A molecular phylogenetic analysis of 25 isolates of fish nodaviruses, the causative agents of viral nervous necrosis of marine fish, was performed based on the nucleotide sequences (427 bases) of the coat protein gene. These fish nodaviruses were classified into four clusters: tiger puffer nervous necrosis virus, striped jack nervous necrosis virus, berfin flounder nervous necrosis virus, and red-spotted grouper nervous necrosis virus.

Recent progress in the molecular biology of taste reception has revealed that in mammals, the heteromeric receptors T1R1/3 and T1R2/3 respond to amino acids and sweeteners, respectively, whereas T2Rs are receptors for bitter tastants. Similar taste receptors have also been characterized in fish, but their ligands have not been identified yet. In the present study, we conducted a series of experiments to identify the fish taste receptor ligands. Facial nerve recordings in zebrafish (Danio rerio) demonstrated that the fish perceived amino acids and even denatonium, which is a representative of aversive bitter compounds for mammals and Drosophila. Calcium imaging analysis of T1Rs in zebrafish and medaka fish (Oryzias latipes) using an HEK293T heterologous expression system revealed that both T1R1/3 and a series of T1R2/3 responded to amino acids but not to sugars. A triple-labeling, in situ hybridization analysis demonstrated that cells expressing T1R1/3 and T1R2/3s exist in PLC␤2-expressing taste bud cells of medaka fish. Functional analysis using T2Rs showed that zfT2R5 and mfT2R1 responded to denatonium. Behavior observations confirmed that zebrafish prefer amino acids and avoid denatonium. These results suggest that, although there may be some fish-specific way of discriminating ligands, vertebrates could have a conserved gustatory mechanism by which T1Rs and T2Rs respond to attractive and aversive tastants, respectively.

Vertebrates receive tastants, such as sugars, amino acids, and nucleotides, via taste bud cells in epithelial tissues. In mammals, two families of G protein-coupled receptors for tastants are expressed in taste bud cells-T1Rs for sweet tastants and umami tastants (l-amino acids) and T2Rs for bitter tastants. Here, we report two families of candidate taste receptors in fish species, fish T1Rs and T2Rs, which show significant identity to mammalian T1Rs and T2Rs, respectively. Fish T1Rs consist of three types: fish T1R1 and T1R3 that show the highest degrees of identity to mammalian T1R1 and T1R3, respectively, and fish T1R2 that shows almost equivalent identity to both mammalian T1R1 and T1R2. Unlike mammalian T1R2, fish T1R2 consists of two or three members in each species. We also identified two fish T2Rs that show low degrees of identity to mammalian T2Rs. In situ hybridization experiments revealed that fish T1R and T2R genes were expressed specifically in taste bud cells, but not in olfactory receptor cells. Fish T1R1 and T1R2 genes were expressed in different subsets of taste bud cells, and fish T1R3 gene was co-expressed with either fish T1R1 or T1R2 gene as in the case of mammals. There were also a significant number of cells expressing fish T1R2 genes only. Fish T2R genes were expressed in different cells from those expressing fish T1R genes. These results suggest that vertebrates commonly have two kinds of taste signaling pathways that are defined by the types of taste receptors expressed in taste receptor cells.

The aim of this study was to evaluate the safety, tolerability and pharmacokinetics of single dose of Melissa officinalis extract which contained rosmarinic acid, including food-effects in healthy individuals. A total of eleven healthy individuals were randomly assigned to treatment arms in the two studies [Study 1 (fasted state) and Study 2 (fed state)]. Rosmarinic acid in serum was measured by a coulometric detection method using High-Performance Liquid Chromatography electrochemical detector. The serum concentration of total rosmarinic acid peaked at 1 hour after administration of Melissa officinalis extract containing 500mg rosmarinic acid in fasted state, with a maximum serum concentration 162.20 nmol/ L. The area under the curve for intact rosmarinic acid was calculated from the serum concentration-time profile to be 832.13 nmol • hour/ L. Food intake increases area under the curve and delayed time at which the maximum serum concentration. Rosmarinic acid supplementation did not affect liver, kidney, or blood cell function parameters. No adverse event was reported by any of the participants due to the study treatment. Single dose of Melissa officinalis extract containing 500 mg rosmarinic acid appears to be safe and tolerable in healthy individuals. Food intake increased the exposure of rosmarinic acid and delayed absorption of rosmarinic acid in healthy individuals.Trial RegistrationTrial Registration: UMIN-CTR UMIN000004997

We conducted a randomized placebo-controlled double-blind 24-week trial using Melissa officinalis (M. officinalis) extract richly containing rosmarinic acid (RA) on patients with mild dementia due to Alzheimer’s disease (AD) with the aim to examine the safety and tolerability (primary endpoint) of RA (500 mg daily) and its clinical effects and disease-related biomarker changes (secondary endpoints). Patients (n = 23) diagnosed with mild dementia due to probable AD were randomized to either the placebo or M. officinalis extract group. No differences in vital signs or physical and neurologic examination results were detected between the M. officinalis and placebo groups. No serious adverse events occurred. There were no significant differences in cognitive measures; however, the mean Neuropsychiatric Inventory Questionnaire (NPI-Q) score improved by 0.5 points in the M. officinalis group and worsened by 0.7 points in the placebo group between the baseline and 24-week visit, indicating a significant difference (P = 0.012). No significant differences were apparent in disease-related biomarkers between the groups. M. officinalis extract containing 500 mg of RA taken daily was safe and well-tolerated by patients with mild dementia due to AD. Our results suggest that RA may help prevent the worsening of AD-related neuropsychiatric symptoms. Trial registration: The registration number for this clinical trial is UMIN000007734 (16/04/2012).

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Isoflavone data concerning the metabolism and permeability on intestinal epithelial cells are scarce, particularly for microbial isoflavone metabolites. This study evaluates the absorption mechanisms for the isoflavones, genistein and daidzein, and their microbial metabolites, dihydrogenistein (DHG) and dihydrodaidzein (DHD). The permeability characteristics of isoflavones were compared by using the Caco-2 human colon adenocarcinoma cell line for a parallel artificial membrane permeability assay, and comparing their physicochemical properties. The data suggest that genistein, DHG and DHD were efficiently transported by passive diffusion according to the pH-partition hypothesis. Genistein was conjugated by phase II metabolizing enzymes and acted as a substrate of the breast cancer resistance protein (BCRP). Daidzein was not conjugated but did act as a substrate for BCRP, multidrug resistance-associated proteins, and P-glycoprotein. In contrast, DHG and DHD were markedly more permeable than their parent isoflavones; they were therefore difficult to transport by the efflux effect, and glucuronidation/sulfation was limited by the flux time.

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