working places. All statistical examinations were carried out by the use of the computer EMC ODRA 1305.

Adolescent↗

Gas-liquid chromatographic determination of solvents in commercial nail lacquer preparations.

A gas-liquid chromatographic (GLC) method has been developed for the determination of 5 solvents (butanol, butyl acetate, ethyl acetate, 2-propanol, and toluene) and camphor in commerical nail lacquer preparations. Resins and pigments are first precipitated from the solvents by dilution with isooctane. n-Propyl acetate is then added as an internal standard to the isooctane solution of solvents and the mixture is analyzed by temperature-programmed GLC. Analyses at 3 concentration levels (4, 20, and 40%) with 4 determinations at each level gave good recoveries.

Chromatography, Gas↗