<Emphasis Type="Italic">cis</Emphasis>-Cinnamoyl Glucoside as a Major Plant Growth Inhibitor Contained in <Emphasis Type="Italic">Spiraea prunifolia</Emphasis>

Crude extracts of the leaves of Spiraea prunifolia Sieb. showed high plant-growth-inhibiting activity comparable to that of S. thunbergii extracts. To isolate the causal compound in S. prunifolia, we performed bioassay-directed purification by monitoring the biological activity per unit weight of the organism containing the bioactive compound (total activity). We isolated 1-O-cis-cinnamoyl-β-D-glucopyranose (cis-CG) and identified it as the most important growth-inhibiting constituent in the crude extracts. We did not detect 6-O-(4′-hydroxy-2′-methylenebutyroyl)-1-O-cis-cinnamoyl-β-D-glucopyranose (cis-BCG) in S. prunifolia, though it is a major plant growth inhibitor in S. thunbergii together with cis-CG. We estimated the cis-CG content in S. prunifolia to be 3.84 mmol kg⁻¹ F.W. This amount is comparable to the cis-CG plus cis-BCG content in S. thunbergii (3.59 mmol kg⁻¹ F.W.). This indicates that S. prunifolia and S. thunbergii have equally high potential to inhibit plant growth, and cis-CG acts as the most important plant-growth inhibitor in S. prunifolia extracts.     

Quantitative analysis of cereulide, an emetic toxin of Bacillus cereus, by using rat liver mitochondria

An emetic toxin cereulide, produced by Bacillus cereus, causes emetic food poisonings, but a method for quantitative measurement of cereulide has not been well established. A current detection method is a bioassay method using the HEp-2 cell vacuolation test, but it was unable to measure an accurate concentration. We established a quantitative assay for cereulide based on its mitochondrial respiratory uncoupling activity. The oxygen consumption in a reaction medium containing rat liver mitochondria was rapid in the presence of cereulide. Thus uncoupling effect of cereulide on mitochondrial respiration was similar to those of uncouplers 2,4-dinitrophenol (DNP), carbonylcyanide m-chlorophenylhydrazone (CCCP), and valinomycin. This method gave constant results over a wide range of cereulide concentrations, ranging from 0.05 to 100 microg/ml. The minimum cereulide concentration to detect uncoupled oxygen consumption was 50 ng/ml and increased dose-dependently to the maximum level. Semi-log relationship between the oxygen consumption rate and the cereulide concentration enables this method to quantify cereulide. The results of this method were highly reproducible as compared with the HEp-2 cell vacuolation test and were in good agreement with those of the HEp-2 cell vacuolation test. The enterotoxin of B. cereus or Staphylococcus aureus did not show any effect on the oxygen consumption, indicating this method is specific for the identification of cereulide as a causative agent of emetic food poisonings.     

Involvement of the Toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus

We have previously shown that mice inoculated intranasally with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus [AcNPV]) are protected from a lethal challenge by influenza virus. However, the precise mechanism of induction of this protective immune response by the AcNPV treatment remained unclear. Here we show that AcNPV activates immune cells via the Toll-like receptor 9 (TLR9)/MyD88-dependent signaling pathway. The production of inflammatory cytokines was severely reduced in peritoneal macrophages (PECs) and splenic CD11c(+) dendritic cells (DCs) derived from mice deficient in MyD88 or TLR9 after cultivation with AcNPV. In contrast, a significant amount of alpha interferon (IFN-alpha) was still detectable in the PECs and DCs of these mice after stimulation with AcNPV, suggesting that a TLR9/MyD88-independent signaling pathway might also participate in the production of IFN-alpha by AcNPV. Since previous work showed that TLR9 ligands include bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides, we also examined the effect of baculoviral DNA on the induction of innate immunity. Transfection of the murine macrophage cell line RAW264.7 with baculoviral DNA resulted in the production of the inflammatory cytokine, while the removal of envelope glycoproteins from viral particles, UV irradiation of the virus, and pretreatment with purified baculovirus envelope proteins or endosomal maturation inhibitors diminished the induction of the immune response by AcNPV. Together, these results indicate that the internalization of viral DNA via membrane fusion mediated by the viral envelope glycoprotein, as well as endosomal maturation, which releases the viral genome into TLR9-expressing cellular compartments, is necessary for the induction of the innate immune response by AcNPV.     

Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production in the transgenic Arabidopsis thaliana by the in vitro evolved highly active mutants of polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae

In this study, the enhancement of photosynthetic PHA production was achieved using the highly active mutants of PHA synthase created by the in vitro evolutionally techniques. The wild-type and mutated PHA synthase genes from Aeromonas caviae were introduced into Arabidopsis thaliana together with the NADPH-dependent acetoacetyl-CoA reductase gene from Ralstonia eutropha. Expression of the highly active mutated PHA synthase genes, N149S and D171G, led to an 8-10-fold increase in PHA content in the T1 transgenic Arabidopsis, compared to plants harboring the wild-type PHA synthase gene. In homozygous T2 progenies, PHA content was further increased up to 6.1 mg/g cell dry weight. GC/MS analysis of the purified PHA from the transformants revealed that these PHAs were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymers consisting of 0.2-0.8 mol % 3HV. The monomer composition of the P(3HB-co-3HV) copolymers synthesized by the wild-type and mutated PHA synthases reflected the substrate specificities observed in Escherichia coli. These results indicate that in vitro evolved PHA synthases can enhance the productivity of PHA and regulate the monomer composition in transgenic plants.     

Time-resolved X-ray diffraction study on poly[(R)-3-hydroxybutyrate] films during two-step-drawing: generation mechanism of planar zigzag structure

Uniaxially oriented films with high tensile strength were processed from ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] (P(3HB)) by a method combining hot-drawing near the melting point of the polymer and two-step-drawing at room temperature. In a two-step-drawn and subsequently annealed film, P(3HB) molecular chains fall into two states: 2/1 helix (alpha-form) and planar zigzag (beta-form) conformations. The mechanism for generating the beta-form during two-step-drawing was investigated by time-resolved synchrotron wide- and small-angle X-ray scattering measurements (WAXD and SAXS), together with the measurement of stress-strain curves. It was found that the improvement of mechanical properties is due to not only the orientation of molecular chains but also the generation of the beta-form during the drawing. The crystal and molecular structures of the alpha-form remained unchanged until the yield point of the stress-strain curve. At the yield point, the long period obtained from SAXS doubled and a new reflection indicative of the beta-form was observed on the equatorial line in WAXD. The intensity of the reflection from the beta-form increased with an increase in the two-step-drawing ratio at room temperature. The SAXS pattern changed from a two-point reflection along the meridian to a cross pattern with streaking on the equatorial line, demonstrating the close alignment of shish-kebab structures. The reflection intensity, crystal orientation and crystal size of the alpha-form decreased during two-step-drawing. Based on these results, the beta-form is mainly introduced from the orientation of free molecular chains in the amorphous regions between alpha-form lamellar crystals, but the structural transformation of molecular chains also occurs from the alpha-form to the beta-form at the deformed lamellar crystals.     

Physical and functional interactions of the lysophosphatidic acid receptors with PDZ domain-containing Rho guanine nucleotide exchange factors (RhoGEFs)

Lysophosphatidic acid (LPA) is a serum-derived phospholipid that induces a variety of biological responses in various cells via heterotrimeric G protein-coupled receptors (GPCRs) including LPA1, LPA2, and LPA3. LPA-induced cytoskeletal changes are mediated by Rho family small GTPases, such as RhoA, Rac1, and Cdc42. One of these small GTPases, RhoA, may be activated via Galpha(12/13)-linked Rho-specific guanine nucleotide exchange factors (RhoGEFs) under LPA stimulation although the detailed mechanisms are poorly understood. Here, we show that the C terminus of LPA1 and LPA2 but not LPA3 interact with the PDZ domains of PDZ domain-containing RhoGEFs, PDZ-RhoGEF, and LARG, which are comprised of PDZ, RGS, Dbl homology (DH), and pleckstrin homology (PH) domains. In LPA1- and LPA2-transfected HEK293 cells, LPA-induced RhoA activation was observed although the C terminus of LPA1 and LPA2 mutants, which failed to interact with the PDZ domains, did not cause LPA-induced RhoA activation. Furthermore, overexpression of the PDZ domains of PDZ domain-containing RhoGEFs served as dominant negative mutants for LPA-induced RhoA activation. Taken together, these results indicate that formation of the LPA receptor/PDZ domain-containing RhoGEF complex plays a pivotal role in LPA-induced RhoA activation.     

Low density lipoprotein receptor-related protein 1 (LRP1) controls endocytosis and c-CBL-mediated ubiquitination of the platelet-derived growth factor receptor beta (PDGFR beta)

The low density lipoprotein receptor-related protein 1 (LRP1) has been implicated in intracellular signaling functions as well as in lipid metabolism. Recent in vivo and in vitro studies suggest that LRP1 is a physiological modulator of the platelet-derived growth factor (PDGF) signaling pathway. Here we show that in mouse fibroblasts LRP1 modulates PDGF-BB signaling by controlling endocytosis and ligand-induced down-regulation of the PDGF receptor beta (PDGFRbeta). In LRP1-deficient fibroblasts, basal PDGFRbeta tyrosine kinase activity was derepressed, and PDGF-BB-induced endocytosis and degradation of PDGFRbeta were accelerated as compared with control cells. This was accompanied by rapid uptake of receptor-bound PDGF-BB into the cells and by attenuated ERK activation in response to PDGF-BB stimulation. Pulse-chase analysis indicated that the steady-state turnover rate of PDGFRbeta was also accelerated in LRP-deficient fibroblasts. The rapid degradation of PDGFRbeta in the LRP1-deficient fibroblasts was prevented by MG132 and chloroquine. Furthermore, the association of PDGFRbeta with c-Cbl, a ubiquitin E3-ligase, as well as the ligand-induced ubiquitination of PDGFRbeta were increased in LRP1-deficient fibroblasts. We show that LRP1 can directly interact with c-Cbl, suggesting a Sprouty-like role for LRP1 in regulating the access of the PDGFRbeta to the ubiquitination machinery. Thus, LRP1 modulates PDGF signaling by controlling ubiquitination and endocytosis of the PDGFRbeta.     

Anatomical and Behavioral Differences Among Threespine Sticklebacks: the Marine Form,the Landlocked Form and Their Hybrids

In order to elucidate the mechanisms and degree of isolation in the landlocked form of the threespine stickleback, Gasterosteus aculeatus (L.), inhabiting the Shinano and Agano Rivers that flow into the Japan Sea, comparative studies on the morphology, behavior and histology were made of the marine form, the landlocked form and their hybrids reared in the laboratory. The body shape, coloration and behavior of reciprocal hybrids were almost intermediate between the two parent forms. From January to February, the testis of the hybrid between the female marine form and the male landlocked form is occupied exclusively by spermatocytes and some spermatids. However, a small number of large cells, identified as macrophages, are found invading the seminiferous tubules, indicating the occurrence of phagocytosis. On the other hand, ripe functional eggs appear in the ovary in May, i.e. the breeding season. The eggs deposited into the nest do not develop even when the hybrids display the same mating behavior as their parents. Functional sperms are found in the testis of the hybrid between female landlocked and male marine forms at the period of breeding. However, only a single female lived until March. In the ovary of this female, all eggs reaching vitellogenesis (= primary yolk globule stage) are collapsed, while the younger oocytes appear in healthy condition. The hybrid female of this pairing seemed also to be sterile.     

Effects of liver failure on the enzymes in the branched-chain amino acid catabolic pathway

Branched-chain alpha-keto acid dehydrogenase (BCKDH) complex catalyzes the committed step of the catabolism of branched-chain amino acids (BCAA). The liver cirrhosis chemically induced in rats raised the activity of hepatic BCKDH complex and decreased plasma BCAA and branched-chain alpha-keto acid concentrations, suggesting that the BCAA requirement is increased in liver cirrhosis. Since the effects of liver cirrhosis on the BCKDH complex in human liver are different from those in rat liver, further studies are needed to clarify the differences between rats and humans. In the valine catabolic pathway, crotonase and beta-hydroxyisobutyryl-CoA hydrolase are very important to regulate the toxic concentration of mitochondrial methacrylyl-CoA, which occurs in the middle part of valine pathway and highly reacts with free thiol compounds. Both enzyme activities in human and rat livers are very high compared to that of BCKDH complex. It has been found that both enzyme activities in human livers were significantly reduced by liver cirrhosis and hepatocellular carcinoma, suggesting a decrease in the capability to dispose methacrylyl-CoA. The findings described here suggest that alterations in hepatic enzyme activities in the BCAA catabolism are associated with liver failure.