Molecular cloning of an elongation factor 1α and its mRNA localization in testis of the Nile tilapia Oreochromis niloticus

During spermatogenesis, many proteins are synthesized in the testis prior to the completion of sperm maturation. Many components are involved in the testicular protein synthesis and one of the components that is implicated in the polypeptide chain elongation is elongation factor 1α. In the present study, the molecular cloning of elongation factor 1α (EF-1α) was conducted from a testis cDNA library of the Nile tilapia. The cDNA for tilapia EF-1α (tEF-1α) contains a complete open reading frame encoding 462 amino acids. The predicted amino acid sequence of EF-1α shows an approximate 90% similarity to those identified in other teleost fish, such as medaka, sea bream and zebrafish. Northern blotting revealed that the gene is expressed in all of the examined tissues and in ovulated eggs. The results of in situ hybridization indicate that the gene is expressed specifically in Leydig cells in testis, suggesting the involvement of EF-1α as an actin-binding protein in the cluster formation of Leydig cells. In the ovary, the gene is expressed in the perinucleolus stage of oocytes, suggesting that EF-1α is also implicated in oocyte growth.     

萜烯化学与利用的新进展(英文)

天然存在的精油中,松节油以其价格低廉平稳、供应充足而明显地被认为是作为精细化学品如:香料、食品添加剂、药物、农用化学品中间体的最重要的有机资源之一。当今迫切需要将基础研究用来对有机资源作重新评价和高水平的应用。本文介绍一系列有价值作为香料物质和食品添加剂的单萜化合物和倍半萜化合物,而这些香料与添加剂近来已发现与天然精油有关,并能由松节油合成。     

Superoxide dismutase and catalase activities in the seminal plasma of normozoospermic and asthenozoospermic Beagles

We measured the blood plasma testosterone (T) levels and superoxide dismutase (SOD) and catalase activities in the seminal plasma of the ejaculates of 5 normal (2-5 years old) and 5 asthenozoospermic (AZ-) (3-5 years old) Beagles. Sperm ejaculated by AZ-dogs was incubated for 3 hr in Eagle's MEM only (controls) or Eagle's MEM containing 100 units/ml of SOD or catalase. Sperm motility was examined during incubation. The mean (+/- SE) plasma T level of the AZ-dogs (1.2 +/- 0.2 ng/ml) was significantly lower than in the normal dogs (2.5 +/- 0.2 ng/ml) (P<0.005). The mean (+/- SE) seminal plasma SOD and catalase activities (18.8 +/- 1.9 and 0.5 +/- 0.1 unit/g protein, respectively) were significantly lower in the AZ-dogs than in the normal dog (43.3 +/- 2.5 and 2.2 +/- 0.4 unit/g protein, respectively) (P<0.001 and 0.01, respectively). The motility of sperm incubated in Eagle's MEM containing SOD or catalase was significantly higher than that of control sperm incubated in only Eagle's MEM after 2 or 3 hr of incubation (P<0.05). The results of this study indicate that poor T secretion by the testes and low antioxidant enzyme activities are related to AZ in the dog.     

Changes in the plasma testosterone level and testicular superoxide dismutase activity of 5 azoospermic beagles after GnRH analogue injections

The peripheral blood plasma testosterone (T) levels and superoxide dismutase (SOD) activity were measured in 5 azoospermic (AZ-) beagles. The mean values in the AZ-dogs were significantly lower than in 7 control beagles (P<0.001). Subcutaneous injections of 1 mug/kg GnRH analogue three times weekly in the AZ-dogs induced significant increases in mean T level and SOD activity (P<0.05) and improvement in spermatogenesis. Thus, spermatogenic function in the dog appears to be maintained by T and normal SOD activity in the testis.     

Seroepidemiology of Toxoplasma gondii and Neospora caninum in seals around Hokkaido, Japan

Serological analysis was performed to detect Toxoplasma gondii and Neospora caninum infection in seals in Hokkaido. Serum samples were collected from 322 Kuril harbor seals (Phoca vitulina stejnegeri) at Nosappu, Akkeshi and Erimo, from 46 spotted seals (P. largha) at Nosappu, Erimo, Yagishiri Island, Hamamasu and Syakotan, and from 4 ribbon seals (P. fasciata) and a bearded seal (Erignathus barbatus) at Nosappu between 1998 and 2006. Recombinant surface antigen of T. gondii (SAG2t) and N. caninum (NcSAG1t) were used as antigens for ELISA to detect antibodies. Antibodies against SAG2t were detected from 4% of 77 Kuril harbor seals at Nosappu in 2005. Antibodies against NcSAG1t were detected from 2% (1/66) in 2003, 5% (4/79) in 2004 and 10% (8/77) in 2005 of Kuril harbor seals and 11% of 9 spotted seals in 2004 sampled at Nosappu. Eight percent of 12 Kuril harbor seals from Akkeshi and 25% of 4 spotted seals from Erimo in 2005 also contained antibodies against NcSAG1t. These suggest sporadic infection of T. gondii and N. caninum in Kuril harbor seals and spotted seals in Hokkaido. Of the ELISA-positive seals, 2 seals having anti-SAG2t antibodies and 3 seals having anti-NcSAG1t antibodies in 2005 were judged to be juveniles that have no maternal antibodies. These suggest that the protozoan infections have occurred in recent years. Infection of terrestrial protozoa such as T. gondii and N. caninum in seals indicates that the sea environment has been contaminated with protozoa.     

Ascophyllan Purified from Ascophyllum nodosum Induces Th1 and Tc1 Immune Responses by Promoting Dendritic Cell Maturation

Marine-derived sulfated polysaccharides have been shown to possess certain anti-virus, anti-tumor, anti-inflammatory and anti-coagulant activities. However, the in vivo immunomodulatory effects of marine-derived pure compounds have been less well characterized. In this study, we investigated the effect of ascophyllan, a sulfated polysaccharide purified from Ascophyllum nodosum, on the maturation of mouse dendritic cells (DCs) in vitro and in vivo. Ascophyllan induced up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in bone marrow-derived DCs (BMDCs). Moreover, in vivo administration of ascophyllan promotes up-regulation of CD40, CD80, CD86, MHC class I and MHC class II and production of IL-6, IL-12 and TNF-α in spleen cDCs. Interestingly, ascophyllan induced a higher degree of co-stimulatory molecule up-regulation and pro-inflammatory cytokine production than fucoidan, a marine-derived polysaccharide with well-defined effect for promoting DC maturation. Ascophyllan also promoted the generation of IFN-γ-producing Th1 and Tc1 cells in the presence of DCs in an IL-12-dependent manner. Finally, myeloid differentiation primary response 88 (MyD88) signaling pathway was essential for DC maturation induced by ascophyllan. Taken together, these results demonstrate that ascophyllan induces DC maturation, and consequently enhances Th1 and Tc1 responses in vivo. This knowledge could facilitate the development of novel therapeutic strategies to combat infectious diseases and cancer.     

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.