2-Aryl-1,3-thiazolidines as masked sulfhydryl agents for inhibition of melanogenesis

As a part of an ongoing project aimed at developing new skin depigmenting agents, the ability of variously substituted 2-aryl-1,3-thiazolidines to inhibit melanogenesis in vitro was investigated. At 0.2 mM concentration 2-(2'-hydroxyphenyl)-1,3-thiazolidine-4-carboxylic acid (Th2), as well as the descarboxy analog (Th1) and, to a lower extent, the 4'-hydroxy isomer (Th3) all proved capable of preventing the tyrosinase catalyzed conversion of 0.2 mM L-tyrosine to melanin. Spectrophotometric monitoring of the reaction course in the presence of Th2 showed the initial formation of a yellow chromophore (lambda max 400 nm) which slowly decayed, being eventually replaced by a new absorption maximum centered at 305 nm. HPLC analysis of the final incubation mixture revealed the presence of a major product (lambda max 306 nm), ninhydrin and ferric chloride positive, which was isolated by gel filtration on Sephadex G-10 and was identified as beta-[7-(3-carboxy-5-hydroxy-3,4-dihydro-2H-1,4-benzothiazinyl)]al anine (DBA) by 1H-NMR spectroscopy. Attempts to isolate the intermediate with lambda max 400 nm were hampered by its marked instability under the usual chromatographic conditions. However, the nature of the chromophore, coupled with mechanistic considerations, suggested for the compound the Schiff base-containing structure 3,4-dihydroxy-5-S-(N-salicylidenecysteinyl)phenylalanine (salcysdopa). This was substantiated by: (i) the formation of a zinc complex (lambda max 349 nm) analogous to that observed with the model Schiff base N-salicylidene leucine; and (ii) detection by 1H-NMR of a Schiff base resonance at delta 8.1 during the yellow chromophoric phase of the reaction. It was concluded that 1,3-thiazolidines inhibit melanin formation by a mechanism that involves the trapping of enzymically generated dopaquinone by the -SH containing Schiff base arising by cleavage of the thiazolidine ring. The salcysdopa adduct thus formed undergoes hydrolysis and subsequent ring closure to give eventually the colorless DBA.     

Psoralens sensitize glutathione photooxidation in vitro

In vitro experiments are reported showing that psoralens and other furocoumarins of current pharmacological interest, e.g., angelicin and 4,6,4'-trimethylangelicin, all have, to a variable extent, the ability to sensitize the photooxidation of glutathione in ethanol/phosphate buffer with pyrex-filtered ultraviolet light. Besides substrate concentration and the nature of the furocoumarin used, the rate of the sensitized reaction is markedly dependent on the partial pressure of oxygen and the pH of the medium, being progressively faster on passing from pH 5 to pH 8.5. Scavengers of superoxide ions (superoxide dismutase), hydrogen peroxide (catalase) and singlet oxygen (sodium azide, diazabicyclooctane, sorbic acid) have little or no inhibitory effect on the reaction rate. These and other data suggest that furocoumarins can directly sensitize glutathione photooxidation by forming a charge transfer complex which is driven to the oxidized products in the presence of oxygen. The possible relevance of these results to the mechanisms of skin melanin hyperpigmentation induced by furocoumarins and ultraviolet light is discussed.     

A correlation between BCL-2 modifying factor,p53 and livin gene expressions in cancer colon patients

Accumulating evidence has revealed that livin gene and BCL-2 modifying factor (BMF) gene are closely associated with the initiation and progression of colon carcinoma by activating or suppressing multiple malignant processes. Those genes that can detect colon - cancer are a promising approach for cancer screening and diagnosis. This study aimed to evaluate correlation between livin, BMF and p53 genes expression in colon cancer tissues of patients included in the study, and their relationship with clinicopathological features and survival outcome in those patients. In this study, 50 pathologically diagnosed early cancer colon patients included and their tissue biopsy with 50 matched adjacent normal tissue, and 50 adenoma tissue specimens were analyzed for livin gene and BMF gene expressions using real time PCR. The relationship of those genes expressions with clinicopathological features, tumor markers, Time to Progression and overall survival for those patients were correlated in cancer colon group. In this study, there was a significant a reciprocal relationship between over expression of livin gene and down regulation of BMF and p53 genes in colon cancer cells. Livin mRNA was significantly higher, while BMF and p53 mRNA were significantly lower in colorectal cancer tissue compared to benign and normal colon tissue specimens (P < 0.001), however, this finding was absent between colon adenomas and normal mucosa. There was a significant association between up regulation of livin and down regulation of BMF and p53 expressions with more aggressive tumor (advanced TNM stage), rapid progression with metastasis and decreased overall survival in cancer colon patients, hence these genes can serve as significant prognostic markers of poor outcome in colon cancer patients. This work highlights the role of livin, BMF and p53 genes in colorectal tumorigenesis and the applicability of using those genes as a diagnostic and prognostic markers in patients with colon carcinoma and as a good target for cancer colon treatment in the future.     

A Bark Necrosis of Grapevine Caused by Pseudomonas syringae V. Hall, in Sardinia

An unusual disorder occurred on ‘Vernaccia’ vine, particularly in the typical cultivation area of the variety, in the province of Oristano. Main symptom is the decaying of wood and bark. This causes the loss of the bark on the circumference of the trunk and causes the plants to die. Degradation can develop very deep in the wood rings. Affected rings can detach from the new formed ones. The infectious process is very serious in the younger plants and can slow their evolution in the years. Furthermore it does not allow the recovery of the affected plants. In some vineyards 4 % of affected plants occur. Mortality can reach up to 20%, of the affected plants and is very high during the early years after infection. A bacterium has been constantly isolated from decaying tissues; for its morphological and physiological characters and the response to serological tests it has been included in the Pseudomonas syringae Van Hall group. Pathogenicity tests carried out on ‘Vernaccia’ vines and other varieties resulted always positive; the bacterium has always been reisolated from artificially affected areas. The latter were much diffused and, sometimes, reproduced the typical symptomatology of the disease observed in nature.     

Pictet-Spengler condensation of the antiparkinsonian drug L-DOPA with D-glyceraldehyde. Opposite kinetic effects of Fe3+ and Cu2+ ions and possible implications for the origin of therapeutic side effects

In 0.05 M phosphate buffer, pH 7.4, and at 37 degrees C. L-DOPA, a widely used antiparkinsonian drug, reacted smoothly with D-glyceraldehyde to afford diastereoisomeric (1R, 1'S,3S)-3-carboxy-1-(1',2'-dihydroxyethyl)-6,7-dihydroxy-1,2,3,4- tetrahydroisoquinoline (1) and (1S,1'5S,3S)-3-carboxy-1-(1',2'-dihydroxyethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (2) in an approx. 3:2 ratio. The prevalent formation of 1 over 2 reflects stereoselective cyclisation of a transient Schiff base in accord with the Felkin-Anh model. Fe3+ ions, present at relatively high levels in parkinsonian brains, markedly accelerated formation of 1 and 2, whereas Cu2+ decreased the reaction rate, due apparently to different sites of chelate formation between L-DOPA and the metal ions. Both metal ions markedly decreased the stereoselectivity of the reaction. Product 1 exhibited chelating properties toward metal ions comparable or stronger than those of L-DOPA. These results throw new light on the effects of transition metal ions on the Pictet-Spengler reaction and suggest a possible role of tetrahydroisoquinoline products from L-DOPA and carbohydrate metabolites in the severe side effects of the drug.     

New regulatory mechanisms in the biosynthesis of pheomelanins: rearrangement vs. redox exchange reaction routes of a transient 2H-1,4-benzothiazine-o-quinonimine intermediate

Pheomelanins, the typical epidermal pigments of red haired, Celtic-type Caucasians, arise from oxidative cyclization of cysteinyldopas, mainly the 5-S-isomer CD, via 1,4-benzothiazines. However, the mechanism and the relative yields of formation of these intermediates have remained poorly defined. We have now examined the course of the oxidation of CD at physiological pHs, under different reaction conditions. Surprisingly, a consumption of CD far exceeding the stoichiometry of the oxidant was observed at low oxidant-to-substrate ratios, low temperatures and high substrate concentrations. The yields of the 3,4-dihydro-1,4-benzothiazine-3-carboxylic acid DHBCA vs. the non-carboxylated analogue DHB in the oxidation mixture, after NaBH₄ reduction, were also found to depend markedly on the reaction conditions. Based on these and other results, a reaction scheme is proposed involving a transient o-quinonimine generated by oxidative cyclization of CD to which three different paths are offered, namely redox exchange with CD to give DHBCA (path A) or intramolecular rearrangement with (path B) or without (path C) decarboxylation, leading to the benzothiazine BTZ and the 3-carboxy analogue BTZCA, respectively. The relative operation of path A vs. path C was assessed by deuterium labeling experiments. These findings point to new mechanisms of regulation of the initial steps of pheomelanogenesis, bearing significant implications on the structure of the final pigment.     

Transient quinonimines and 1,4-benzothiazines of pheomelanogenesis: new pulse radiolytic and spectrophotometric evidence.

Biosynthetic and model in vitro studies have shown that pheomelanins, the distinctive pigments of red human hair, arise by oxidative cyclization of cysteinyldopas mainly 5-S-cysteinyldopa (1) via a critical o-quinonimine intermediate, which rearranges to unstable 1,4-benzothiazines. To get new evidence for these labile species, fast time resolution pulse radiolytic oxidation by dibromide radical anion of a suitable precursor, the dihydro-1,4-benzothiazine-3-carboxylic acid 7 was performed in comparison with that of 1. In the case of 7, dibromide radical anion oxidation leads over a few microseconds (k = 2.1 x 10(9) M(-1) s(-1)) to a phenoxyl radical (lambda(max) 330 nm, epsilon = 6300 M(-1) cm(-1)) which within tens of milliseconds gives rise with second-order kinetics (2k = 2.7 x 10(7) M(-1) s(-1)) to a species exhibiting an absorption maximum at 540 nm (epsilon = 2200 M(-1) cm(-1)). This was formulated as the o-quinonimine 3 arising from disproportionation of the initial radical. The quinonimine chromophore is converted over hundreds of milliseconds (k = 6.0 s(-1)) to a broad maximum at around 330 nm interpreted as due to a 1,4-benzothiazine or a mixture of 1,4-benzothiazines, which as expected are unstable and subsequently decay over a few seconds (k = 0.5 s(-1)). Interestingly, the quinonimine is observed as a labile intermediate also in the alternative reaction route examined, involving cyclization of the o-quinone (lambda(max) 390 nm, epsilon = 6900 M(-1) cm(-1)) arising by disproportionation (2k = 1.7 x 10(8) M(-1) s(-1)) of an o-semiquinone (lambda(max) 320 nm, epsilon = 4700 M(-1) cm(-1)) directly generated by dibromide radical anion oxidation of 1. Structural formulation of the 540 nm species as an o-quinonimine was further supported by rapid scanning diode array spectrophotometric monitoring of the ferricyanide oxidation of a series of model dihydrobenzothiazines.