Glycosylation of trans-Resveratrol by Plant-Cultured Cells

Plant-cultured cells of Catharanthus roseus converted trans-resveratrol into its 3-O-β-D-glucopyranoside, 4'-O-β-D-glucopyranoside, 3-O-(6-O-β-D-xylopyranosyl)-β-D-glucopyranoside, and 3-O-(6-O-α-L-arabinopyranosyl)-β-D-glucopyranoside. The 3-O-(6-O-β-D-xylopyranosyl)-β-D-glucopyranoside and 3-O-(6-O-α-L-arabinopyranosyl)-β-D-glucopyranoside compounds of trans-resveratrol are both new. Incubation of plant-cultured cells of Ipomoea batatas and Strophanthus gratus with trans-resveratrol gave trans-resveratrol 3-O-β-D-glucopyranoside and trans-resveratrol 4'-O-β-D-glucopyranoside.     

The effect of genetically enriched (E)-β-ocimene and the role of floral scent in the attraction of the predatory mite Phytoseiulus persimilis to spider mite-induced volatile blends of torenia

Plants under herbivore attack emit mixtures of volatiles (herbivore-induced plant volatiles, HIPVs) that can attract predators of the herbivores. Although the composition of HIPVs should be critical for the attraction, most studies of transgenic plant-emitted volatiles have simply addressed the effect of trans-volatiles without embedding in other endogenous plant volatiles. We investigated the abilities of transgenic wishbone flower plants (Torenia hybrida and Torenia fournieri) infested with spider mites, emitting a trans-volatile ((E)-β-ocimene) in the presence or absence of endogenous volatiles (natural HIPVs and/or floral volatiles), to attract predatory mites (Phytoseiulus persimilis). In both olfactory- and glasshouse-based assays, P. persimilis females were attracted to natural HIPVs from infested wildtype (wt) plants of T. hybrida but not to those of T. fournieri. The trans-volatile enhanced the ability to attract P. persimilis only when added to an active HIPV blend from the infested transgenic T. hybrida plants, in comparison with the attraction by infested wt plants. Intriguingly, floral volatiles abolished the enhanced attractive ability of T. hybrida transformants, although floral volatiles themselves did not elicit any attraction or avoidance behavior. Predator responses to trans-volatiles were found to depend on various background volatiles (e.g. natural HIPVs and floral volatiles) endogenously emitted by the transgenic plants.     

Kinase-dependent activation of voltage-gated Ca2+ channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia

Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ[Ca(2+)](i)) occurring through activation of voltage-dependent Ca(2+) channels (VDCC) even though ET-1-induced depolarization via inhibition of K(+) channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ[Ca(2+)](i) in PASMCs from rats exposed to CH (10% O(2), 3 wk) using the Ca(2+)-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ[Ca(2+)](i) by >70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ[Ca(2+)](i) to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ[Ca(2+)](i) in PASMCs occurs via Ca(2+) influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.     

An inducible RNA interference system in Physcomitrella patens reveals a dominant role of augmin in phragmoplast microtubule generation

Mitosis is a fundamental process of eukaryotic cell proliferation. However, the molecular mechanisms underlying mitosis remain poorly understood in plants partly because of the lack of an appropriate model cell system in which loss-of-function analyses can be easily combined with high-resolution microscopy. Here, we developed an inducible RNA interference (RNAi) system and three-dimensional time-lapse confocal microscopy in the moss Physcomitrella patens that allowed in-depth phenotype characterization of the moss genes essential for cell division. We applied this technique to two microtubule regulators, augmin and γ-tubulin complexes, whose mitotic roles remain obscure in plant cells. Live imaging of caulonemal cells showed that they proceed through mitosis with continual generation and self-organization of acentrosomal microtubules. We demonstrated that augmin plays an important role in γ-tubulin localization and microtubule generation from prometaphase to cytokinesis. Most evidently, microtubule formation in phragmoplasts was severely compromised after RNAi knockdown of an augmin subunit, leading to incomplete expansion of phragmoplasts and cytokinesis failure. Knockdown of the γ-tubulin complex affected microtubule formation throughout mitosis. We conclude that postanaphase microtubule generation is predominantly stimulated by the augmin/γ-tubulin machinery in moss and further propose that this RNAi system serves as a powerful tool to dissect the molecular mechanisms underlying mitosis in land plants.     

ACPA-negative RA consists of two genetically distinct subsets based on RF positivity in Japanese

HLA-DRB1, especially the shared epitope (SE), is strongly associated with rheumatoid arthritis (RA). However, recent studies have shown that SE is at most weakly associated with RA without anti-citrullinated peptide/protein antibody (ACPA). We have recently reported that ACPA-negative RA is associated with specific HLA-DRB1 alleles and diplotypes. Here, we attempted to detect genetically different subsets of ACPA-negative RA by classifying ACPA-negative RA patients into two groups based on their positivity for rheumatoid factor (RF). HLA-DRB1 genotyping data for totally 954 ACPA-negative RA patients and 2,008 healthy individuals in two independent sets were used. HLA-DRB1 allele and diplotype frequencies were compared among the ACPA-negative RF-positive RA patients, ACPA-negative RF-negative RA patients, and controls in each set. Combined results were also analyzed. A similar analysis was performed in 685 ACPA-positive RA patients classified according to their RF positivity. As a result, HLA-DRB1*04:05 and *09:01 showed strong associations with ACPA-negative RF-positive RA in the combined analysis (p = 8.8×10⁻⁶ and 0.0011, OR: 1.57 (1.28–1.91) and 1.37 (1.13–1.65), respectively). We also found that HLA-DR14 and the HLA-DR8 homozygote were associated with ACPA-negative RF-negative RA (p = 0.00022 and 0.00013, OR: 1.52 (1.21–1.89) and 3.08 (1.68–5.64), respectively). These association tendencies were found in each set. On the contrary, we could not detect any significant differences between ACPA-positive RA subsets. As a conclusion, ACPA-negative RA includes two genetically distinct subsets according to RF positivity in Japan, which display different associations with HLA-DRB1. ACPA-negative RF-positive RA is strongly associated with HLA-DRB1*04:05 and *09:01. ACPA-negative RF-negative RA is associated with DR14 and the HLA-DR8 homozygote.     

Rapid recruitment of BRCA1 to DNA double-strand breaks is dependent on its association with Ku80

BRCA1 is the first susceptibility gene to be linked to breast and ovarian cancers. Although mounting evidence has indicated that BRCA1 participates in DNA double-strand break (DSB) repair pathways, its precise mechanism is still unclear. Here, we analyzed the in situ response of BRCA1 at DSBs produced by laser microirradiation. The amino (N)- and carboxyl (C)-terminal fragments of BRCA1 accumulated independently at DSBs with distinct kinetics. The N-terminal BRCA1 fragment accumulated immediately after laser irradiation at DSBs and dissociated rapidly. In contrast, the C-terminal fragment of BRCA1 accumulated more slowly at DSBs but remained at the sites. Interestingly, rapid accumulation of the BRCA1 N terminus, but not the C terminus, at DSBs depended on Ku80, which functions in the nonhomologous end-joining (NHEJ) pathway, independently of BARD1, which binds to the N terminus of BRCA1. Two small regions in the N terminus of BRCA1 independently accumulated at DSBs and interacted with Ku80. Missense mutations found within the N terminus of BRCA1 in cancers significantly changed the kinetics of its accumulation at DSBs. A P142H mutant failed to associate with Ku80 and restore resistance to irradiation in BRCA1-deficient cells. These might provide a molecular basis of the involvement of BRCA1 in the NHEJ pathway of the DSB repair process.     

Enzyme inactivation and quality preservation of sake by high-pressure carbonation at a moderate temperature

The effect of a high-pressure carbonation treatment on the change in quality of sake during storage was investigated. Measurements of the amino acidity and isovaleraldehyde content of carbonated sake (20 MPa pressure at 40, 45 and 50 degrees C for 7, 21 and 33 min, respectively) as well as of heat-treated sake (reaching temperature of 65 degrees C and immediately cooled) were almost unchanged during storage at 3 and 20 degrees C. Glucose in the sake subjected to these treatments was retained at an almost constant under the same storage conditions, except for the sake carbonated at 40 degrees C and stored at 20 degrees C. In contrast, the amino acidity, and glucose and isovaleraldehyde contents of non-pasteurized (fresh) sake increased during storage at both temperatures. The sake samples subjected to the carbonation treatment and heat treatment both gave better sensory scores than the fresh sake sample after 6 month of storage at 3 and 20 degrees C, especially at 3 degrees C for the flavor. These results suggest that the high-pressure carbonation treatment is an effective new technique for preserving the quality of sake.     

Biotransformation of phenolic compounds by the cultured cells of Catharanthus roseus

The cultured cells of Catharanthus roseus were able to convert 2-, 3-, and 4-hydroxybenzyl alcohols into their corresponding hydroxybenzyl-β- -glucopyranosides or β- -glucopyranosylbenzyl alcohols, and then convert 2- and 3-hydroxybenzyl-β- -glucopyranosides into primeverosides and vicianosides. Further, the C. roseus cells were capable of hydroxylation of 2-hydroxybenzoic acid to afford 2,5-dihydroxybenzoic acid and then glucosylation of the newly introduced phenolic hydroxyl group.     

Phytoestrogens from the roots of Polygonum cuspidatum (Polygonaceae): structure-requirement of hydroxyanthraquinones for estrogenic activity.

The methanolic extract from the roots of Polygonum (P.) cuspidatum was found to enhance cell proliferation at 30 or 100 microg/mL in MCF-7, an estrogen-sensitive cell line. By bioassay-guided separation from P. cuspidatum with the most potent activity, emodin and emodin 8-O-beta-D-glucopyranoside were isolated as active principles. The methanolic extracts from Polygonum, Cassia, Aloe, and Rheum species, which were known to contain anthraquinones, also showed the MCF-7 proliferation. As a result of the evaluation of various anthraquinones from plant sources and synthetic anthraquinones, aloe-emodin, chrysophanol, chrysophanol 8-O-beta-D-glucopyranoside, and 1,8-dihydroxyanthraquinone showed weak activity. On the other hand, alizalin and 2,6-dihydroxyanthraquinone as well as emodin having the 2- and/or 6-hydroxyl groups showed potent activity. These results show that the unchelated hydroxyl group is essential for strong activity. Emodin and 2,6-dihydroxyanthraquinone also inhibited 17beta-estradiol binding to human estrogen receptors (ERs) with K(i) values of 0.77 and 0.31microM for ERalpha and 1.5 and 0.69 microM for ERbeta. These findings indicate that hydroxyanthraquinones such as emodin are phytoestrogens with an affinity to human estrogen receptors.