Quantification of leuco-indigo in indigo-dye-fermenting suspension by normal pulse voltammetry.

Quantification of leuco-indigo is most important for Aizome, Japanese indigo-dyeing; however, there has been no convenient quantitative method. This study demonstrated that normal pulse voltammetry under quiescent conditions can be used to detect leuco-indigo. As a result of quantification of leuco-indigo in the depth direction in fermenting suspensions, the steady-state concentrations of leuco-indigo showed sigmoidal profiles in the depth direction. The steady state is caused by competitive reactions of microbial reduction of indigo and autoxidation of leuco-indigo by O2 dissolved from the air interface of the suspension. In addition, we investigated the effects of stirring the suspension and adding some nutrients to the concentration profile. The weakened activity was partially recovered by the addition of ethanol and remarkably recovered by the addition of hipolypepton or glucose. Knowledge is essential for the proper management of indigo-dye-fermenting suspensions.

Isolation and characterization of indigo-reducing bacteria and analysis of microbiota from indigo fermentation suspensions.

In natural indigo dyeing, the water-insoluble indigo included in the composted indigo leaves called sukumo is converted to water-soluble leuco-indigo through the reduction activities of microorganisms under alkaline conditions. To understand the relationship between indigo reduction and microorganisms in indigo-fermentation suspensions, we isolated and identified the microorganisms that reduce indigo and analyzed the microbiota in indigo-fermentation suspensions. Indigo-reducing microorganisms, which were not isolated by means of a conventional indigo carmine-reduction assay method, were isolated by using indigo as a direct substrate and further identified and characterized. We succeeded in isolating bacteria closely related to Corynebacterium glutamicum, Chryseomicrobium aureum, and Enterococcus sp. for the first time. Anthraquinone was found to be an effective mediator that facilitated the indigo-reduction activity of the isolated strains. On analysis of the microbiota in indigo-fermentation suspensions, the ratio of indigo-reducing bacteria and others was found to be important for maintaining the indigo-reduction activity.

Voltammetric in-situ monitoring of leuco-indigo in indigo-fermenting suspensions.

Cyclic voltammetry was successfully applied to in-vivo monitoring of leuco-indigo in indigo-fermenting suspensions under quiescent conditions without deoxygenation; the working and counter electrodes were kept on the surface of each suspension by a polyethylene vinyl alcohol tube holder. The anodic peak current was used as a measure of the leuco-indigo concentration. The voltammetric wave shape suggested partial solubilization of the indigo with some macromolecules in the fermenting suspensions, which lead to an in-situ method without any electrode surface pretreatment. The anodic peak current well reflected the dyeing activity of a suspensions. The results obtained for laboratory-level fermentation systems clarified the number of days required for dye fermentation, the effectiveness of addition of old suspension as an additive for preparing fresh fermenting suspensions, and the importance of addition of a nitrogen-based nutrient as well as a glucose-based one to recover the indigo-reducing activity. The method can also be applied to determine the amounts of indigo in used dye suspensions and extracts of fermented indigo leaves (sukumo) by adding a chemical reduction pretreatment.

DNA Methylation Suppresses Leptin Gene in 3T3-L1 Adipocytes.

Leptin is a key regulator of energy intake and expenditure. This peptide hormone is expressed in mouse white adipose tissue, but hardly expressed in 3T3-L1 adipocytes. Using bisulfite sequencing, we found that CpG islands in the leptin promoter are highly methylated in 3T3-L1cells. 5-azacytidine, an inhibitor of DNA methyltransferase, markedly increased leptin expression as pre-adipocytes matured into adipocytes. Remarkably, leptin expression was stimulated by insulin in adipocytes derived from precursor cells exposed to 5-azacytidine, but suppressed by thiazolidinedione and dexamethasone. In contrast, adipocytes derived from untreated precursor cells were unresponsive to both 5-azacytidine and hormonal stimuli, although lipid accumulation was sufficient to boost leptin expression in the absence of demethylation. Taken together, the results suggest that leptin expression in 3T3-L1 cells requires DNA demethylation prior to adipogenesis, transcriptional activation during adipogenesis, and lipid accumulation after adipogenesis.