Photooxidative Damage in Photosynthetic Activities of Chromatium vinosum

The capacity of photosynthetic CO₂ fixation in the anaerobic purple-sulfur bacterium, Chromatium vinosum is markedly impaired by strong illumination (9 × 10⁴ lux) in the presence of 100% O₂. In the absence of HCO₃⁻, decline in activity occurred gradually, with about 40% of the initial activity remaining after a 1-hour incubation. The addition of 50 millimolar HCO₃⁻ to the incubation medium resulted in a measurable delay (about 30 minutes) of the inactivation process. Ribulose-1,5-bisphosphate carboxylase activity and light-dependent O₂ uptake (electron flow) or crude extracts prepared after pretreatment of the bacterial cells with O₂ and light were not affected but the photophosphorylation capacity of either bacterial cells or chromatophores was drastically reduced. The inhibition of photophos-phorylation in the chromatophore preparations was significantly reduced by the addition of either an O₂⁻ scavenger, Tiron, or an ¹O₂ scavenger, α-tocopherol. These results suggest that the active O₂ species, O₂⁻ or ¹O₂, might take part in the observed inactivation.

The pretreatment of the bacteria with O₂ and light inhibited CO₂ assimilation through the Calvin-Benson cycle, while relatively stimulating the formation of aspartate and glutamate. It also inhibited the conversion of glycolate to glycine, resulting in a sustained extracellular excretion of glycolate. The inactivation of photosynthetic CO₂ fixation by intact cells was enhanced by low temperature, KCN, or methylviologen addition during the pretreatment with O₂ and light. The mechanism(s) of O₂-dependent photoinactivation of photosynthetic activities in Chromatium are discussed in relation to the possible role of photorespiration as a means of producing CO₂ in the photosynthetic system.

     

Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.

An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13. The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive. Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10. However, H-296, which contained both H-271 and CS1, was almost inactive with BHK. CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, which was produced by the deletion of H-271 from CH-296. Thus, the cell-binding domain was active with both kinds of cells. The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1. CS1 was specific for the adhesion of B16-F10 but was not essential.     

Engineering of artificial cell adhesion proteins by grafting the Arg-Gly-Asp cell adhesive signal to a calpastatin segment.

A new artificial cell adhesive protein was engineered by grafting the Arg-Gly-Asp (RGD) sequence, the minimal recognition signal of fibronectin for interaction with integrins, to a calpastatin segment by in vitro mutagenesis. The mutagenized protein showed cell adhesive activity in addition to calpain inhibitory activity. The RGD signal grafted to the calpastatin segment was recognized by the vitronectin receptor but not by the fibronectin receptor.     

Development of Enzymes Involved in Photosynthetic Carbon Assimilation in Greening Seedlings of Maize (Zea mays)

Upon illumination of dark-grown maize seedlings (5 days old) with incandescent light, there occurred a nearly simultaneous increase, after a certain lag period, in the activities of enzymes engaged in the C₄ pathway and the Calvin-Benson cycle. The light-induced biosynthesis of chlorophyll (a and b) precedes the increase in enzyme activities and proceeds without lag phase. A diphasic feature in the elevation of enzyme activities as a function of the intensities of light provided was observed; the increase in enzyme activities was enhanced by light intensities greater than 10³ ergs per square centimeter per second in comparison with light of lower intensities. Under light intensities greater than 10³ ergs per square centimeter per second, the simultaneous addition of levulinic acid, which inhibited chlorophyll formation, markedly reduced the increase of enzyme activities. However, neither the diphasic light effect nor the inhibitory effect of levulinic acid was observed with ribulose-1,5-bisphosphate carboxylase. The enzyme activities in the dark-grown maize seedlings were enhanced by a brief irradiation with the red light and the red light effect was reversed by the following far red light treatment. The red light-induced increase in the enzyme activities did not accompany chlorophyll synthesis, and was completely inhibited by cycloheximide, indicating that enzyme synthesis rather than activation might be involved. Light may play a dual role in enzyme induction; one is as an energy source through the photosystems at high intensities and the other is presumably as a signal mediated by phytochrome at low intensities.     

Diacylglycerol kinase η colocalizes and interacts with apoptosis signal-regulating kinase 3 in response to osmotic shock

Diacylglycerol kinase (DGK) η translocates from the cytoplasm to punctate vehicles via osmotic shock. Apoptosis signal-regulating kinase (ASK) 3 (MAP kinase kinase kinase (MAPKKK) 15) is also reported to respond to osmotic shock. Therefore, in the present study, we examined the subcellular localization of DGKη and ASK3 expressed in COS-7 cells under osmotic stress. We found that DGKη was almost completely colocalized with ASK3 in punctate structures in response to osmotic shock. In contrast, DGKδ, which is closely related to DGKη structurally, was not colocalized with ASK3, and DGKη failed to colocalize with another MAPKKK, C-Raf, even under osmotic stress. The structures in which DGKη and ASK3 localized were not stained with stress granule makers. Notably, DGKη strongly interacted with ASK3 in an osmotic shock-dependent manner. These results indicate that DGKη and ASK3 undergo osmotic shock-dependent colocalization and associate with each other in specialized structures.     

Fibroblasts-dependent invasion of podoplanin-positive cancer stem cells in squamous cell carcinoma

To clear whether podoplanin-positive cancer stem cells in squamous cell carcinoma have higher invasion activity during a fibroblasts-dependent invasion. A collagen gel invasion assay was performed using fluorescent ubiquitination-based cell cycle indicator-labeled A431 cells. The total number and number of invading cells in S/G2/M phase were counted using time-lapse imaging cocultured with fibroblasts. There was no significant difference between the number of invading podoplanin-positive and negative A431 cells when fibroblasts did not exist. On the contrary, the number of invading podoplanin-positive cells was significantly higher when fibroblasts existed. The frequency of cells in S/G2/M phase among invasion was no difference. Knockdown of podoplanin decreased the number of invaded A431 cells significantly when fibroblasts existed. Podoplanin-positive A431 cells display higher invasion activity when fibroblasts exist, suggesting that some biological functions of cancer stem cells might become evident only within the fibrous tumor microenvironment.     

Molecular cloning and biochemical characterization of two novel Neu3 sialidases, neu3a and neu3b, from medaka (Oryzias latipes)

Mammalian Neu3 sialidases are involved in various biological processes, such as cell death and differentiation, through desialylation of gangliosides. The enzymatic profile of Neu3 seems to be highly conserved from birds to mammals. In fish, the functional properties of Neu3 sialidase are not clearly understood, with the partial exception of the zebrafish form. To cast further light on the molecular evolution of Neu3 sialidase, we identified the encoding genes in the medaka Oryzias latipes and investigated the properties of the enzyme. PCR amplification using medaka brain cDNA allowed identification of two novel medaka Neu3 genes, neu3a and neu3b. The YRIP, VGPG motif and Asp-Box, characteristic of consensus motifs of sialidases, were well conserved in the both medaka Neu3 sialidases. When each gene was transfected into HEK293 to allow cell lysates for the use of enzymatic characterization, two Neu3 sialidases showed strict substrate specificity toward gangliosides, similar to mammalian Neu3. The optimal pH values were at pH 4.2 and pH 4.0, respectively, and neu3b in particular showed a broad optimum. Immunofluorescence assays indicated neu3a localization at plasma membranes, while neu3b was found in cytosol. The tissue distribution of two genes was then investigated by estimation of mRNA expression and sialidase activity, both being dominantly expressed in the brain. In neu3a gene-transfected neuroblastoma cells, the enzyme was found to positively regulate retinoic acid-induced differentiation with the elongation of axon length. On the other hand, neu3b did not affect neurite formation. These results and phylogenetic analysis suggested that the medaka neu3a is an evolutionally conserved sialidase with regard to enzymatic properties, whereas neu3b is likely to have originally evolved in medaka.     

Enzymatic Formation of β-Citraurin from β-Cryptoxanthin and Zeaxanthin by Carotenoid Cleavage Dioxygenase4 in the Flavedo of Citrus Fruit

In this study, the pathway of β-citraurin biosynthesis, carotenoid contents and the expression of genes related to carotenoid metabolism were investigated in two varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates β-citraurin predominantly, and Miyagawa-wase, which does not accumulate β-citraurin. The results suggested that CitCCD4 (for Carotenoid Cleavage Dioxygenase4) was a key gene contributing to the biosynthesis of β-citraurin. In the flavedo of Yamashitabeni-wase, the expression of CitCCD4 increased rapidly from September, which was consistent with the accumulation of β-citraurin. In the flavedo of Miyagawa-wase, the expression of CitCCD4 remained at an extremely low level during the ripening process, which was consistent with the absence of β-citraurin. Functional analysis showed that the CitCCD4 enzyme exhibited substrate specificity. It cleaved β-cryptoxanthin and zeaxanthin at the 7,8 or 7′,8′ position. But other carotenoids tested in this study (lycopene, α-carotene, β-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin) were not cleaved by the CitCCD4 enzyme. The cleavage of β-cryptoxanthin and zeaxanthin by CitCCD4 led to the formation of β-citraurin. Additionally, with ethylene and red light-emitting diode light treatments, the gene expression of CitCCD4 was up-regulated in the flavedo of Yamashitabeni-wase. These increases in the expression of CitCCD4 were consistent with the accumulation of β-citraurin in the two treatments. These results might provide new strategies to improve the carotenoid contents and compositions of citrus fruits.Carotenoids, a diverse group of pigments widely distributed in nature, fulfill a variety of important functions in plants and play a critical role in human nutrition and health (Schwartz et al., 1997; Cunningham and Gantt, 1998; Havaux, 1998; Krinsky et al., 2003; Ledford and Niyogi, 2005). The pathway of carotenoid biosynthesis has been well documented in various plant species, including Arabidopsis (Arabidopsis thaliana; Park et al., 2002), tomato (Lycopersicon esculentum; Isaacson et al., 2002), pepper (Capsicum annuum; Bouvier et al., 1998), citrus (Citrus spp.; Kato et al., 2004, 2006; Rodrigo et al., 2004; Rodrigo and Zacarías, 2007; Kato, 2012; Zhang et al., 2012a), and apricot (Prunus armenaica; Kita et al., 2007). Genes encoding the enzymes in the carotenoid biosynthetic pathway have been cloned, and their expression profiles have also been characterized (Fig. 1). As carotenoids contain a series of conjugated double bonds in the central chain, they can be oxidatively cleaved in a site-specific manner (Mein et al., 2011). The oxidative cleavage of carotenoids not only regulates their accumulation but also produces a range of apocarotenoids (Walter et al., 2010). In higher plants, many different apocarotenoids derive from the cleavage of carotenoids and have important metabolic functions, such as plant hormones, pigments, aroma and scent compounds, as well as signaling compounds (Fig. 1). A well-known example is abscisic acid, which is a C₁₅ compound derived from the cleavage of the 11,12 double bond of 9-cis-violaxanthin and 9′-cis-neoxanthin (Schwartz et al., 1997; Tan et al., 1997; Cutler and Krochko, 1999; Chernys and Zeevaart, 2000; Giuliano et al., 2003).Open in a separate windowFigure 1.Carotenoid and apocarotenoid metabolic pathway in plants. GGPP, Geranylgeranyl diphosphate. Enzymes, listed here from top to bottom, are named according to the designation of their genes: PSY, phytoene synthase; PDS, Phytoene desaturase; ZDS, ζ-carotene desaturase; ZISO, 15-cis-ζ-carotene isomerase; CRTISO, carotenoid isomerase; LCYb, lycopene β-cyclase; LCYe, lycopene ε-cyclase; HYe, ε-ring hydroxylase; HYb, β-ring hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin deepoxidase; NCED, 9-cis-epoxycarotenoid dioxygenase.Carotenoid cleavage dioxygenases (CCDs) are a group of enzymes that catalyze the oxidative cleavage of carotenoids (Ryle and Hausinger, 2002). CCDs are nonheme iron enzymes present in plants, bacteria, and animals. In plants, CCDs belong to an ancient and highly heterogenous family (CCD1, CCD4, CCD7, CCD8, and 9-cis-epoxycarotenoid dioxygenases [NCEDs]). The similarity among the different members is very low apart from four strictly conserved His residues and a few Glu residues (Kloer and Schulz, 2006; Walter et al., 2010). In Arabidopsis, the CCD family contains nine members (CCD1, NCED2, NCED3, CCD4, NCED5, NCED6, CCD7, CCD8, and NCED9), and orthologs in other plant species are typically named according to their homology with an Arabidopsis CCD (Huang et al., 2009). In our previous study, the functions of CitCCD1, CitNCED2, and CitNCED3 were investigated in citrus fruits (Kato et al., 2006). The recombinant CitCCD1 protein cleaved β-cryptoxanthin, zeaxanthin, and all-trans-violaxanthin at the 9,10 and 9′,10′ positions and 9-cis-violaxanthin at the 9′,10′ position. The recombinant CitNCED2 and CitNCED3 proteins cleaved 9-cis-violaxanthin at the 11,12 position to form xanthoxin, a precursor of abscisic acid (Kato et al., 2006). To date, information on the functions of other CCDs in citrus fruits remains limited, while the functions of CCD7 and CCD8, as well as NCED5, NCED6, and NCED9, in Arabidopsis have been characterized (Kloer and Schulz, 2006; Walter et al., 2010). In Arabidopsis, CCD7 cleaves all-trans-β-carotene at the 9′,10′ position to form all-trans-β-apo-10′-carotenal. All-trans-β-apo-10′-carotenal is further shortened by AtCCD8 at the 13,14 position to produce β-apo-13-carotenone (Alder et al., 2012). NCED5, NCED6, and NCED9 cleave 9-cis-violaxanthin at the 11,12 position to form xanthoxin (Tan et al., 2003). Compared with other CCDs, the function of CCD4 is poorly understood. In Chrysanthemum morifolium, CmCCD4a contributed to the white color formation by cleaving carotenoids into colorless compounds (Ohmiya et al., 2006). Recently, it has been reported that CsCCD4, CmCCD4a, and MdCCD4 could cleave β-carotene to yield β-ionone (Rubio et al., 2008; Huang et al., 2009).β-Citraurin, a C₃₀ apocarotenoid, is a color-imparting pigment responsible for the reddish color of citrus fruits (Farin et al., 1983). In 1936, it was first discovered in Sicilian oranges (Cual, 1965). In citrus fruits, the accumulation of β-citraurin is not a common event; it is only observed in the flavedos of some varieties during fruit ripening. The citrus varieties accumulating β-citraurin are considered more attractive because of their red-orange color (Ríos et al., 2010). Although more than 70 years have passed since β-citraurin was first identified, the pathway of its biosynthesis is still unknown. As its structure is similar to that of β-cryptoxanthin and zeaxanthin, β-citraurin was presumed to be a degradation product of β-cryptoxanthin or zeaxanthin (Oberholster et al., 2001; Rodrigo et al., 2004; Ríos et al., 2010; Fig. 1). To date, however, the specific cleavage reaction producing β-citraurin has not been elucidated. In this study, we found that the CitCCD4 gene was involved in the synthesis of β-citraurin, using two citrus varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates β-citraurin predominantly, and Miyagawa-wase, which does not accumulate β-citraurin. To confirm the role of the CitCCD4 gene further, functional analyses of the CitCCD4 enzyme were performed in vivo and in vitro. Additionally, the regulation of β-citraurin content and CitCCD4 gene expression in response to ethylene and red light-emitting diode (LED) light treatments was also examined. This study, to our knowledge, is the first to investigate the biosynthesis of β-citraurin in citrus fruits. The results might provide new strategies to enhance the nutritional and commercial qualities of citrus fruits.     

Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains

A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.     

Chemical screening of an inhibitor for gibberellin receptors based on a yeast two-hybrid system

We applied a yeast two-hybrid (Y2H) system to the high-throughput monitoring of two proteins’ interaction, a receptor for phytohormone gibberellin (GA) and its direct signal transducer DELLA. With this system, we screened inhibitors to the interaction. As a result, we discovered a chemical, 3-(2-thienylsulfonyl)pyrazine-2-carbonitrile (TSPC), and we confirmed that TSPC is an inhibitor for GA perception by in vitro and in planta evaluations.