Evaluation of the bitterness of green tea catechins by a cell-based assay with the human bitter taste receptor hTAS2R39

Catechins have a broad range of physiological functions and act as the main taste ingredient of green tea. Although catechins show a strong bitterness, the bitter taste receptor for catechins has not been fully understood. The objective of this study was to identify the receptor for the major green tea catechins such as (−)-epicatechin (EC), (−)-epicatechin gallate (ECg), (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCg). By the cell-based assay using cultured cells expressing human bitter taste receptor, a clear response of hTAS2R39-expressing cells was observed to 300 μM of either ECg or EGCg, which elicit a strong bitterness in humans. The response of hTAS2R39-expressing cells to ECg was the strongest among the tested catechins, followed by EGCg. Because the cellular response to EC and EGC is much weaker than those of ECg and EGCg, galloyl groups was strongly supposed to be involved in the bitter intensity. This finding is similar to the observations of taste intensity obtained from a human sensory study. Our results suggest the participation of hTAS2R39 in the detection of catechins in humans, indicating the possibility that bitterness of tea catechins can be evaluated by using cells expressing hTAS2R39.     

Molecular Functions of Oxygen-Evolving Complex Family Proteins in Photosynthetic Electron Flow

Oxygen-evolving complex (OEC) protein is the original name for membrane-peripheral subunits of photosystem (PS) Ⅱ. Recently,multiple isoforms and homologs for OEC proteins have been identified in the chloroplast thylakoid lumen, indicating that functional diversification has occurred in the OEC family. Gene expression profiles suggest that the Arabidopsis OEC proteins are roughly categorized into three groups: the authentic OEC group, the stressresponsive group, and the group including proteins related to the chloroplast NAD(P)H dehydrogenase (NDH) complex involved in cyclic electron transport around PSI. Based on the above gene expression profiles, molecular functions of the OEC family proteins are discussed together with our current knowledge about their functions.     

Polygalacturonase mRNA of Tomato: Size and Content in Ripe Fruits

In ripe tomato fruits, polygalacturonase (PG) mRNA comprisedabout 1% of the translatable RNAs in the poly(A)(+)RNA fraction.Sucrose density gradient centrifugation showed that this PGmRNA is similar in size to 18S rRNA, which suggests the presenceof a non-coding region. (Received June 19, 1984; Accepted October 23, 1984)     

Novel Rana keratin genes and their expression during larval to adult epidermal conversion in bullfrog tadpoles

The conversion of the larval to adult epidermis during metamorphosis of tadpoles of bullfrog, Rana catesbeiana, was investigated utilizing newly cloned Rana keratin cDNAs as probes. Rana larval keratin (RLK) cDNA (rlk) was cloned using highly specific antisera against Xenopus larval keratin (XLK). Tail skin proteins of bullfrog tadpoles were separated by 2-dimensional gel electrophoresis and subjected to Western blot analysis with anti-XLK antisera. The Rana antigen detected by this method was sequenced and identified as a type II keratin. We cloned rlk from tadpole skin by PCR utilizing primers designed from these peptide sequences of RLK. RLK predicted by nucleotide sequences of rlk was a 549 amino acid -long type II keratin. Subtractive cloning between the body and the tail skin of bullfrog tadpole yielded a cDNA (rak) of Rana adult keratin (RAK). RAK was a 433 amino acid-long type I keratin. We also cloned a Rana keratin 8 (RK8) cDNA (rk8) from bullfrog tadpole epidermis. RK8 was 502 amino acid-long and homologous to cytokeratin 8. Northern blot analyses and in situ hybridization experiments showed that rlk was actively expressed through prometamorphosis in larva-specific epidermal cells called skein cells and became completely inactive at the climax stage of metamorphosis and in the adult skin. RAK mRNA was expressed in basal cells of the tadpole epidermis and germinative cells in the adult epidermis. The expression of rlk and rak was down- and up-regulated by thyroid hormone (TH), respectively. In contrast, there was no change in the expression of RK8 during spontaneous and TH-induced metamorphosis. RK8 mRNA was exclusively expressed in apical cells of the larval epidermis. These patterns of keratin gene expression indicated that the expression of keratin genes is differently regulated by TH depending on the type of larval epidermal cells. The present study demonstrated the usefulness of these genes for the study of molecular mechanism of postembryonic epidermal development and differentiation.     

SCRAPPER-dependent ubiquitination of active zone protein RIM1 regulates synaptic vesicle release

Little is known about how synaptic activity is modulated in the central nervous system. We have identified SCRAPPER, a synapse-localized E3 ubiquitin ligase, which regulates neural transmission. SCRAPPER directly binds and ubiquitinates RIM1, a modulator of presynaptic plasticity. In neurons from Scrapper-knockout (SCR-KO) mice, RIM1 had a longer half-life with significant reduction in ubiquitination, indicating that SCRAPPER is the predominant ubiquitin ligase that mediates RIM1 degradation. As anticipated in a RIM1 degradation defect mutant, SCR-KO mice displayed altered electrophysiological synaptic activity, i.e., increased frequency of miniature excitatory postsynaptic currents. This phenotype of SCR-KO mice was phenocopied by RIM1 overexpression and could be rescued by re-expression of SCRAPPER or knockdown of RIM1. The acute effects of proteasome inhibitors, such as upregulation of RIM1 and the release probability, were blocked by the impairment of SCRAPPER. Thus, SCRAPPER has an essential function in regulating proteasome-mediated degradation of RIM1 required for synaptic tuning.     

Electrochemical reduction of ferrous alpha-verdoheme in complex with heme oxygenase-1

The heme oxygenase (HO) reaction consists of three successive oxygenation reactions, i.e. heme to alpha-hydroxyheme, alpha-hydroxyheme to verdoheme, and verdoheme to biliverdin-iron chelate. Of these, the least understood step is the conversion of verdoheme to biliverdin-iron chelate. For the cleavage of the oxaporphyrin ring of ferrous verdoheme, involvement of a verdoheme pi-neutral radical has been proposed. To probe this hypothetical mechanism in the HO reaction, we performed electrochemical reduction of ferrous verdoheme complexed with rat HO-1 under anaerobic conditions. On the basis of the electrochemical spectral changes, the midpoint potential for the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme was found to be -0.47+/-0.01 V vs the normal hydrogen electrode (NHE). Because this potential is far lower than those of both flavins of NADPH-cytochrome P450 reductase, and of NADPH, it is concluded that the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme is unlikely to occur and that the formation of the pi-neutral radical cannot be the initial step in the degradation of verdoheme by HO. Rather, it appears more reasonable to consider an alternative mechanism in which binding of O(2) to the ferrous iron of verdoheme is the first step in the degradation of verdoheme.     

Bowman-birk proteinase inhibitor confers heavy metal and multiple drug tolerance in yeast

Cultured Coptis japonica cells show tolerance to various toxic compounds. By yeast functional screening of cadmium (Cd) plates with its cDNA library, we isolated a gene encoding Bowman-Birk proteinase inhibitor (CjBBI). The yeast transformant of CjBBI showed multiple tolerance to various drugs adding to Cd, and revealed reduced Cd accumulation in cells. Preferential organs for Cjbbi expression were aerial parts of intact plants, and the subcellular localization of CjBBI was shown, using its green fluorescent protein fusion, to be the apoplast. Induction of Cjbbi expression by Cd treatment suggested that CjBBI was responsible for the tolerance to Cd observed in C. japonica cells.