Floral fragrances in two closely related fruit fly orchids,Bulbophyllum hortorum and B. macranthoides (Orchidaceae): assortments of phenylbutanoids to attract tephritid fruit fly males

Floral chemical components are important cues used by plants to attract pollinators. One outstanding case is “fruit fly orchids” in the genus of Bulbophyllum to attract their pollinators by releasing characteristic fragrances. Dacini fruit flies are main or exclusive pollinators which are strongly attracted to certain natural chemicals, either methyl eugenol (ME: a phenylpropanoid) or raspberry ketone (RK: a phenylbutanoid). Furthermore, zingerone (ZN: a phenylbutanoid) has been characterized as the attractant for both ME- and RK-sensitive fruit fly species. In the present study, we examined chemical profiles of two closely related Bulbophyllum orchids—B. hortorum, and B. macranthoides subsp. tollenoniferum—distributed in Papua New Guinea and the Southeast Asian countries, respectively. We first observed that RK-sensitive flies were attracted to these orchids by ex situ cultivation in Penang, Malaysia. These Bulbophyllum orchids contained RK and/or ZN as their main floral components. Other than these attractants, multiple phenylbutanoids including potential attractants for RK-sensitive species were identified from these orchids. Therefore, we examined attractiveness of potential phenylbutanoid attractants to an RK-sensitive melon fly, Zeugodacus cucurbitae, using laboratory-reared flies. Furthermore, we analyzed molecular phylogenetic relationships among phenylpropanoid- or phenylbutanoid-producing orchids to see a relation between chemical profiles and phylogenetic classification in the related species.     

Effect of Diglucosamine on the Entrapment of Protein into Liposomes

Liposomes, which had entrapped bovine serum albumin (BSA), were modified with diglucosamine by two methods. The liposome was prepared by a freeze-thawing method in the presence of the disaccharide, or the disaccharide was added to the liposome prepared in advance without it. To examine the effects of diglucosamine, the morphology, mean particle size, and zeta potential of both liposomes were compared with those of BSA-entrapping liposome prepared without the disaccharide. Diglucosamine caused no remarkable change in shape and no aggregation of the liposome. The presence of the disaccharide was confirmed on the surfaces of modified liposomes, and the entrapment of BSA into the liposomes was increased by the disaccharide. The entrapment behavior was affected by the way the disaccharide was added, and the difference in the way the BSA was entrapped was also indicated.     

Biological nitrification inhibition (BNI) activity in sorghum and its characterization

Aims

The ability to suppress soil nitrification through the release of nitrification inhibitors from plant roots is termed ‘biological nitrification inhibition’ (BNI). Here, we aimed at the quantification and characterization of the BNI function in sorghum that includes inhibitor production, their chemical identity, functionality and factors regulating their release.

Methods

Sorghum was grown in solution culture and root exudate was collected using aerated NH₄Cl solutions. A bioluminescence assay using recombinant Nitrosomonas europaea was employed to determine the BNI activity. Activity-guided chromatographic fractionation was used to isolate biological nitrification inhibitors (BNIs). The chemical structure was analyzed using NMR and mass spectrometry; pH-stat systems were deployed to analyze the role of rhizosphere pH on BNIs release.

Results

Sorghum roots released two categories of BNIs: hydrophilic- and hydrophobic-BNIs. The release rates for hydrophilic- and hydrophobic- BNIs ranged from 10 to 25 ATU?g^?1 root dwt. d^?1. Addition of hydrophilic BNIs (10 ATU?g^?1 soil) significantly inhibited soil nitrification (40 % inhibition) during a 30-d incubation test. Two BNI compounds isolated are: sakuranetin (ED₈₀ 0.6 μM; isolated from hydrophilic-BNIs fraction) and sorgoleone (ED₈₀ 13.0 μM; isolated from hydrophobic-BNIs fraction), which inhibited Nitrosomonas by blocking AMO and HAO enzymatic pathways. The BNIs release required the presence of NH ₄ ⁺ in the root environment and the stimulatory effect of NH ₄ ⁺ lasted 24 h. Unlike the hydrophobic-BNIs, the release of hydrophilic-BNIs declined at a rhizosphere pH >5.0; nearly 80 % of hydrophilic-BNI release was suppressed at pH ≥7.0. The released hydrophilic-BNIs were functionally stable within a pH range of 5.0 to 9.0. Sakuranetin showed a stronger inhibitory activity (ED₅₀ 0.2 μM) than methyl 3-(4-hydroxyphenyl) propionate (MHPP) (ED₅₀ 100 μM) (isolated from hydrophilic-BNIs fraction) in the in vitro culture-bioassay, but the activity was non-functional and ineffective in the soil-assay.

Conclusions

There is an urgent need to identify sorghum genetic stocks with high potential to release functional-BNIs for suppressing nitrification and to improve nitrogen use efficiency in sorghum-based production systems.     

Induction of Anti-influenza Immunity by Modified Green Fluorescent Protein (GFP) Carrying Hemagglutinin-derived Epitope Structure

The development of vaccination methods that can overcome the emergence of new types of influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel β-sheet structure. Our previous study identified this β-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this β-sheet region was considered a good target to induce broadly reactive immunity against the influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of enhanced green fluorescent protein (EGFP), whose surface is mostly composed of β-sheets. Through site-directed mutagenesis, several modified EGFPs with an epitope-mimicking structure embedded in their surface were prepared. Two EGFP variants, differing from wild-type (parental) EGFP by only five and nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus. Moreover, three of five mice immunized with each of these EGFP variants followed by a booster with equivalent mCherry variants acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage. In contrast to conventional methods, such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains.     

Fate of organic carbon during decomposition of different litter types in Japan

Carbon dynamics during litter decomposition have been described in a variety of forest ecosystems and provided insights into carbon flow in soils. To quantitatively assess how decomposition processes vary between litter types, solid-state ¹³C cross-polarization and magic-angle spinning nuclear magnetic resonance (CPMAS NMR) technique was applied to analyze conifer (cedar, cypress) and hardwood (chinquapin, beech, oak, birch) litter which had degraded during a 3 year litterbag experiment throughout Japan. The results were used to identify compositional changes and estimate decomposition constants (k values) in exponential equations. Total litter and carbon type mass losses during decomposition varied significantly between litter types, being affected by the initial physicochemical litter quality. Concomitant increases and decreases in carbonyl and O/N-alkyl C compositions, respectively, were observed for all litter types, but aromatic and aliphatic C dynamics were less consistent. In hardwoods, [aromatic/aliphatic C ratio] was generally stable during decomposition, suggesting that, in hardwoods, the decomposabilities of aromatic and aliphatic C were similar. In the conifers, an increasing [aromatic/aliphatic C ratio] during decomposition suggested that aromatic C was more recalcitrant than aliphatic C. These results suggest that different decomposition processes between litter types might be related to different aromatic and aliphatic C behaviors, as affected by lignin stability and lipid leachability and biosynthesis. Variations in the k values for total litter and carbon types were not obvious between litter types, although the mass loss patterns differed significantly. The k values estimated in this study may contribute to predictions of soil carbon dynamics and the validation of carbon compartment models in forest ecosystems.     

Detection of eggs in the living white grub beetle Dasylepida ishigakiensis (Coleoptera: Scarabaeidae) by magnetic resonance imaging

The use of magnetic resonance imaging (MRI) as a tool for in vivo detection of eggs in living Dasylepida ishigakiensis Niijima et Kinoshita, a major pest of sugarcane, was explored using females with an ovary at different developmental stages. MRI measurements of beetles were performed at 13 °C to avoid motion artifacts on the MR images. Spin–lattice relaxation time-weighted images allowed the observation of eggs at short acquisition times (2 min, 8 s). By comparing MR images with dissection data, criteria for determining mature eggs in MR images were a clear circular or ellipsoidal shape surrounded by a relatively bright rim and a size typically larger than 1.3 mm in the minor axis. Although small oocytes could not be detected, females with a developed or undeveloped ovary could be clearly distinguished based on MR images. The possibility of confusing the digestive tract as eggs in a female with a less developed ovary can be eliminated using a proton density weighted image.     

Syntheses and antitumor activities of N′1,N′3-dialkyl-N′1,N′3-di-(alkylcarbonothioyl) malonohydrazide: The discovery of elesclomol

A series of N′¹,N′³-dialkyl-N′¹,N′³-di(alkylcarbonothioyl) malonohydrazides have been designed and synthesized as anticancer agents by targeting oxidative stress and Hsp70 induction. Structure–activity relationship (SAR) studies lead to the discovery of STA-4783 (elesclomol), a novel small molecule that has been evaluated in a number of clinical trials as an anticancer agent in combination with Taxol.     

Transnitrosation of alicyclic N-nitrosamines containing a sulfur atom

Aromatic and aliphatic nitrosamines are known to transfer a nitrosonium ion to another amine. The transnitrosation of alicyclic N-nitroso compounds generates S-nitrosothiols, which are potential nitric oxide donors in vivo. In this study, certain alicyclic N-nitroso compounds based on non-mutagenic N-nitrosoproline or N-nitrosothioproline were synthesised, and the formation of S-nitrosoglutathione (GSNO) was quantified under acidic conditions. We then investigated the effect of a sulfur atom as the substituent and as a ring component on the GSNO formation. In the presence of thiourea under acidic conditions, GSNO was formed from N-nitrosoproline and glutathione, and an N-nitroso compound containing a sulfur atom and glutathione produced GSNO without thiourea. The quantity of GSNO derived from the reaction of the N-nitrosamines containing a sulfur atom and glutathione was higher than that from the N-nitrosoproline and glutathione plus thiourea. Among the analogues that contained a sulfur atom either in the ring or as a substituent, the thiazolidines produced a slightly higher quantity of GSNO than the analogue with a thioamide group. A compound containing sulfur atoms both in the ring and as a substituent exhibited the highest activity for GSNO formation among the alicyclic N-nitrosamines tested. The results indicate that the intramolecular sulfur atom plays an important role in the transnitrosation via alicyclic N-nitroso compounds to form GSNO.     

A series of nonsecosteroidal vitamin D receptor agonists for osteoporosis therapy

In an extension of our study on gamma hydroxy carboxylic acid analogs, we explored a series of nonsecosteroidal vitamin D receptor (VDR) agonists in which 1,3-diol of 1,25(OH)₂D₃ had been replaced by aryl acetic acid. These analogs showed very potent activity in vitro compared with 1,25(OH)₂D₃. An X-ray analysis of 8d showed that the inserted phenyl ring well mimicked the folded methylene linker of the gamma hydroxy carboxylic acid moiety but the carboxylic acid of 8d interacted with VDR in a different manner from gamma hydroxy carboxylic acids. Through our in vivo screening in an osteoporosis rat model using immature rats, we identified a potent active vitamin D₃ analog, compound 7e. In mature rats of the same model, compound 7e also showed good PK profiling and excellent ability to prevent bone mineral density loss without severe hypercalcemia. Our nonsecosteroidal VDR agonist 7e (CH5036249) could be a possible new drug candidate for treating osteoporosis in human.