β-Glucosidase activities and HCN liberation in unimbibed and imbibed seeds, and the induction of cocklebur seed germination by cyanogenic glycosides

In many seed species, the major source of HCN evolved during water imbibition is cyanogenic glycosides. The present investigation was performed to elucidate the role of endogenous cyanogenic glycosides in the control of seed germination and to examine the involvment of β-glucosidase in this process. All seed species used here contained some activities of β-glucosidase already in the dry state before imbibition. in the decreasing order of Malus pumila, Daucus carota, Hordeum vulgare, Chenopodium album and so on. β-Gluosidase activity in upper and lower seeds of cocklebur (Xanthium pennsylvanicum Wallr.) decreased with imbibition, and in lower seeds the activity disappeared when they germinated. On the contrary, in caryopses of rice (Oryza sativa L. cv. Sasanishiki) β-glucosidase increased during imbibition, and this increase continued even after germination. β-Glucosidase in cocklebur seeds was more active in the axial than in the cotyledonary tissue. Amygdalin, prunasin and linamarin could all serve as substrattes for the β-glucosidase(s) from both cocklebur and rice. Amygdalin, prunasin and linamarin as well as KCN, were effective in stimulating the germination of upper cocklebur seeds. The seeds evolved much more free HCN gas when they were exposed to the cyanogenic glycosides than when the glycosides were absent. Moreover, the application of the cyanogenic glycosides or of KCN caused accumulation of bound HCN in the seeds. Carbon monoxide, which stimulated cocklebur seed germination only slightly, did not cause accumulation of bound HCN. We suggest that a balance between the cytochrome and the alternative respiration pathways, which is adequate for germination (Esashi et al. 1987. Plant Cell Physiol. 28: 141–150), may be brought about by the action of endogenous HCN; a large portion of which is liberated from cyanogenic glycosides via the action of β-glucosidase. In addition to the partial suppression of the cytochrome path and unlike carbon monoxide, the HCN thus produced may act to supply cyanide group(s) to unknown compounds necessary for germination.     

Marine Hydroquinone Zonarol Prevents Inflammation and Apoptosis in Dextran Sulfate Sodium-Induced Mice Ulcerative Colitis

Background and Aim

We previously identified an anti-inflammatory compound, zonarol, a hydroquinone isolated from the brown algae Dictyopteris undulata as a marine natural product. To ascertain the in vivo functions of zonarol, we examined the pharmacological effects of zonarol administration on dextran sulfate sodium (DSS)-induced inflammation in a mouse model of ulcerative colitis (UC). Our goal is to establish a safe and effective cure for inflammatory bowel disease (IBD) using zonarol.

Methods and Results

We subjected Slc:ICR mice to the administration of 2% DSS in drinking water for 14 days. At the same time, 5-aminosalicylic acid (5-ASA) at a dose of 50 mg/kg (positive control) and zonarol at doses of 10 and 20 mg/kg, were given orally once a day. DSS-treated animals developed symptoms similar to those of human UC, such as severe bloody diarrhea, which were evaluated by the disease activity index (DAI). Treatment with 20 mg/kg of zonarol, as well as 5-ASA, significantly suppressed the DAI score, and also led to a reduced colonic ulcer length and/or mucosal inflammatory infiltration by various immune cells, especially macrophages. Zonarol treatment significantly reduced the expression of pro-inflammatory signaling molecules, and prevented the apoptosis of intestinal epithelial cells. Finally, zonarol protected against in vitro lipopolysaccharide (LPS)-induced activation in the RAW264.7 mouse macrophage cell line.

Conclusions

This is the first report that a marine bioproduct protects against experimental UC via the inhibition of both inflammation and apoptosis, very similar to the standard-of-care sulfasalazine, a well-known prodrug that releases 5-ASA. We believe that the oral administration of zonarol might offer a better treatment for human IBDs than 5-ASA, or may be useful as an alternative/additive therapeutic strategy against UC, without any evidence of side effects.     

A Novel Peroxisome Proliferator-activated Receptor (PPAR)α Agonist and PPARγ Antagonist,Z-551, Ameliorates High-fat Diet-induced Obesity and Metabolic Disorders in Mice

A novel peroxisome proliferator-activated receptor (PPAR) modulator, Z-551, having both PPARα agonistic and PPARγ antagonistic activities, has been developed for the treatment of obesity and obesity-related metabolic disorders. We examined the effects of Z-551 on obesity and the metabolic disorders in wild-type mice on the high-fat diet (HFD). In mice on the HFD, Z-551 significantly suppressed body weight gain and ameliorated insulin resistance and abnormal glucose and lipid metabolisms. Z-551 inhibited visceral fat mass gain and adipocyte hypertrophy, and reduced molecules involved in fatty acid uptake and synthesis, macrophage infiltration, and inflammation in adipose tissue. Z-551 increased molecules involved in fatty acid combustion, while reduced molecules associated with gluconeogenesis in the liver. Furthermore, Z-551 significantly reduced fasting plasma levels of glucose, triglyceride, free fatty acid, insulin, and leptin. To elucidate the significance of the PPAR combination, we examined the effects of Z-551 in PPARα-deficient mice and those of a synthetic PPARγ antagonist in wild-type mice on the HFD. Both drugs showed similar, but weaker effects on body weight, insulin resistance and specific events provoked in adipose tissue compared with those of Z-551 as described above, except for lack of effects on fasting plasma triglyceride and free fatty acid levels. These findings suggest that Z-551 ameliorates HFD-induced obesity, insulin resistance, and impairment of glucose and lipid metabolisms by PPARα agonistic and PPARγ antagonistic activities, and therefore, might be clinically useful for preventing or treating obesity and obesity-related metabolic disorders such as insulin resistance, type 2 diabetes, and dyslipidemia.     

Solution Structures of Cytosolic RNA Sensor MDA5 and LGP2 C-terminal Domains: IDENTIFICATION OF THE RNA RECOGNITION LOOP IN RIG-I-LIKE RECEPTORS*

The RIG-I like receptor (RLR) comprises three homologues: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2). Each RLR senses different viral infections by recognizing replicating viral RNA in the cytoplasm. The RLR contains a conserved C-terminal domain (CTD), which is responsible for the binding specificity to the viral RNAs, including double-stranded RNA (dsRNA) and 5′-triphosphated single-stranded RNA (5′ppp-ssRNA). Here, the solution structures of the MDA5 and LGP2 CTD domains were solved by NMR and compared with those of RIG-I CTD. The CTD domains each have a similar fold and a similar basic surface but there is the distinct structural feature of a RNA binding loop; The LGP2 and RIG-I CTD domains have a large basic surface, one bank of which is formed by the RNA binding loop. MDA5 also has a large basic surface that is extensively flat due to open conformation of the RNA binding loop. The NMR chemical shift perturbation study showed that dsRNA and 5′ppp-ssRNA are bound to the basic surface of LGP2 CTD, whereas dsRNA is bound to the basic surface of MDA5 CTD but much more weakly, indicating that the conformation of the RNA binding loop is responsible for the sensitivity to dsRNA and 5′ppp-ssRNA. Mutation study of the basic surface and the RNA binding loop supports the conclusion from the structure studies. Thus, the CTD is responsible for the binding affinity to the viral RNAs.     

No association between complement factor H gene polymorphism and exudative age-related macular degeneration in Japanese

Age-related macular degeneration (ARMD) is the leading cause of blindness in the elderly population not only Western but also Asian industrial countries. In Caucasian, a polymorphism of the complement factor H gene (CFH), the C allele of rs1061170 (Y402H), was established as the first strong genetic factor for excursively exudative type of ARMD. In this study, we performed an extensive sequencing of the 22 exons in the CFH gene by recruiting 146 exudative ARMD patients and 105 normal controls of Japanese origin and identified 61 polymorphisms. We found that the frequency of the C allele of rs1061170 (Y402H) is much lower (0.04) in Japanese controls than in Caucasians (0.45). No case disease susceptibility to exudative ARMD was noted for rs1061170 (Y402H) (χ ² = 3.19, P _corr = 0.423), or other 12 single nucleotide polymorphisms (SNPs) whose frequency is greater than 0.05. When haplotypes were inferred for 13 SNPs (these 12 SNPs with a frequency greater than 0.05 and rs1061170), three haplotypes whose pattern was similar to those in Caucasians were identified but with substantial difference in frequency. Again we failed to identify genetic association between Japanese exudative ARMD and any of the haplotypes including the J1 haplotype which was shown to be susceptible to ARMD in Caucasians (χ ² = 3.92, P _corr = 0.157). CFH does not appear to be a primary hereditary contributor to ARMD in Japanese. The absence of CFH contribution to ARMD in Japanese may correlate with the findings in ethnic differences of ARMD phenotypes.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.This work was accomplished by equal contribution of two groups organized by the last two authors.     

Polyamines promote the development of embryonal-suspensor masses and the formation of somatic embryos in <Emphasis Type="Italic">Picea glehnii</Emphasis>

Effects of polyamines on the development of embryonal-suspensor masses (ESMs) and the formation of somatic embryos were examined in an effort to improve the frequency of somatic embryogenesis in Picea glehnii. Exogenous spermidine at 100 and 300 μM promoted the elongation of suspensors and the formation of somatic embryos. Exogenous putrescine at 100 μM increased the number of somatic embryos and decreased the time required for formation of somatic embryos. Thus, exogenous polyamines improved the maturation capacity of ESMs and promoted the formation of somatic embryos in P. glehnii. Light microscopy revealed that ESMs with high maturation capacity consisted of the three following cells: round or oval cells, elongated cells, and protean cells. These components might be related to the capacity for formation of somatic embryos.     

Transient gene expression system established in Porphyra yezoensis is widely applicable in Bangiophycean algae

The establishment of transient gene expression systems in the marine red macroalga Porphyra yezoensis has been useful for the molecular analysis of cellular processes in this species. However, there has been no successful report about the expression of foreign genes in other red macroalgae, which has impeded the broader understanding of the molecular biology of these species. We therefore examined whether the P. yezoensis transient gene expression system was applicable to other red macroalgae. The results indicated that a codon-optimized GUS, designated PyGUS, and plant-adapted sGFP(S65T) were successfully expressed under the control of the P. yezoensis PyAct1 promoter in gametophytic cells of six Porphyra species and also in Bangia fuscopurpurea, all of which are classified as Bangiophyceae. In contrast, there were no reporter-expressing cells in the Florideophycean algae examined. These results indicate the availability of PyGUS and sGFP as reporters and the 5' upstream region of the PyAct1 gene as a heterologous promoter for transient gene expression in Bangiophycean algae, which could provide a clue to the efficient expression of foreign genes and transformation in marine red macroalgae.     

Mycoplasma fermentans glycolipid-antigen as a pathogen of rheumatoid arthritis

Mycoplasma fermentans has been suspected as one of the causative pathogenic microorganisms of rheumatoid arthritis (RA) however, the pathogenic mechanism is still unclear. We, previously, reported that glycolipid-antigens (GGPL-I and III) are the major antigens of M. fermentans. Monoclonal antibody against the GGPL-III could detect the existence of the GGPL-III antigens in synovial tissues from RA patients. GGPL-III antigens were detected in 38.1% (32/84) of RA patient’s tissues, but not in osteoarthritis (OA) and normal synovial tissues. Immunoelectron microscopy revealed that a part of GGPL-III antigens are located at endoplasmic reticulum. GGPL-III significantly induced TNF-α and IL-6 production from peripheral blood mononulear cells, and also proliferation of synovial fibroblasts. Further study is necessary to prove that M. fermentans is a causative microorganism of RA; however, the new mechanisms of disease pathogenesis provides hope for the development of effective and safe immunotherapeutic strategies based on the lipid-antigen, GGPL-III, in the near future.     

Antimicrobial substances from rhizomes of the giant knotweed Polygonum sachalinense against the fish pathogen Photobacterium damselae subsp. piscicida

The antimicrobial compounds against the fish pathogen Photobacterium damselae subsp. piscicida were isolated from Polygonum sachalinense rhizomes. The structures of the antimicrobial compounds 1 and 2 were determined by 1H and 13C NMR, 2D-NMR (COSY, HSQC, HMBC and ROESY) and FAB-MS to be phenylpropanoid glycosides, vanicoside A and B, respectively. Both compounds have feruloyl and p-coumaroyl groups bonded to a sucrose moiety in their structures. Vanicoside A also has an acetyl group in the sucrose moiety. The MIC values for vanicoside A and B against Ph. damselae subsp. piscicida DPp-1 were 32 and 64 microg/ml, respectively. The antimicrobial activities of these vanicosides were modest, in contrast to higher activities (MICs at < 4 microg/ml) of antibiotics, florphenicol, ampicillin and amoxicillin, which have been generally used for treating pasteurellosis. The activities of the vanicosides, however, were higher than those (MICs at 256 microg/ml) of ferulic acid and p-coumaric acid. It was suggested that the structure of phenylpropanoids esterified with sucrose was essential for higher antimicrobial activity of vanicosides and also acetylation of sucrose might affect the activity against the bacterium.