Effects of exogenous methyl jasmonate and light condition on grape berry coloration and endogenous abscisic acid content.

Suitable postharvest treatment methods were investigated to improve the color of grape berries. Culture solutions containing jasmonic acid (JA), methyl jasmonate (MeJA), and prohydrojasmon (PDJ) enhanced the skin coloration of grape berries ('Pione') harvested at the initial stage of coloration. MeJA vapor treatment under sealed conditions increased anthocyanin accumulation in grape berries ('AkiQueen' and 'Pione') harvested at the early stage of skin coloration. Furthermore, promoting skin coloration by MeJA vapor treatment was as effective in mature clusters as it was in detached berries. These effects were confirmed in light conditions but not in constant darkness. Our results showed that postharvest MeJA vapor treatment improved skin coloration in grapes. In addition, postharvest treatment with MeJA was found to have no effect on the endogenous abscisic acid content of grape berry skins. Therefore, we suggest that MeJA vapor treatment can be a useful and labor-saving method for the horticultural industry.

Genetic knockout and pharmacologic inhibition of NCX1 attenuate hypoxia-induced pulmonary arterial hypertension.

The Na+/Ca2+ exchanger type-1 (NCX1) is a bidirectional transporter that is controlled by membrane potential and transmembrane gradients of Na+ and Ca2+. Vascular smooth muscle NCX1 plays an important role in intracellular Ca2+ homeostasis and Ca2+ signaling. We found that NCX1 was upregulated in the pulmonary arteries of mice exposed to chronic hypoxia (10% O2 for 4 weeks). Hence, we investigated the pathophysiological role of NCX1 in hypoxia-induced pulmonary arterial hypertension (PAH), using NCX1-heterozygous (NCX1+/-) mice, in which NCX1 expression is reduced by half, and SEA0400, a specific NCX1 inhibitor. NCX1+/- mice exhibited attenuation of hypoxia-induced PAH and right ventricular (RV) hypertrophy compared with wild-type mice. Furthermore, continuous administration of SEA0400 (0.5 mg/kg/day for 4 weeks) to wild-type mice by osmotic pumps significantly suppressed hypoxia-induced PAH and pulmonary vessel muscularization, with a slight reduction in RV hypertrophy. These findings indicate that the upregulation of NCX1 contributes to the development of hypoxia-induced PAH, suggesting that NCX1 inhibition might be a novel approach for the treatment of PAH.

Insertion of a transposon-like sequence in the 5'-flanking region of the YUCCA gene causes the stony hard phenotype.

Melting-flesh peaches produce large amounts of ethylene, resulting in rapid fruit softening at the late-ripening stage. In contrast, stony hard peaches do not soften and produce little ethylene. The indole-3-acetic acid (IAA) level in stony hard peaches is low at the late-ripening stage, resulting in low ethylene production and inhibition of fruit softening. To elucidate the mechanism of low IAA concentration in stony hard peaches, endogenous levels of IAA and IAA intermediates or metabolites were analysed by ultra-performance liquid chromatography-tandem mass spectrometry. Although the IAA level was low, the indole-3-pyruvic acid (IPyA) level was high in stony hard peaches at the ripening stage. These results indicate that YUCCA activity is reduced in ripening stony hard peaches. The expression of one of the YUCCA isogenes in peach, PpYUC11, was suppressed in ripening stony hard peaches. Furthermore, an insertion of a transposon-like sequence was found upstream of the PpYUC11 gene in the 5'-flanking region. Analyses of the segregation ratio of the stony hard phenotype and genotype in F1 progenies indicated that the transposon-inserted allele of PpYUC11, hd-t, correlated with the stony hard phenotype. On the basis of the above findings, we propose that the IPyA pathway (YUCCA pathway) is the main auxin biosynthetic pathway in ripening peaches of 'Akatsuki' and 'Manami' cultivars. Because IAA is not supplied from storage forms, IAAde novo synthesis via the IPyA pathway (YUCCA pathway) in mesocarp tissues is responsible for auxin generation to support fruit softening, and its disruption can lead to the stony hard phenotype.

Abscisic acid affects expression of citrus FT homologs upon floral induction by low temperature in Satsuma mandarin (Citrus unshiu Marc.).

After a long juvenile period, citrus trees undergo seasonal flowering cycles. Under natural conditions, citrus flowering is regulated mainly by low ambient temperatures around 15-20 °C and water deficit stress. Recent studies have revealed that fluctuations in the expression of citrus homologs of FLOWERING LOCUS T (FT, encoding a flowering integrator) are correlated with their presumed role as flower-promoting signals. Previous ectopic expression analyses have demonstrated the flower-promoting function of citrus FT homologs. In this study, we examined whether abscisic acid (ABA) affects the expression of FT homologs and the flowering induced by low ambient temperatures. Application of exogenous ABA to potted Satsuma mandarin (Citrus unshiu Marc.) trees resulted in transient accumulation of citrus FT homolog transcripts. The promoter of one citrus FT homolog, CiFT3, was active in transgenic A. thaliana (Arabidopsis thaliana) and responded to exogenous and endogenous ABA. CiFT3 is preferentially expressed in shoots, and its expression was affected by flower-inductive treatments. Endogenous ABA accumulated in mandarin shoots during the floral induction period at 15 °C and under field conditions. The accumulation of ABA was correlated with the accumulation of FT homolog transcripts and flowering intensity. It was consistent with changes in the expression of genes related to ABA metabolism. The abundance of carotenoid precursors that serve as substrates for ABA biosynthesis decreased in leaves during the accumulation of ABA. Our data indicate that ABA and carotenoid precursors in leaves influence the flowering of mandarin trees induced by low temperature.

Overexpression of a citrus basic helix-loop-helix transcription factor (CubHLH1), which is homologous to Arabidopsis activation-tagged bri1 suppressor 1 interacting factor genes, modulates carotenoid metabolism in transgenic tomato.

To explore the transcription factors associated with carotenoid metabolism in citrus fruit, one transcription factor (CubHLH1) was selected through microarray screening in Satsuma mandarin (Citrus unshiu Marc.) fruit, which was treated with exogenous ethylene or gibberellin (GA), accelerating or retarding carotenoid accumulation in peel, respectively. The amino acid sequence of CubHLH1 has homology to Arabidopsis activation-tagged bri1 suppressor 1 (ATBS1) interacting factor (AIF), which is functionally characterized as a negative regulator of the brassinolide (BR) signalling pathway. Yeast two-hybrid analysis revealed that protein for CubHLH1 could interact with Arabidopsis and tomato ATBS1. Overexpression of CubHLH1 caused a dwarf phenotype in transgenic tomato (Solanum lycopersicum L.), suggesting that CubHLH1 has a similar function to Arabidopsis AIF. In the transgenic tomato fruit at ripening stage, the lycopene content was reduced along with the changes in carotenoid biosynthetic gene expression. The abscisic acid (ABA) content of all the transgenic tomato fruit was higher than that of the wild type. These results implied that CubHLH1 is considered to have a similar function to Arabidopsis AIFs and might be directly involved in carotenoid metabolism in mature citrus fruit.

Antimyeloma activity of NK012, a micelle-forming macromolecular prodrug of SN-38, in an orthotopic model.

NK012 is a micelle-forming macromolecular prodrug of 7-ethyl-10-hydroxy camptothecin (SN-38), an active metabolite of irinotecan. It is accumulated and retained in tumor tissues and gradually releases SN-38 in an enzyme-independent manner. NK012 was previously demonstrated to have stronger antitumor activity than irinotecan in a broad range of human solid-tumor xenograft models. In our study, we used an orthotopic multiple myeloma (MM) model created by injecting CD138-positive U266B1, a myeloma cell line that produces human IgE lambda light chain (monoclonal protein, M protein), into immunodeficient NOD/Shi-scid, IL-2Rγc (null) mice. This model shows typical bone marrow infiltration by the human myeloma cells. We evaluated the antimyeloma activity of intravenously administered NK012 in this model and showed that it suppressed the M protein concentration in the plasma and proliferation of myeloma cells in the bone marrow in a dose-dependent manner. NK012 suppressed the progression of hind-leg paralysis and prolonged the survival time of the mice compared to the untreated control group. In combination with bortezomib (BTZ), NK012 increased the median survival time compared to that with BTZ alone. In conclusion, these results suggest that NK012 is a potential candidate for use-alone and in combination-in the treatment of MM in humans.

Increased levels of IAA are required for system 2 ethylene synthesis causing fruit softening in peach (Prunus persica L. Batsch).

The fruit of melting-flesh peach (Prunus persica L. Batsch) cultivars produce high levels of ethylene caused by high expression of PpACS1 (an isogene of 1-aminocyclopropane-1-carboxylic acid synthase), resulting in rapid fruit softening at the late-ripening stage. In contrast, the fruit of stony hard peach cultivars do not soften and produce little ethylene due to low expression of PpACS1. To elucidate the mechanism for suppressing PpACS1 expression in stony hard peaches, a microarray analysis was performed. Several genes that displayed similar expression patterns as PpACS1 were identified and shown to be indole-3-acetic acid (IAA)-inducible genes (Aux/IAA, SAUR). That is, expression of IAA-inducible genes increased at the late-ripening stage in melting flesh peaches; however, these transcripts were low in mature fruit of stony hard peaches. The IAA concentration increased suddenly just before harvest time in melting flesh peaches exactly coinciding with system 2 ethylene production. In contrast, the IAA concentration did not increase in stony hard peaches. Application of 1-naphthalene acetic acid, a synthetic auxin, to stony hard peaches induced a high level of PpACS1 expression, a large amount of ethylene production and softening. Application of an anti-auxin, α-(phenylethyl-2-one)-IAA, to melting flesh peaches reduced levels of PpACS1 expression and ethylene production. These observations indicate that suppression of PpACS1 expression at the late-ripening stage of stony hard peach may result from a low level of IAA and that a high concentration of IAA is required to generate a large amount of system 2 ethylene in peaches.

Evaluation of new affinity chromatography resins for polyclonal, oligoclonal and monoclonal antibody pharmaceuticals.

The performance of MabSelect SuRe and IgSelect affinity chromatography resins designed for process-scale purification of antibodies was investigated. Various antibodies (4 human monoclonal, 1 human polyclonal and 1 bovine polyclonal antibody and 1 Fc-fusion protein) were used to evaluate the elution pH and dynamic binding capacity of the resins. The elution pH for each human antibody was similar on MabSelect SuRe and IgSelect (pH 3.5-3.8). No significant differences in dynamic binding capacity were observed among human antibodies on MabSelect SuRe ( approximately 20-40 mg/mL resin) and IgSelect (approximately 10-30 mg/mL resin). The binding capacity order for the human antibodies was the same on MabSelect SuRe and IgSelect. Using a linear pH gradient, both resins were able to partially separate monomeric and aggregated forms of the antibodies. The results indicate that these new affinity resins are powerful tools for the purification of human polyclonal antibodies from transgenic animals and oligoclonal antibodies from CHO cell cultures.

The Japanese value of harmony and nursing ethics.

Harmony is one of the most fundamental Japanese values. It is derived from Confucianism and encompasses a state of mind, an action process and outcomes of the action. This article draws on research data and discusses Japanese nurses' perceptions of harmony as reflected in their everyday practice. The most important virtues for these nurses were reported as politeness and respect for other persons. The outcome from the nurses' harmonious practice, it is claimed, benefited patients and created peaceful, harmonious relationships for all. Because of the unique link between harmony and the location of interaction, the ideal 'workplace harmony' threatened some nurses' professional decision making. These nurses confused harmony with conformity by superficial agreement. The Japanese seniority system could be a major factor contributing to this problem. Ethics education that includes traditional values and concepts in Japanese culture is strongly urged.

Effect of postharvest temperature and ethylene on carotenoid accumulation in the Flavedo and juice sacs of Satsuma Mandarin ( Citrus unshiu Marc.) fruit.

The effect of postharvest temperature (5, 20, and 30 degrees C) and ethylene at different temperatures (20 and 5 degrees C) on carotenoid content and composition and on the expression of the carotenoid biosynthesis-related genes was investigated in the flavedo and juice sacs of Satsuma mandarin ( Citrus unshiu Marc.) fruit. Under an ethylene-free atmosphere, storage at 20 degrees C rapidly increased the carotenoid content in flavedo and maintained the content in juice sacs. In contrast, storage at 5 and 30 degrees C gradually decreased the content in juice sacs but slowly increased that in flavedo. Under an ethylene atmosphere, storage at 20 degrees C enhanced the carotenoid accumulation in flavedo more dramatically than found under an ethylene-free atmosphere with distinct changes in the carotenoid composition but did not noticeably change the content and composition in juice sacs. In contrast, storage at 5 degrees C under an ethylene atmosphere repressed carotenoid accumulation with changes in the carotenoid composition in flavedo but did not clearly change the carotenoid content in juice sacs. Under an ethylene-free atmosphere, differences in the gene expression profile among the temperatures were observed but were not well-correlated with those in the carotenoid content in flavedo and juice sacs. Under an ethylene atmosphere, in flavedo, the gene expression of phytoene synthase (PSY) and phytoene desaturase (PDS) was slightly higher at 20 degrees C but lower at 5 degrees C than under an ethylene-free atmosphere. At 20 degrees C, the gene expression of several carotenoid biosynthetic enzymes promoted by ethylene seemed to be responsible for the enhanced accumulation of carotenoid in flavedo. In contrast, at 5 degrees C, the repressed gene expression of PSY and PDS by ethylene seemed to be primarily responsible for the repressed accumulation of carotenoid in flavedo. In juice sacs, the small response of the gene expression to ethylene seemed to be responsible for small changes in carotenoid accumulation under an ethylene atmosphere.

Quantification of carotenoids in citrus fruit by LC-MS and comparison of patterns of seasonal changes for carotenoids among citrus varieties.

To quantify the 18 carotenoids on the basic routes of the carotenoid biosynthesis in plants simultaneously, a method for liquid chromatography-mass spectrometry (LC-MS) using atmospheric pressure chemical ionization was developed. With this method, the seasonal changes of carotenoids in the flavedo and juice sacs of 39 citrus varieties were analyzed. On the basis of the patterns of seasonal changes of carotenoids in both flavedo and juice sacs, 39 citrus varieties were classified. In flavedo, 39 varieties were classified into 5 clusters, in which the carotenoid profiles were carotenoid-poor, phytoene-abundant, violaxanthin-abundant, violaxanthin- and beta-cryptoxanthin-abundant, and phytoene-, violaxanthin-, and beta-cryptoxanthin-abundant, respectively. In juice sacs, they were classified into 4 clusters, in which the carotenoid profiles were carotenoid-poor, violaxanthin-abundant, violaxanthin- and phytoene-abundant, and violaxanthin-, phytoene-, and beta-cryptoxanthin-abundant, respectively. In flavedo, many citrus varieties, except for the carotenoid-poor and phytoene-abundant varieties, massively accumulated beta,epsilon-carotenoids (e.g., lutein), beta,beta-carotenoids (e.g., beta-cryptoxanthin and violaxanthin), and phytoene, in that order. In juice sacs, the accumulation order among beta,beta-carotenoids was observed. Violaxanthin accumulation preceded beta-cryptoxanthin accumulation in violaxanthin-, phytoene-, and beta-cryptoxanthin-abundant varieties. In each variety, the carotenoid profiles of the flavedo and juice sacs on the basis of the concentration in violaxanthin and beta-cryptoxanthin were similar, with the exception of a few varieties.

Arabidopsis Aux/IAA genes are involved in brassinosteroid-mediated growth responses in a manner dependent on organ type.

We examined whether auxin/indole-3-acetic acid (Aux/IAA) proteins, which are key players in auxin-signal transduction, are involved in brassinosteroid (BR) responses. iaa7/axr2-1 and iaa17/axr3-3 mutants showed aberrant BR sensitivity and aberrant BR-induced gene expression in an organ-dependent manner. Two auxin inhibitors were tested in terms of BR responses. Yokonolide B inhibited BR responses, whereas p-chlorophenoxyisobutyric acid did not inhibit BR responses. DNA microarray analysis revealed that 108 genes were up-regulated, while only eight genes were down-regulated in iaa7. Among the genes that were up- or down-regulated in axr2, 22% were brassinolide-inducible genes, 20% were auxin-inducible genes, and the majority were sensitive neither to BR nor to auxin. An inhibitor of BR biosynthesis, brassinazole, inhibited auxin induction of the DR5-GUS gene, which consists of a synthetic auxin-response element, a minimum promoter, and a beta-glucuronidase. These results suggest that Aux/IAA proteins function in auxin- and BR-signaling pathways, and that IAA proteins function as the signaling components modulating BR sensitivity in a manner dependent on organ type.

Activation of cell proliferation by brassinolide application in tobacco BY-2 cells: effects of brassinolide on cell multiplication, cell-cycle-related gene expression, and organellar DNA contents.

Brassinosteroids (BRs) are steroidal phytohormones that are essential for many processes in plant growth and development, such as cell expansion, vascular differentiation, and responses to stress. The effects of BRs on cell division are unclear, as attested by contradictory published results. To determine the effect of BRs on cell division, the tobacco (Nicotiana tabacum) BY-2 cell line, which is a widely-used model system in plant cell biology, was used. It was found that brassinolide (BL) promoted cell division only during the early phase of culture and in the absence of auxin (2,4-D). This promotion of cell division was confirmed by RNA gel blot analyses using cell-cycle-related gene probes. At later stages in the culturing periods of BL-supplied and 2,4-D-supplied BY-2 cells, differences in cell multiplication and cell-cycle-related gene expression were observed. Moreover, the BL-treated BY-2 cells had morphological differences from the 2,4-D-treated cells. To determine whether suppressed organellar DNA replication limited this promotion of cell division during the early culture phase, this replication was examined and it was found that BL treatment had no effect on activating organellar (plastid- and mitochondrial-) DNA synthesis. As preferential organellar DNA synthesis, which is activated by 2,4-D, is necessary during successive cell divisions in BY-2 cells, these data suggest that the mechanism of the promotion of cell division by BL treatment is distinct from that regulated by the balance of auxin and cytokinin.

Biosynthesis of cholestanol in higher plants.

To understand the early steps of C(27) brassinosteroid biosynthesis, metabolic experiments were performed with Arabidopsis thaliana and Nicotiana tabacum seedlings, and with cultured Catharanthus roseus cells. [26, 28-2H(6)]Campestanol, [26-2H(3)]cholesterol, and [26-2H(3)]cholestanol were administered to each plant, and the resulting metabolites were analyzed by gas chromatography-mass spectrometry. In all the species examined, [2H(3)]cholestanol was identified as a metabolite of [2H(6)]campestanol, and [2H(3)]cholest-4-en-3-one and [2H(3)]cholestanol were identified as metabolites of [2H(3)]cholesterol. This study revealed that cholestanol (C(27) sterol) was biosynthesized from both cholesterol (C(27) sterol) and campestanol (C(28) sterol). It was also demonstrated that cholestanol was converted to 6-oxocholestanol, and campestanol was converted to 6-oxocampestanol.