Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents.

We previously reported that bakuchiol, a phenolic isoprenoid anticancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney cell lysates and purified the specific bakuchiol-binding proteins with SDS-PAGE. Using nanoLC-MS/MS analysis, we identified prohibitin (PHB) 2, voltage-dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using Western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and colocalized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein expression levels and viral titers in the conditioned medium of virus-infected Madin-Darby canine kidney cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.

Validation of the three-chamber strategy for studying mate choice in medaka.

The three-chamber experiment, in which one test animal can choose between two animals placed in physically inaccessible compartments, is a widely adopted strategy for studying sexual preference in animals. Medaka, a small freshwater teleost, is an emerging model for dissecting the neurological/physiological mechanisms underlying mate choice for which intriguing findings have been accumulating. The three-chamber strategy has rarely been adopted in this species; therefore, here we investigated its validity using medaka colour variants that mate assortatively. First, a total of 551 movies, in which a test male and two choice females interacted for 30 min under a free-swimming condition, were manually analysed. The sexual preference of the males, calculated as a courtship ratio, was highly consistent between human observers (r > 0.96), supporting the objectivity of this manual-counting strategy. Second, we tested two types of three-chamber apparatuses, in which choice fish were presented in either a face-to-face or side-by-side location. Test fish (regardless of sex) spent most of the time associating with choice fish in the compartments. However, their sexual preference, calculated as an association ratio, was poorly reproduced when the locations of the choice fish were swapped. Third, the sexual preferences of males quantified using the manual-counting and either of the three-chamber strategies did not correlate (r = 0.147 or 0.297). Hence, we concluded that, even for individuals of a species like medaka, which spawn every day, sexual preference could not be reliably evaluated using the three-chamber strategy. Optimization of the protocol may solve this problem; however, the explanation for the observation that animals that are ready for spawning persist with never-accessible mating partners must be reconsidered.

Development of a Screening System for Targeting Carriers Using Peptide-Modified Liposomes and Tissue Sections.

Liposomes have been used as targeting carriers for drug delivery systems (DDSs), and the carriers are able to be modified with targeting ligands, such as antibodies and peptides. To evaluate the targetability of DDS carriers modified with a targeting ligand, culture cells expressing the targeting molecules as well as small animals are used. Furthermore, in vitro and in vivo screening analyses must be repeatedly performed. Therefore, it is important to establish an easy and high-precision screening system for targeting carriers. With this aim, we focused that whether this ex vivo system could easily support assessment of interaction between targeting ligand and its receptor under physiological environment and further screen the DDS carrier-modified with targeting moiety. We examined targeting ability via in vitro, ex vivo, and in vivo analyses using integrin αvß3-targeting C16Y-L. For the in vitro analysis, the cellular uptake of C16Y-L was higher than that of control liposomes in colon26 cells. For the ex vivo analysis, we performed an immunohistochemical analysis using colon26 tumor sections. C16Y-L was specifically attached to the tumor sections, as found in the in vitro analysis. Moreover, to evaluate the ex vivo-in vivo correlation, we examined the intratumoral localization of C16Y-L. This result showed that C16Y-L was accumulated not only in the tumor tissue but also in the tumor vasculature after the intravenous injection of C16Y-L, suggesting that the ex vivo peptide-modified liposomal analysis was correlated with the in vivo analysis. Thus, the ex vivo peptide-modified liposomal analysis may be an easy and rapid screening system with high-precision and for consideration in in vivo conditions.

Carnitine in severely disabled patients: relation to anthropometric, biochemical variables, and nutritional intake.

BACKGROUND: Carnitine plays a pivotal role in a variety of cellular functions. Carnitine deficiency often occurs in severely disabled patients, especially under valproic acid administration. However, the possible causative factors underlying carnitine deficiency have not been fully identified. The present study aimed at clarifying the association of various anthropometric and biochemical variables, including dietary intake of carnitine, with carnitine levels in severely disabled patients. METHODS: Twenty-six severely disabled patients (mean age: 14.1 years; s.d. 7.8) were enrolled. Plasma carnitine levels were evaluated by an enzyme cycling assay. Estimation of the dietary intake of carnitine was made based on dietary records over a 3-day period. RESULTS: Plasma total and free carnitine levels in patients were significantly lower than those in controls obtained from the previous report. However, the ratios of free carnitine to total carnitine did not change significantly. Free carnitine levels were well correlated with a nutritional intake of carnitine. Administration of not only valproic acid but also other anti-epileptic drugs was found to cause a significant decrease of free carnitine levels after adjusting the nutritional intake of carnitine. Among various anthropometric or biochemical variables, albumin and uric acid showed a significant correlation with free carnitine levels. CONCLUSIONS: Physicians should be aware of the fact that severely disabled patients are at risk for carnitine deficiency even in the absence of valproic acid administration, and pay more attention to the nutritional intake of carnitine.

Modification of the C16Y peptide on nanoparticles is an effective approach to target endothelial and cancer cells via the integrin receptor.

Liposomes have been explored as potential drug and gene-delivery particles. In recent years, tumor-targeted liposomes have been developed to improve the efficacy of antitumor treatment. The C16Y peptide is a modified C16 peptide, which is derived from the laminin γ1 chain, and binds to integrins α(v)ß3 and α5ß1 on endothelial cells. In this study, we prepared integrin-targeted C16Y peptide-modified liposomes (C16Y-L) to enhance the intracellular uptake of drugs and genes specifically into tumor tissues. The selectivity of C16Y-L for endothelial cells and cancer cells, which strongly express integrins α(v)ß3 and α5ß1, was assessed by flow cytometry and confocal microscopy. The cellular uptake of C16Y-L by both cell types was higher than uptake of the un-labeled and scramble peptide-modified liposomes. Next, to ascertain the involvement of receptor-mediated endocytosis in the process, these cells were treated with C16Y-L for 1h at 37°C or at 4°C. We found that uptake was also dependent on the temperature. Moreover, to evaluate whether the uptake depended on an integrin-ligand interaction, we examined the inhibition of C16Y-L uptake using recombinant integrin αVß3 and found that the cellular uptake of C16Y-L treated with αVß3 integrin decreased. This result suggests that C16Y-L can selectively target cells that highly express integrin αVß3. Thus, the modification of the C16Y peptide on a Drug Delivery System (DDS) carrier may be a feasible approach for drug or gene delivery into tumors.