Hinokitiol-induced decreases of tyrosinase and microphthalmia-associated transcription factor are mediated by the endoplasmic reticulum-associated degradation pathway in human melanoma cells.

Tyrosinase (TYR) is a key enzyme for melanin production. We previously showed that hinokitiol, a naturally occurring seven-membered ring terpenoid, potently inhibits human TYR activity. Interestingly, hinokitiol was recently reported to decrease expression of TYR and microphthalmia-associated transcription factor (MITF), which is a main transcription factor of the TYR gene, in murine melanoma cells. However, the mechanisms by which hinokitiol decreases the intracellular levels of TYR and MITF have not been fully elucidated. Here, we investigated the underlying mechanisms of the decreases using cultured human melanoma cells. As a result, hinokitiol treatment decreased TYR protein level in a time- and dose-dependent manner in G361 human melanoma cells, while MITF protein level was decreased only at higher concentrations after 3 days treatment. Notably, the mRNA levels of TYR and MITF were slightly increased by hinokitiol treatment. Therefore, we focused on the degradation of TYR and MITF in endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Importantly, co-treatment of ERAD inhibitor with hinokitiol restored the protein levels of TYR and MITF to approximately 30% and 20% of total those in untreated control cells, respectively. Hinokitiol affected the ER homeostasis as well as degradation of TYR and MITF in two human melanoma cell lines, G361 and HT-144, but the changes of ER-stress markers under the hinokitiol treatment were different in the two human melanoma cell lines. Taken together, these observations indicate that hinokitiol may induce ER stress and trigger the degradation of unfolded newly synthesizing TYR and MITF via the ERAD pathway.

Highly Sensitive Labeling Reagents for Scarce Natural Products.

Scarce natural products that possess unique biological activities have been ideal drug leads for decades. However, their identification and structural determinations are problematic owing to sample amount limitation. Inspired by an extremely rare natural product yaku'amide B (10), highly sensitive labeling reagents that would be powerful tools for scarce natural product chemistry were designed and synthesized in this study. By fusion with the key structural motif for the structural revision of 10, the detection sensitivities of amino acid labeling reagents were drastically enhanced in LC-MS analysis. These advanced labeling reagents enabled the detection of infinitesimal amounts of amino acids and peptide hydrolysates. This sensitivity-enhancement design concept was also applicable to reagents for labeling saccharides and reactivity-guided isolation of electrophilic natural products. Details of these reagents, including their practical preparations and extended applications, are also provided.

Intraclass correlations of seizure duration by wavelet transform, sample entropy, and visual determination in electroconvulsive therapy.

Although electroconvulsive therapy seizure duration has been shown to have limited relevance to efficacy, seizure duration remains important for clinically valid stimulus efficiency. There has been no report on seizure duration using sample entropy with Thymatron (Somatics, Inc), which is widely used in Japan. Furthermore, wavelet transform analysis is also suitable for a seizure because of the wide range of dominant frequencies. Therefore, in this study with Thymatron, the intraclass correlations of seizure duration determined by sample entropy, wavelet transform, and visual determination were investigated to determine whether these methods were applicable for clinical use. Wavelet transform, sample entropy, and the human rater had high intraclass correlations for seizure duration. The present results indicate that wavelet transform and sample entropy can be useful in the clinical electroconvulsive therapy setting, and they may also be suitable for clinical research into the mechanisms of the generalized tonic-clonic seizures related to the efficacy of electroconvulsive therapy.