Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.     

Involvement of acetaldehyde in seed deterioration of some recalcitrant woody species through the acceleration of aerobic respiration

The rate of acetaldehyde (Ald) evolution in the deterioration of recalcitrant woody seeds was investigated. Four plant species, Ligustrum japonicum, Quercus serrata, Quercus myrsinaefolia and Camellia japonica, were used for the experiments. Similar to orthodox seeds, all of the recalcitrant seeds used contained Ald in addition to methanol and ethanol, although the amount of Ald in Camellia, a typical oil seed, was very small. These volatiles were accumulated in a container in which Ligustrum and Q. serrata seeds were stored for a short period. Moreover, all of the seeds that had been previously exposed to Ald for only 6 d at 3 or 13 degrees C lost their vigor rapidly in proportion to the concentration of Ald. The occasional removal by decompression of Ald accumulated in the container prolonged the life span of Q. serrata seeds from 4 to 6 months. These findings suggest that a short life span of the hydrated recalcitrant seeds may involve Ald synthesis as in the orthodox seeds. However, the action mechanism of Ald in Ligustrum and Quercus seeds in which storage substances were polysaccharides seems to differ slightly from that in orthodox seeds, because their aerobic respiration was significantly stimulated by exposure to exogenously applied Ald. It was, therefore, thought that the rapid deterioration of some recalcitrant seeds in woody species may result from a decline in vigor, not only due to the denaturation of functional proteins by Ald as in the orthodox seeds but also due to the rapid consumption of direct substrates for the Ald-stimulated aerobic respiration and related co-enzymes within seeds. In contrast, in the oil-bearing Camellia seeds, Ald was slightly produced and their aerobic respiration was not enhanced by Ald, although they were very sensitive to Ald. Desiccation storage of Camellia seeds caused the deterioration of their outer part, which was accelerated by exogenously applied Ald, which suggests that in Camellia Ald acts only to denature the functional proteins as in orthodox seeds. Thus, the short longevity of these woody recalcitrant seeds is discussed in relation to the actions of Ald produced endogenously.     

Characterization of a xyloglucan endotransglucosylase gene that is up-regulated by gibberellin in rice

Xyloglucan endotransglucosylases/hydrolases (XTHs) that mediate cleavage and rejoining of the beta (1-4)-xyloglucans of the primary cell wall are considered to play an important role in the construction and restructuring of xyloglucan cross-links. A novel rice (Oryza sativa) XTH-related gene, OsXTH8, was cloned and characterized after being identified by cDNA microarray analysis of gibberellin-induced changes in gene expression in rice seedlings. OsXTH8 was a single copy gene; its full-length cDNA was 1,298 bp encoding a predicted protein of 290 amino acids. Phylogenetic analysis revealed that OsXTH8 falls outside of the three established subfamilies of XTH-related genes. OsXTH8 was preferentially expressed in rice leaf sheath in response to gibberellic acid. In situ hybridization and OsXTH8 promoter GUS fusion analysis revealed that OsXTH8 was highly expressed in vascular bundles of leaf sheath and young nodal roots where the cells are actively undergoing elongation and differentiation. OsXTH8 gene expression was up-regulated by gibberellic acid and there was very little effect of other hormones. In two genetic mutants of rice with abnormal height, the expression of OsXTH8 positively correlated with the height of the mutants. Transgenic rice expressing an RNAi construct of OsXTH8 exhibited repressed growth. These results indicate that OsXTH8 is differentially expressed in rice leaf sheath in relation to gibberellin and potentially involved in cell elongation processes.     

An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer's disease

Various types of stress, such as disruption of calcium homeostasis, inhibition of protein glycosylation and reduction of disulfide bonds, result in accumulation of misfolded proteins in the endoplasmic reticulum (ER). The initial cellular response involves removal of such proteins by the ER, but excessive and/or long-term stress results in apoptosis. In this study, we used a randomized ribozyme library and ER stress-mediated apoptosis (tunicamycin-induced apoptosis) in SK-N-SH human neuroblastoma cells as a selective phenotype to identify factors involved in this process. We identified a double-stranded RNA-dependent protein kinase (PKR) as one of the participants in this process. The level of nuclear PKR was elevated, but the level of cytoplasmic PKR barely changed in tunicamycin-treated SK-N-SH cells. Furthermore, tunicamycin also raised levels of phosphorylated PKR in the nucleus. We also detected the accumulation of phosphorylated PKR in the nuclei of autopsied brain tissues in Alzheimer's disease. Thus, PKR might play a role in ER stress-induced apoptosis and in Alzheimer's disease.     

Regrowth of the stalk of the sea lily, Metacrinus rotundus (Echinodermata: Crinoidea)

Sea lilies are critical to understanding the evolution of the echinoderm body plan, because they are the only extant group whose adults possess a stalk, a prevalent feature in the radiation of a number of primitive echinoderm lineages. Extensive crown regeneration ability has been reported in Metacrinus rotundus, but the regenerative potential of the stalk has never been determined in any species of sea lilies. In this study, we show that M. rotundus whose stalks have been completely excised are capable of stalk regeneration. The process is similar to the growth of the original stalk, but much slower, and the regenerated stalks are not morphologically identical to the original stalk. Since stalk regeneration, in contrast to well-studied regeneration events, probably requires little additional activation of morphogenetic programs, we refer to the stalk regeneration phenomenon as "stalk regrowth" to distinguish it as a special form of regeneration. Since specimens whose entire stalk below the basal plates had been removed were able to regrow, the basal plates, and probably the aboral nerve center within them, are essential for stalk regrowth. Sea lily stalk regrowth is described in detail, and the evolution of feather stars is discussed in light of the growth pattern of the sea lily stalk.     

Infrequent cavity-forming fluctuations in HPr from Staphylococcus carnosus revealed by pressure- and temperature-dependent tyrosine ring flips

Infrequent structural fluctuations of a globular protein is seldom detected and studied in detail. One tyrosine ring of HPr from Staphylococcus carnosus, an 88-residue phosphocarrier protein with no disulfide bonds, undergoes a very slow ring flip, the pressure and temperature dependence of which is studied in detail using the on-line cell high-pressure nuclear magnetic resonance technique in the pressure range from 3 MPa to 200 MPa and in the temperature range from 257 K to 313 K. The ring of Tyr6 is buried sandwiched between a beta-sheet and alpha-helices (the water-accessible area is less than 0.26 nm2), its hydroxyl proton being involved in an internal hydrogen bond. The ring flip rates 10(1)-10(5) s(-1) were determined from the line shape analysis of H(delta1, delta2) and H(epsilon1,epsilon2) of Tyr6, giving an activation volume DeltaV++ of 0.044 +/- 0.008 nm3 (27 mL mol(-1)), an activation enthalpy DeltaH++ of 89 +/- 10 kJ mol(-1), and an activation entropy DeltaS++ of 16 +/- 2 JK(-1) mol(-1). The DeltaV++) and DeltaH++ values for HPr found previously for Tyr and Phe ring flips of BPTI and cytochrome c fall within the range of DeltaV(double dagger) of 28 to 51 mL mol(-1) and DeltaH++ of 71 to 155 kJ mol(-1). The fairly common DeltaV++ and DeltaH++ values are considered to represent the extra space or cavity required for the ring flip and the extra energy required to create a cavity, respectively, in the core part of a globular protein. Nearly complete cold denaturation was found to take place at 200 MPa and 257 K independently from the ring reorientation process.     

Synthesis and biological activity of novel alpha-substituted beta-phenylpropionic acids having pyridin-2-ylphenyl moiety as antihyperglycemic agents

We previously reported the identification of novel oximes having 5-benzyl-2,4-thiazolidinedione with antihyperglycemic activity. We now report the synthesis and biological activity of a novel series of oximes and amides having alpha-substituted-beta-phenylpropionic acids. In this series, we obtained potent PPAR alpha/gamma dual agonist (S)-9d, with which activation of PPAR alpha and PPAR gamma was considerably more potent than that of the reference compounds GW9578 22 and rosiglitazone 3, respectively. This means (S)-9d is of the strongest class of PPAR alpha/gamma dual agonists. In the course of this study, we also obtained 8h, which indicated potent plasma glucose lowering effect in spite of weak PPAR alpha/gamma agonistic activity.     

Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

Image shift due to beam-induced specimen charging has become the most severe problem in electron microscopy for imaging two-dimensional (2D) crystals of biological macromolecules, especially in the case of highly tilted specimens. Image shift causes diffraction spots perpendicular to the tilt axis to disappear even at medium or low resolution. The yield of good images from tilted specimens prepared on a single layer of continuous carbon support film is therefore very low. In this paper, we have used 2D crystals of aquaporin-4 to investigate the effect of a carbon sandwich preparation method on specimen charging. We find that a larger number of images show sharp diffraction spots perpendicular to the tilt axis if crystals are placed in between two sheets of carbon film as compared to images taken from specimens prepared by the conventional single carbon support film technique. Our results demonstrate that the reproducible carbon sandwich preparation technique overcomes the severe specimen charging problem and thus has the potential to significantly speed up structure analysis by electron crystallography.