Comparison of PCR assays for detection and quantification of Phomopsis sclerotioides in plant and soil

We developed a real-time PCR assay using a TaqMan probe (TM-qPCR) for specific detection and quantification of Phomopsis sclerotioides, causal agent of black root rot of cucurbit crops. The design of the primer sets and hybridization probe was based on the internal transcribed spacer region of the ribosomal DNA. The TM-qPCR assay was compared with a conventional, standard PCR (sPCR) assay and on a quantitative real-time PCR (SG-qPCR) assay based on SYBR Green I. The TM-qPCR assay had a detection limit of ca. 0.4 fg of P. sclerotioides DNA, which was approximately 100 times more sensitive than the sPCR assay and almost equivalent to the SG-qPCR assay. The TM-qPCR and SG-qPCR assays both were able to detect various quantities of P. sclerotioides DNA from diseased plants and infested soils, including DNA levels that were not detectable by the sPCR assay. However, the TM-qPCR was advantageous for samples containing PCR-inhibiting substances because its multiplex real-time PCR function allows the adjustment of cycle threshold values with an internal control. Based on the high specificity and sensitivity required for analyzing DNA in natural samples, the newly developed TM-qPCR assay was the most reliable tool for rapidly detecting and quantifying P. sclerotioides in plant and soil samples.     

Induction of Apoptotic Cell Death in Human Leukemia U937 Cells by C18 Hydroxy Unsaturated Fatty Acid Isolated from Red Alga Tricleocarpa jejuensis

Our previous studies have found that (±)-(E)-12-hydroxyoctadec-10-enoic acid (HOEA) isolated from the red alga Tricleocarpa jejuensis showed cytotoxic effects on various living organisms including harmful microalgae, Gram-positive bacteria, and mammalian tumor cells. Since natural products with apoptosis-inducing ability can be promising anti-cancer agents, in this study, we investigated the cytotoxic mechanism of HOEA on U937 cells focusing on apoptosis induction. HOEA showed much stronger cytotoxic and cytolytic effects on U937 cells than elaidic acid, which has similar structure but no 12-hydroxy group, suggesting that hydroxy group is important for the cytotoxicity of HOEA. HOEA induced apoptotic nuclear morphological changes, DNA fragmentation, and decrease in mitochondrial membrane potential. Furthermore, time-dependent increase in annexin V+/PI+ cell population in HOEA-treated U937 cells was detected. Among the apoptosis-related reagents, caspase-family inhibitor almost completely inhibited HOEA-induced DNA fragmentation. In the analyses using specific caspase-substrates, extremely high cleavage activity toward caspase-3/7/8 substrate was observed in HOEA-treated U937 cells, and weak activities of caspase-1 and -3 were detected. Analyses using specific caspase inhibitors suggested that caspase-3 and caspase-8 might be predominantly responsible for the cleavage activity. Activation of these caspases were also confirmed by western blotting in which significant levels of cleaved forms of caspase 3, caspase 8, and PARP were detected in HOEA-treated U937 cells. Our results suggest that HOEA is capable of inducing apoptosis in U937 cells in which caspase-3 and caspase-8 might play important roles. Since the cytotoxic effect of HOEA is not strictly specific to tumor cells, development of appropriate drug delivery system for selective tumor targeting is necessary for the clinical applications to reduce the possible side effects.     

Ammonia Emission from a Young Larch Ecosystem Afforested after Clear-Cutting of a Pristine Forest in Northernmost Japan

The present study aimed to elucidate the atmosphere–forest exchange of ammoniacal nitrogen (NH_X-N) at a young larch ecosystem. NH_X-N exchanges were measured at a remote site in northernmost Japan where 4-year-old larches were growing after a pristine forest had been clear-cut and subsequent dense dwarf bamboo (Sasa) had been strip-cut. The site was a clean area for atmospheric ammonia with mean concentrations of 0.38 and 0.11 μg N m^?3 in snowless and snow seasons, respectively. However, there was a general net emission of NH_X-N. The annual estimated emission of NH_X-N of 4.8 kg N ha^?1 year^?1 exceeded the annual wet deposition of 2.4 kg N ha^?1 year^?1, but the weekly exchange fluxes may have been underestimated by 28–60%. The main cause of the ammonia loss from the young larch ecosystem was probably enhanced nitrogen supply stimulated by the cutting of the pristine forest and Sasa, in particular, the Sasa.     

Flowering behaviors of the inflorescences of an alien plant (Plantago asiatica), an alpine plant (Plantago hakusanensis), and their hybrids on Mt. Hakusan,Japan

In the subalpine zone on Mt. Hakusan, Japan, Plantago asiatica, an alien plant, and Plantago hakusanensis, a native alpine species, grow sympatrically along with their putative hybrids. Here, their flowering behavior, which affects the frequency of hybridization and the colonizing ability of P. asiatica and its hybrids, is described. The flowering behavior of each species and of two F₁ hybrids from different seed parents was determined based on the position of the flower in the inflorescence by using a generalized linear mixed model. The percentage fruit set of individually bagged inflorescences was calculated to corroborate the assumptions of the opportunities for self‐pollination. All the flowers were protogynous; however, many P. asiatica anthers dehisced before browning of the stigma in the flower and the sex presentations in the inflorescence were asynchronous. The percentage of fruit set was high. Consequently, P. asiatica has the opportunity for self‐pollination within the flower and in the inflorescence. In contrast, the P. hakusanensis anthers dehisced after browning of the stigma in the flower; their sex presentation was synchronous in the inflorescence, showing negligible opportunities for self‐pollination, and the fruit set was low. Accordingly, in the field, P. hakusanensis might require pollination among the inflorescences for seed production and be actively outcrossed, while P. asiatica is able to outcross in the early flowering phase. Therefore, P. asiatica and P. hakusanensis have opportunities for hybridization. The F₁ hybrids exhibited intermediate flowering behavior and produced fruits, demonstrating the potential to reproduce by selfing.     

Antiangiogenic activity of brown algae fucoxanthin and its deacetylated product, fucoxanthinol

The antiangiogenic effects of fucoxanthin and a deacetylated product, fucoxanthinol, were examined. Fucoxanthin significantly suppressed HUVEC proliferation and tube formation at more than 10 microM, but it had no significant effect on HUVEC chemotaxis. The formation of blood vessel-like structures from CD31-positive cells was evaluated using embryonic stem cell-derived embryoid bodies. Fucoxanthin effectively suppressed the development of these structures at 10-20 microM, suggesting that it could suppress differentiation of endothelial progenitor cells into endothelial cells involving new blood vessel formation. Fucoxanthin and fucoxanthinol suppressed microvessel outgrowth in an ex vivo angiogenesis assay using a rat aortic ring, in a dose-dependent manner. These results imply that fucoxanthin having antiangiogenic activity might be useful in preventing angiogenesis-related diseases.     

柚木黑色条纹心材的颜色和化学特性

印尼岛的柚木心材在年轮周围显示了不规则的黑色条纹变化。本研究调查柚木黑色条纹心材的颜色和化学特性。利用色坐标（CIELAB）测定颜色特性（pH值、无机物含量、抽出物含量和特性）。结果表明：黑色条纹的颜色较对照少12-15个亮度单位。因此,黑色条纹的颜色与对照比较,有较多的红色,较少的黄色、色彩和色度。黑色条纹心材部分的pH值灰度含量和钙含量都比对照的高。黑色条纹的心材抽出物含量较对照的高。推测当生物活性物质（杀虫剂）明显增加时,心材显示发黑的过程可能是一种防御机制。     

Synthesis and herbicidal activity of 3-substituted toxoflavin analogs

We investigated the synthesis and herbicidal activity of 23 toxoflavin analogs, 1a–w, in which aromatic rings (R) were introduced into the C-3 position. In paddy field conditions, 1k (R=2-CF₃–C₆H₄) and 1w (R=2-thienyl) showed excellent herbicidal activity. Under upland field conditions, we found that toxoflavin analogs 1a (R=C₆H₅), 1n (R=2-CH₃O–C₆H₄), and 1p (R=4-CH₃O–C₆H₄) exhibited wide herbicidal spectrum against Echinochloa crus-galli (L) var. crus-galli (ECHCG), Chenopodium album, and Amaranthus viridis (AMAVI). The analog with the 2-fluoro group on benzene ring 1b also showed high herbicidal activity against both ECHCG and AMAVI.     

Lipophilic extractives of the inner and outer barks from six different Pinus species grown in Indonesia

The chemical compositions of the dichloromethane extracts of inner and outer barks from six Pinus species(P.elliotii,P.oocarpa,P.caribeae,P.merkusii,P.montezumae,and P.insularis) grown in Indonesia were investigated by GC and GC–MS.Generally,the amounts of extractive contents were higher in the inner bark than in the outer bark except for P.merksuii.Fatty acids,monoterpenes,sesquiterpenes,resin acids,triterpenoids,and steroids were detected and quantified.Inner and outer barks differed not only in content of these compounds but also in their composition.Fatty acids and alcohols were the major classes of lipophilic compounds in the outer bark of P.caribeae, P.insularis,and P.montezumae.Steroids and triterpenoids were the dominant compounds identified in the inner bark of P.elliotii,P.insularis,and P.merkusii.Resin acids were the most abundant group in the inner bark of P.oocarpa whereas monoterpenes and sesquiterpenes were recorded in minor quantities in both bark layers of all species.     

Analysis of the triple gene block sequence in an important melon pathogen,Melon yellowing-associated virus

A novel viral disease of melon, caused by Melon yellowing-associated virus (MYaV), has become one of the most serious problems for melon production in Brazil. Despite its importance, little is known about the genome sequence of the virus. In this study, the triple gene block genomic region including the 3′-end region of an isolate of MYaV was elucidated (ca. 3.1 kb). Sweet potato chlorotic fleck virus, a newly suggested carlavirus species, was the most closely related virus with 62% nucleotide identity. The presence of two heptanucleotide-motifs for subgenomic RNA synthesis was confirmed in this sequence.