Search

It is essential to understand the combined effects of elevated CO2 and temperature on phytoplankton-zooplankton link when attempting to predict climate change responses of freshwater ecosystems. Phytoplankton species differ in stoichiometric and fatty acids composition, and this may result in phytoplankton-mediated effect on zooplankton at elevated CO2 and temperature. Beyond the isolated analysis of CO2 or temperature effect, few studies have assessed zooplankton growth under the phytoplankton-mediated effects of elevated CO2 and temperature. In this study, three algal species (green alga, diatom, cyanobacteria) were fed on zooplankton Daphnia magna, under the conditions of CO2 concentrations of ambient (390 ppm) and elevated (1000 ppm) levels and temperatures at 20, 25 and 30 °C. Elevated CO2 increased the algal biomass, while it reduced the phosphorus (P) and ω3 polyunsaturated fatty acids (ω3 PUFAs) to carbon (C) ratios. Elevated temperature decreased the P/C ratios in all algal cultures and ω3 PUFAs/C ratios in the diatom and the cyanobacteria cultures. Phytoplankton-mediated effect of elevated CO2 reduced the growth of zooplankton fed on the green and the mixed three algae culture. The stimulation of zooplankton fed on the diatom and the cyanobacteria by elevated temperature can be offset by decreasing food P and ω3 PUFAs contents. The combined effects of elevated CO2 and temperature on the growth of daphnids were mainly mediated by ω3 PUFAs/C ratios in the phytoplankton. Rising temperature as a combined direct and indirectly phytoplankton-mediated effect on zooplankton may be able to ameliorate the negative effects of elevated CO2. The results indicated that the combined effects of increased CO2 and temperature increased the fatty acid content of the green alga but not the other algae. This study highlighted that climate change with simultaneously increasing temperature and CO2 may entangle the carbon transfer in freshwater planktonic communities.

We present stratigraphic, geochemical (CNS elemental analysis), and micropaleontological (ostracode assemblages) evidence for the 2011 Tohoku-oki tsunami impact in Matsushima Bay, northeastern Japan, and examine the recovery process after the impact to the bay environment using short sediment cores from two different topographic settings at a water depth of approximately 4 m. At both sites, tsunami deposits are composed of two sedimentary layers that recorded the first tsunami run-up and backwash. At the western site near the channel that connects the bay with the Pacific Ocean, the first tsunami wave eroded the bay floor sediments and left the tsunami deposits composed of medium sand rich in shell fragments, but with sparse numbers of meiobenthic ostracodes. By contrast, at the eastern site, which is surrounded by many small islands, the first wave eroded very little of the bay floor sediments and left tsunami deposits consisting of sandy mud, rich in exotic ostracodes, such as phytal species, shallow marine sand dwelling species, and brackish water species. Overlying post-tsunami deposits are composed mainly of organic-rich mud in which organic matter was derived primarily from marine plankton. Ostracode assemblages in the tsunami deposits lack the offshore species that live in water depths >50 m and are dominated by the species from Matsushima Bay and its adjacent nearshore, upper sublittoral areas, and brackish water environments. The distance from deep-water offshore areas to the core sites is too far to transport ostracode valves by tsunami waves.

Rising temperature enhances the algal growth, which in turn increases the water pH. Ecotoxicity studies have suggested that copper becomes more toxic to microalgae species by increasing the temperature (within 20–30 °C) and pH. In this study, the joined effect of pH and temperature on copper toxicity to the microalgae Raphidocelis subcapitata was investigated using acclimated cells. Algal growth and toxicity tests were conducted using the medium recommended by the Organisation for Economic Co-operation and Development (OECD medium) at pH 6, 7, and 8 units from 15 to 30 °C, spaced by 3 °C. The specific growth rate of R. subcapitata increased by raising the pH and temperature, attributed to the higher membrane permeability and metabolism. The ecotoxicity tests showed that temperature changes the effect of pH on copper toxicity. Copper became less toxic when rising the temperature from 15 to 18 °C and from 6 to 8 pH-unit, suggesting that high pH controls copper bioavailability and toxicity. In contrast, from 21 to 30 °C, the effect of copper was not significantly altered by temperature, but it became more toxic at high pH. Results of this study warn about the higher risk of copper in cold seasons rather than warm conditions.

The characteristics of nitrous oxide, N2O, from a sewage treatment plant, which conducts nitrogen removal, and the river that receives its effluent water, were investigated by intensive daily surveys in summer and winter. N2O production in the sewage treatment plant was promoted in winter when nitrite accumulated in the reaction tank. The dissolved N2O concentration in the effluent water was also high in winter, which caused the dissolved N2O concentration to increase in the river downstream. In contrast, the N2O production inside the plant and the dissolved N2O emission through the effluent water, the dissolved N2O discharge, was controlled in summer when the nitrogen removal was more complete and there was no-nitrite accumulation. The dissolved N2O in the effluent water was rapidly lost after leaving the plant by as much as 26% in summer and 59% in winter. Additionally, the amount of the dissolved N2O discharge in winter was almost equal to that of the indirect N2O emission. When the nitrogen removal proceeded successfully, the amount of dissolved N2O discharge was small. In contrast, when the nitrogen removal was insufficient, the dissolved N2O discharge became an important N2O source.

Highly unsaturated fatty acids such as 20:5n3 (EPA) are both hormone precursors and cell membrane components, making them important nutrients for aquatic animals. Many animals must obtain EPA from their diets because they cannot synthesize enough EPA to meet their requirements, and algae are the main source of EPA in aquatic ecosystems. In a previous study, we detected EPA in the faeces of Danio rerio, a freshwater fish, even though the fish consumed a green algae diet that did not contain EPA. The objective of this study was to determine why EPA was detected in fish faeces. A significant positive relationship was detected between the number of heterotrophic protozoa and the concentration of EPA in the faeces, which suggests that this EPA was of protozoan origin. In addition, another experiment showed that protozoa adhered to faeces far more than the green algal diet remnants, which indicates that protozoa preferred to swarm on faeces. Furthermore, we cultured protozoa in an EPA-free medium and fed them a bacterial diet also lacking EPA, and found that Cyclidium sp. synthesized EPA de novo. The results demonstrate that protozoa produce essential fatty acids and enhance the nutritional quality of animal faeces in detritus-based food webs in freshwater ecosystems.

Nitrous oxide (N2O), a strong greenhouse and ozone depleting gas, is known to be generated in the river environment. However, the impact of sewage treated water on the production mechanism has not been clarified. In this study, N2O production in the upper reach of a river was evaluated by field survey and activity test. The results demonstrated that the N2O production activity of the river pebbles increased with the inflow of the sewage treated water, which was supported by field survey results, such as the dissolved N2O concentrations and water quality. The emission factors of N2O were determined to be 0.02–0.05% in nitrification and 0.01–0.025% in denitrification. Our study shows that combining a field survey and an activity test improves the reliability of the results and leads to the appropriate quantitative evaluation. From a perspective of controlling the N2O emissions from the sewage treatment plant, N2O generation inside the plant is critical. However, appropriate nitrogen removal in the treatment plant is connected to the reduction of N2O generation in the river environment.

We investigated the composition of fatty acids and oxygen consumption rate (OCR) of the settling particulate organic matter (POM) and its potential sources (suspended POM, oyster biodeposits, fouling mussel biodeposits, POM attached on oysters and farm gears) in oyster farms, and estimated their relative contribution to the settling POM using a stable isotope mixing model. The settling POM was lower in both the content of algal-originating fatty acids and OCR compared with those of the potential sources. Furthermore, the content of fatty acids and OCR of settling POM were lower inside than outside the oyster farms. The settling flux of POC was 2–18 times higher inside than outside the oyster farms, with the settling POM estimated to consume on average 5 times more oxygen in the bottom layers. The contribution of oyster biodeposits to the settling POM inside the farms was equivalent to that of other potential sources. Our study highlighted that preventing recruitment and removing sessile organisms from the cultivated organisms and farm facilities such as mussels and microorganisms could be an effective strategy for reducing the amount of POM settling inside the farms. Furthermore, we showed that the fatty acids marker would be a good indicator for predicting the potential of oxygen consumption of various POM sources.

In this study, two double-anode microbial fuel cells (MFCs) were connected in series for degradation of the azo dye reactive brilliant red X-3B. After the series connection, the electricity generation of one of the MFCs decreased, and the other was not affected too much. Due to the special structure in the double-anode MFC reduced the imbalanced performance between the MFC units, the occurrence of voltage reversal was limited. The removal efficiencies in two MFC reactors were not consistent after the series connection, the results showed that the MFC with the reduced electricity generation had the higher removal efficiencies, it was 12.90, 11.66, and 40.05% higher than in the MFC in which the power generation capacity was not affected after the series connection, the MFC without serial connection, and the control group, respectively. Meanwhile, the microbial communities related to the degradation of refractory organic compounds increased and related to electricity generation decreased in the MFC with the reduced electricity generation, the changes of the microbial communities were consistent with its electricity generation and the removal efficiencies. The degradation products in the effluent from two MFC units showed that had the products generated from the MFC with the reduced electricity generation had simpler structures comparing the other MFC unit.

The effects of watershed land cover on the biogeochemical properties of estuarine tidal flat sediment were examined in estuarine tidal flats of 16 watersheds in a densely populated, subtropical island of Japan. Despite the small sizes of the watersheds (

Summary Primordial germ cells (PGCs) are specified from epiblast cells in mice. Genes associated with naive pluripotency are repressed in the transition from inner cell mass to epiblast cells, followed by upregulation after PGC specification. However, the molecular mechanisms underlying the reactivation of pluripotency genes are poorly characterized. Here, we exploited the in vitro differentiation of epiblast-like cells (EpiLCs) from embryonic stem cells (ESCs) to elucidate the molecular and epigenetic functions of PR domain-containing 14 (PRDM14). We found that Prdm14 overexpression in EpiLCs induced their conversion to ESC-like cells even in the absence of leukemia inhibitory factor in adherent culture. This was impaired by the loss of Kruppel-like factor 2 and ten-eleven translocation (TET) proteins. Furthermore, PRDM14 recruited OCT3/4 to the enhancer regions of naive pluripotency genes via TET-base excision repair-mediated demethylation. Our results provide evidence that PRDM14 establishes a transcriptional network for naive pluripotency via active DNA demethylation., Graphical Abstract, Highlights • PRDM14 converts from EpiLCs to ESCs in adherent culture • EpiLC to ESC conversion by PRMD14 depends on KLF2 and TET-base excision repair pathway • PRDM14 enhances the recruitments of OCT3/4 at pluripotent genes, In this article, Seki and colleagues exploit an in vitro differentiation system of epiblast-like cells (EpiLCs) from embryonic stem cells (ESCs) to investigate the function of PRDM14. Overexpression of PRDM14 in EpiLCs induces the conversion from EpiLCs to ESCs through active DNA demethylation-mediated OCT3/4 recruitment at pluripotent genes in adherent culture.

Differentiation of pluripotent stem cells (PSCs) requires transposon silencing throughout the process. PIWIs, best known as key factors in germline transposon silencing, are also known to act in somatic differentiation of planarian PSCs (neoblasts). However, how PIWIs control the latter process remains elusive. Here, using Dugesia japonica, we show that a nuclear PIWI, DjPiwiB, was bound to PIWI-interacting RNAs (generally key mediators of PIWI-dependent transposon silencing), and was detected in not only neoblasts but also their descendant somatic cells, which do not express piwi. In contrast, cytoplasmic DjPiwiA and DjPiwiC were detected only in neoblasts, in accord with their transcription there. DjPiwiB was indispensable for regeneration, but dispensable for transposon silencing in neoblasts. However, transposons were derepressed at the onset of differentiation in DjPiwiB-knockdown planarians. Thus, DjPiwiB appears to be inherited by descendant somatic cells of neoblasts to ensure transposon silencing in those cells, which are unable to produce PIWI proteins.

Dense algal growth on shells of the freshwater mud snail species, Bellamya chinensis , is commonly found. In rice paddy fields of Northeastern Japan, fatty acid biomarkers and carbon stable isotope composition were analyzed to test whether B. chinensis grazes on shell-attached algae. The carbon stable isotope ratio of B. chinensis was positively related to that of shell-attached algae. B. chinensis also assimilated substantial amounts of omega-6 fatty acids, which were abundant in shell-attached algae. Furthermore, the effect of assimilating shell-attached algae on B. chinensis growth was examined in a field experiment. Individuals feeding on shell-attached algae exhibited faster shell growth than those with no access to shell-attached algae of other individuals. Our results demonstrate that B. chinensis growth is enhanced by algal fouling on their own shells, which provides them with a nutritious food source, although very few studies have documented benefits conferred to an organism that hosts an epibiotic species.

Heavy metal pollution of marine sediments has attracted a great deal of attention because of its persistence, bioaccumulation, and toxicity. To evaluate the effects of mega-tsunami, anthropogenic activities, and redox conditions on heavy metal accumulation in coastal areas, sediments from Matsushima Bay, Miyagi Prefecture, Japan, were sampled to test variations in heavy metal spatial distribution on the bay floor during 4 years following the 2011 Tohoku Earthquake tsunami. Cluster analysis and principal component analysis were performed to assess the influencing factors and potential sources of heavy metal enrichment in the sediments of the bay. Additionally, the sediment enrichment levels of heavy metals were assessed on the basis of the enrichment factor (EF). The results of multivariate statistical analyses showed that the Ti, Fe, V, Pb, and Zn contents in Matsushima Bay sediments, which were transported mainly from Sendai Bay, depended on the mud content. The value of EF 2 for Fe, V, Pb, and Zn indicated that these elements were not enriched. The value of EF 7 for Cu suggested that the contamination levels in western Matsushima Bay were moderate to severe in every sampling year from 2012 to 2016 by anthropogenic activities. From the values of EF 5 for U and Mo during 2012 and 2014, the severe enrichment of both elements in these periods may be explained by contamination with 2011 tsunami deposits; the improvement in 2015-2016 suggests that there was recovery of the tsunami-affected sediment composition to its original state. The values of EF 3 for Mn and As indicated moderate to severe contamination with these heavy metals in the bay mouth area during 2015. This was likely explained by more oxic bottom conditions in the mouth of Matsushima Bay during that year.

SummaryThe identification of molecular networks at the system level in mammals is accelerated by next-generation mammalian genetics without crossing, which requires both the efficient production of whole-body biallelic knockout (KO) mice in a single generation and high-performance phenotype analyses. Here, we show that the triple targeting of a single gene using the CRISPR/Cas9 system achieves almost perfect KO efficiency (96%–100%). In addition, we developed a respiration-based fully automated non-invasive sleep phenotyping system, the Snappy Sleep Stager (SSS), for high-performance (95.3% accuracy) sleep/wake staging. Using the triple-target CRISPR and SSS in tandem, we reliably obtained sleep/wake phenotypes, even in double-KO mice. By using this system to comprehensively analyze all of the N-methyl-D-aspartate (NMDA) receptor family members, we found Nr3a as a short-sleeper gene, which is verified by an independent set of triple-target CRISPR. These results demonstrate the application of mammalian reverse genetics without crossing to organism-level systems biology in sleep research.

The seasonal variety of greenhouse gas (GHGs) emissions and the main emission source in a sewage treatment plant were investigated. The emission coefficient to treated wastewater was 291gCO2m(-3). The main source of GHGs was CO2 from the consumption of electricity, nitrous oxide from the sludge incineration process, and methane from the water treatment process. They accounted for 43.4%, 41.7% and 8.3% of the total amount of GHGs emissions, respectively. The amount of methane was plotted as a function of water temperature ranging between 13.3 and 27.3°C. An aeration tank was the main source of methane emission from all the units. Almost all the methane was emitted from the aeration tank, which accounted for 86.4% of the total gaseous methane emission. However, 18.4% of the methane was produced in sewage lines, 15.4% in the primary sedimentation tank, and 60.0% in the aeration tank.

Despite of the importance for tracking sources and fates of organics in determining carbon cycling and assessing the overall health of lacustrine ecosystems, little is known of organics behaviors in shallow eutrophic lakes. In this study, for relating the benthic organics to pelagic sources, an ecosystem-level investigation was performed in hypereutrophic Lake Taihu, China, by using source-specific fatty acid biomarkers on the molecular level. Results exhibited the unexpected asymmetric phenomena of organics between surface water and sediments. In the abundance, the dominant organics shifted from cyanobacteria in surface water to terrestrial plants in sediments. In the spatial pattern, as opposed to terrestrial plants, cyanobacteria were found not site-to-site symmetrical, although both of their spatial distributions varied strongly. Essentially, these asymmetric phenomena ascribed to a more considerable loss of cyanobacteria compared to terrestrial plants during sinking, caused by aquatic animal consumption and microbial utilization with different degree. Combined dual stable isotopes (δ13C and δ15N) and fatty acid biomarkers revealed that there were only subtle differences in the diets of benthic and pelagic animals and cyanobacteria were their main food source. Concomitantly, results of δ13C of bacteria-specific fatty acids demonstrated that bacteria equally and profoundly affected organics accumulation or preservation in the sediments, because they preferentially utilized labile cyanobacteria as their carbon source instead of terrestrial plants (>95% within these two sources). Consequently, these novel findings clarify that not only in deep lakes, but also shallow eutrophic ones, the extensive losses of autochthonous organic matter can be expected during sedimentation coupling with the dramatical modifications of biogeochemical processes.

A lab-scale UASB reactor was operated for the treatment of p-acetamidobenzene sulfonyl chloride (p-ASC)-containing wastewater with and without ethanol for 210 days. The influence of the organic loading rate on the performance of reactor by step-decreasing HRT reduction/step-increasing the concentration of p-ASC was evaluated. Almost complete degradation of 50 mg/l p-ASC and COD removal efficiency (79.5%) was noticed with a COD removal of 79.5% at HRT of 0.5 day or longer and an applied p-ASC loading rate below 120 mg/l/day. No inhibition to the performance of this reactor was observed even at a p-ASC concentration up to 1000 mg/I. Of interest that despite the absence of sulfate in the feed, a certain quantity of sulfate and sulfide was simultaneously generated in the effluent. The trial result in the absence of ethanol demonstrated that p-ASC could be used as the sole source of carbon and energy. Clone libraries for the archaeal and bacterial communities were constructed for a biomass sample taken on day 190. The majority of bacterial clones were represented by Proteobacteria, followed by Thermotogae, Bacteroidetes and Firmicutes. Bacterial groups within the phyla Clostridia might be responsible for the desulfonation of p-ASC. (C) 2014 Elsevier Ltd. All rights reserved.

Although an understanding of the quantity and quality of sedimentary organic matter (SOM) pools is necessary to design sound environmental management strategies for lacustrine systems, the characterization of organic matter sources and the assessment of their relative contributions to eutrophic and inland lake sediments remain insufficient. In this study, the contribution of potential organic matter sources to sediments in shallow and hypereutrophic lake Taihu, China was assessed on the molecular level using source-specific fatty acid biomarkers. The results indicated that SOM was composed mainly of terrestrial plants with a maximal contribution of 45.3 ± 2.4% to the total organic carbon, which accounted for approximately 66% among the determined organic matter sources. Evidence suggests the terrestrial plants remained in a fresh state in surface sediments: the correlation ( R 2 = 0.62, p R 2 = 0.60, p

The feasibility of treating of chemical synthesis-based pharmaceutical wastewater containing rich organic sulfur compounds and sulfate using an upflow anaerobic sludge blanket (UASB) was investigated. The initial COD/SO42- ratio of 8 in the wastewater varied from 5 to 1.5 after adding sulfate. Of interest that under the condition of COD/SO42- at 8 and 5, despite the simultaneous generation of sulfide and hydrogen sulfide, the sulfate concentration in the effluent was higher than in the influent. This is due to the sulfur release in the form of sulfate during the degradation of these organic sulfur compounds in this reactor. Conversely, at COD/SO42- of 1.5, influent sulfate was higher than effluent sulfate due to reduction of more sulfates in the reactor. At COD/SO42- of 8, for practical application, the optimum OLR was found to be 8 kg COD/m(3)/d, where a nearly 70% COD reduction occurred with biogas containing 63% methane. In this stage, the distribution of the archaea and bacterial community varied greatly with altered OLR (accompanied with prolonged operation time). Some species, such as Lysinibacillus sphaericus, Clostridium cellulovorans were expected to be partly responsible for S release from some organic sulfur compounds in the reactor. By increasing the sulfate loading at a COD/SO42- ratio up to 1.5 resulted in a light inhibition of methanogenesis due to the high sulfide concentration (1212 SO42- - S mg/L) with no obvious suppression of sulfidogenesis. (C) 2014 Elsevier B.V. All rights reserved.

The quantity and quality of sedimentary organic matter (SOM) in relation to material and energy flows are crucial for understanding the current state and future development of lake systems, yet, characterization of organic matter sources and assessment of their relative contributions in different trophic-state lakes caused by anthropogenic impacts are scarcely known. In this study, for obtaining information concerning the source of SOM and its compositional diversity along different trophic gradients, a total of thirty-one sampling sites from four freshwater lakes located in China and Japan were performed by the molecular level analysis using source-specific fatty acid biomarkers. Results indicated that SOM in these lakes was composed of microalgae-, aquatic plant-, terrestrial plant- and bacteria-derived organic matters based on their fatty acid profiles. The scatter plot matrix exhibited correlations between these sources, however, only terrestrial plant-derived organic carbon was a well predictor for sediment TOC with strong, spatiotemporal dynamics. The source and composition of SOM were evidently influenced by lake trophic state with redundancy analysis. Moreover, increase of lake trophic state led to the relatively higher contribution of aquatic organic matter sources to SOM pool compared with terrigenous sources, as evidenced by significant correlations between the trophic state index [TSI (TP)] and the ratio of terrigenous to aquatic fatty acids (TARFA ratio). Yet, this changing trend became more gradual with higher trophic state and prevented the occurrence of regime shift from allochthonous to autochthonous dominant state by a threshold (0.683) of TARFA ratio. Together, a conceptual diagram was proposed, which highlighted the prevailing state of allochthonous source and implicated sedimentary organics in biogeochemistry cycle within freshwater lakes.

Cyanobacterial blooms and their corresponding toxins have resulted in the reduction of heterogeneity within aquatic ecosystems, and vice versa. Yet much remain to be known whether high heterogeneity can lead to effective elimination of cyanobacteria and microcystin (MC). In this work, by introducing biota (macrophyte and bivalve) and abiota (artificial substrate) to increase heterogeneity, cyanobacterial and MC elimination were enhanced. With increasing heterogeneity, the mean removal efficiencies of cyanobacteria increased from 58.86% to 79.11%. Correspondingly, the mean MC removal efficiencies enhanced from 42.38% to 74.16%. The biofilm was shown to be the most efficient for removing cyanobacteria and MC, whereas the bivalve was less efficient for cyanobacterial removal than the macrophyte and had no effect on MC removal. Furthermore, crustaceans were significantly enriched in all the heterogeneous systems; the introduced bivalve increased respiratory activity in the substrate biofilm. Conclusively, increasing heterogeneity appears to be a not only viable solution for improving the quality of water in protected sources but also potential criterion for restoring and managing aquatic ecosystems.

In this study, a continuous-flow microbial fuel cell (MFC) system was constructed to treat azo dye wastewater for 400 days. The electrical properties of the MFC after 400-day operation and the removal efficiencies of the MFC after long- and short-term operation were analyzed with respect to co-substrate concentrations. The power output of the MFC system decreased from 586 to 330 mV with increasing operating time, and the removal efficiencies of the MFC remained stable after long-term operation in comparison to those after short-term operation, even when the co-substrate concentration was reduced. Analysis of the degradation products showed that products generated from long-term operation of the MFC were present at low concentrations. The microbial community analysis revealed that the relative abundance of microorganisms related to the degradation of organics in the MFC increased after long-term operation, and microorganisms related to electricity generation decreased.

SummaryAdipose tissue secretes adipokines that mediate insulin resistance, a characteristic feature of obesity and type 2 diabetes. By differential proteome analysis of cellular models of insulin resistance, we identified progranulin (PGRN) as an adipokine induced by TNF-α and dexamethasone. PGRN in blood and adipose tissues was markedly increased in obese mouse models and was normalized with treatment of pioglitazone, an insulin-sensitizing agent. Ablation of PGRN (Grn−/−) prevented mice from high fat diet (HFD)-induced insulin resistance, adipocyte hypertrophy, and obesity. Grn deficiency blocked elevation of IL-6, an inflammatory cytokine, induced by HFD in blood and adipose tissues. Insulin resistance induced by chronic administration of PGRN was suppressed by neutralizing IL-6 in vivo. Thus, PGRN is a key adipokine that mediates HFD-induced insulin resistance and obesity through production of IL-6 in adipose tissue, and may be a promising therapeutic target for obesity.

Melanization is one of the major immune responses in arthropods. Prophenoloxidases (proPOs) catalyze the oxidation of mono- or o-diphenols, a reaction that is the key initial step of melanin formation. Well-characterized proPOs from crustaceans are synthesized in haemocytes and are released into plasma in response to microbial attack. However, PO activity does exist in the plasma of haemolymph without pathogenic infections. Here, we demonstrate that a novel type of proPO contributes to such PO activity in the plasma fraction of haemolymph of crustaceans. The novel enzyme, which was purified from the plasma of the kuruma prawn (Marsupenaeus japonicus), possessed strong and specific monophenol and o-diphenol oxidation activity compared with that of known haemocyte-type proPO. Amino acid sequence analyses indicated that this enzyme was distinct from the known proPO. The cDNA sequence and deduced amino acid sequence of this enzyme has a putative binuclear copper center, and showed approximately 30% and 20% identity with the primary structures of reported proPO and haemocyanin sequences of the kuruma prawn, respectively. Reverse transcription PCR analysis showed that this enzyme was synthesized in the hepatopancreas rather than in haemocytes. Although the primary structure and enzymatic properties of this novel enzyme suggested that it is a phenoloxidase, its biogenesis, tissue distribution, and oligomeric state resemble those of haemocyanin, which belongs to the same protein family (type III copper protein). This novel proPO enzyme may share a role with the already characterized version, itself a major component of the innate immune system in crustaceans.

Many eukaryotic proteins are blocked at the α-amino group of their N-terminal with various modifications, thereby making it difficult to determine their N-terminal sequence by protein sequencer. We propose a novel method for selectively isolating the blocked N-terminal peptide from the peptide mixture generated by endoproteinase AspN digestion of N-blocked protein. This method is based on removal of all peptides other than the N-terminal one (non-N-terminal peptides) through their carbonyl group introduced by a chemical transamination reaction. The transamination reaction converts the free α-amino group of the non-N-terminal peptides to a carbonyl group, whereas the blocked N-terminal peptide, which lacks only the free α-amino group, remains unchanged. Silica functionalized with the tosylhydrazino group effectively captures non-N-terminal peptides through their carbonyl group; thus, the blocked N-terminal peptide is selectively recovered in the supernatant. This method was applied to several model proteins, and their N-terminal peptides were successfully isolated and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, the method was extended to N-terminal analysis of N-free protein by artificially blocking the free α-amino group of its N-terminal with N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide reagent.

The thermophilic oxic process (TOP) is a composting process that enables simultaneous complete decomposition and evaporation of organic waste under high temperature conditions supported by well-balanced calorific value control. To develop the simulation model for TOP, three-dimensional relationships among decomposition rate constant, temperature (20-70 °C) and moisture content (30-70%) were determined for swine waste and cooking oil based on the oxygen consumption rate during a thermophilic oxic decomposition reaction. The decomposition rate of swine waste and cooking oil under various moisture contents was described by the Arrhenius equation. The optimal temperature and moisture content were 60 °C and 60% for swine waste and 60 °C and 50% for cooking oil, respectively. The simulation model for TOP was constructed on the basis of the carbon, heat, and moisture balance. The validation of the simulation model was examined by comparing the measured temperature in the TOP reactor to that estimated by the simulation. The simulation model was proven by comparing experimental and calculated values. The relationship between the injection calorific value and the process mechanism of TOP was interpreted by the simulation model. On the basis of their relationship during TOP, the appropriate process conditions were discussed.

Background Left main coronary artery (LMCA) stenosis (≥50%) has historically been recognized as a risk factor among patients undergoing coronary artery bypass grafting. Methods From January 2002 to December 2008, a total of 665 patients, 268 of whom had significant LMCA disease, underwent isolated off-pump coronary artery bypass surgery at Shiga Medical University Hospital. We compared the clinical results in the 237 patients with LMCA stenosis (LMCA group) with those in the propensity score-matched 237 patients without LMCA stenosis (non-LMCA group). We performed off-pump surgery in all coronary artery bypass grafting cases with no exclusion criteria. Results All procedures were performed by off-pump technique without conversion to on-pump. Two patients in the LMCA group (2 of 237; 0.8%) and four in the non-LMCA group (4 of 237; 1.7%) died within 30 days after surgery. Follow-up was completed in 96.2% of the patients. The rates of six-year freedom from all cause death were 87.3% and 60.7% in the LMCA group and non-LMCA group, respectively (p = 0.17), and the corresponding rates for the combined endpoint of cardiac death, myocardial infarction, angina pectoris, repeat coronary intervention, and heart failure were 80.4% and 70.4% (p = 0.98). Multivariate Cox regression analysis revealed chronic renal failure as a statistically significant predictor for late cardiac event. Conclusions Off-pump coronary artery bypass grafting is feasible and safe in patients with critical LMCA stenosis and LMCA disease is not recognized as a risk factor after off-pump coronary artery bypass grafting in either the short or the long term.

Background We compared the outcomes in propensity score-matched patients with diabetes undergoing off-pump coronary artery bypass grafting using skeletonized bilateral or single internal thoracic artery (ITA) and assessed any benefit of bilateral ITA grafting for outcomes. Methods Among 770 consecutive patients undergoing isolated coronary artery bypass graft surgery (99.2% by off-pump technique without conversion to cardiopulmonary bypass), 423 patients had diabetes mellitus. After excluding patients who were older than 85 years of age (n = 10) or had only one target vessel at the left coronary area (n = 9), 170 pairs were matched using propensity scores created on the basis of 12 preoperative factors. Results Except for 1 patient, bilateral ITA was anastomosed to the left coronary system. Postoperative serum glucose was well controlled in both groups. The rate of deep sternal infection was similar between the groups. The mean observation period was 3.2 years. The 5-year survival free from overall death in bilateral versus single ITA grafting was 87.5% versus 70.8% (log-rank test p = 0.01). For freedom from cardiac death, the respective rate was 92.1% versus 78.7% (p = 0.01). For freedom from cardiac event, the respective rate was 91.0% versus 72.6% (p = 0.01). In multivariate Cox models, bilateral ITA grafting was significantly associated with a lower risk for overall death (hazard ratio, 0.45; 95% confidence interval, 0.22 to 0.89; p = 0.02), cardiac death (hazard ratio, 0.43; 95% confidence interval, 0.21 to 0.87; p = 0.02), and cardiac event (hazard ratio, 0.42; 95% confidence interval, 0.20 to 0.85; p = 0.02). Conclusions Off-pump skeletonized left-sided bilateral ITA grafting is associated with better mid-term outcomes than single ITA grafting, without increasing the risk of deep sternal infection.

Planarian regeneration depends on the presence and precise regulation of pluripotent adult somatic stem cells named neoblasts, which differentiate to replace cells of any missing tissue. A characteristic feature of neoblasts is the presence of large perinuclear nonmembranous organelles named “chromatoid bodies”, which are comparable to ribonucleoprotein structures found in germ cells of organisms across different phyla. In order to better understand regulation of gene expression in neoblasts, and potentially the function and composition of chromatoid bodies, we characterized homologues to known germ and soma ribonucleoprotein granule components from other organisms and analyzed their function during regeneration of the planarian Dugesia japonica. Expression in neoblasts was detected for 49 of 55 analyzed genes, highlighting the prevalence of post-transcriptional regulation in planarian stem cells. RNAi-mediated knockdown of two factors [ago-2 and bruli] lead to loss of neoblasts, and consequently loss of regeneration, corroborating with results previously reported for a bruli ortholog in the planarian Schmidtea mediterranea (Guo et al., 2006). Conversely, depletion mRNA turnover factors [edc-4 or upf-1], exoribonucleases [xrn-1 or xrn-2], or DEAD box RNA helicases [Djcbc-1 or vas-1] inhibited planarian regeneration, but did not reduce neoblast proliferation or abundance. We also found that depletion of cap-dependent translation initiation factors eIF-3A or eIF-2A interrupted cell cycle progression outside the M-phase of mitosis. Our results show that a set of post-transcriptional regulators is required to maintain the stem cell identity in neoblasts, while another facilitates proper differentiation. We propose that planarian neoblasts maintain pluripotency by employing mechanisms of post-transcriptional regulation exhibited in germ cells and early development of most metazoans.

Planted floating-beds can be used for treating eutrophic water in a simple and cost-effective manner, the performance of which is, however, unavoidably restricted by the growth rate and limited stand biomass of the plant. A novel approach is reported here to enhance the performance of traditional planted floating-bed by introduction of filter-feeding bivalve and biofilm carrier. The objective of the present study was to prove that the co-existence of the three key components of the ecological floating-bed is necessary for the integrated ecological floating-bed (IEFB) and for the evaluation of the influence of the water exchange period on the water purification efficacy of IEFB. The mesocosm experiments were carried out at the shore of Meiliang Bay, north part of Lake Taihu, China. The IEFB concurrently employing plant (Ipomoea aquatica), freshwater clams (Corbicula fluminea) and biofilm carrier (an artificial semi-soft assembly medium) performed better than the other two kinds of floating-beds, one of which was constructed with freshwater clams and biofilm carrier, while the other one consisted of plant and biofilm carrier. Moreover, percentage reductions of pollutants increased with extended water exchange period. With a water exchange period of 7 d, the removal efficiencies of IEFB for total nitrogen (TN), ammonium nitrogen (NH4+-N), total phosphorus (TP), total organic carbon (TOC), chlorophyll-a (Chl-a), total microcystin-LR and extracellular microcystin-LR were 52.7%, 33.7%, 54.5%, 49.2%, 80.2%, 77.4% and 68.0%, respectively.

Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

A novel method for selectively labeling and isolating N-terminal peptide from protein has been developed. An N(alpha)-amino group of protein was converted to a carbonyl group through transamination reaction and the resulting carbonyl group was modified with O-(4-nitrobenzyl)hydroxylamine (NBHA). After proteolytic digestion using Grifola frondosa metalloendopeptidase (LysN), the modified N-terminal peptide remained unbound in the following treatment using amino-reactive p-phenylenediisothiocyanate (DITC) glass, whereas peptides other than the N-terminal peptide were effectively scavenged from the supernatant solution. The modified N-terminal peptide was thus successfully isolated and sequenced by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) analysis.

Recent studies indicate that the balance between cell survival and proapoptotic signals determines which cells commit to life or death. We have shown that the balance between follicle-stimulating hormone and prolactin determines differentiation or apoptosis in 7th generation spermatogonia during newt spermatogenesis; however, the molecular mechanisms specifying their fate are poorly understood. Here we show that the newt RNA-binding protein (nRBP) plays a critical role in determining their fate. nRBP was identified as a clone whose mRNA is decreased by prolactin, resulting in the reduction of the protein, which is otherwise expressed predominantly in the spermatogonia. nRBP protein associated with the mRNA for newt programmed cell death protein 4 (nPdcd4) at the 3′-untranslated region. nRBP reduction increased nPdcd4 mRNA but decreased its protein. In a cell-free system, cytoplasmic extracts containing reduced amounts of nRBP and nPdcd4 protein induced apoptosis, whereas adding nRBP protein to the extracts blocked apoptosis. Furthermore, overexpression of nRBP protected cells from apoptosis, stabilized the chimeric transcript containing the nPdcd4 3′-untranslated region, and accelerated its translation. These data suggest that, in the absence of nRBP, nPdcd4 mRNA is not stabilized and its translation is suppressed, leading to apoptosis in the spermatogonia.

SummaryVertebrate Wnt proteins activate several distinct pathways. Intrinsic differences among Wnt ligands and Frizzled (Fzd) receptors, and the availability of pathway-specific coreceptors, LRP5/6, and Ror2, affect pathway selection. Here, we show that a secreted glycoprotein, Cthrc1, is involved in selective activation of the planar cell polarity (PCP) pathway by Wnt proteins. Although Cthrc1 null mutant mice appeared normal, the introduction of a heterozygous mutation of a PCP gene, Vangl2, resulted in abnormalities characteristic of PCP mutants. In HEK293T cells, Cthrc1 activated the PCP pathway but suppressed the canonical pathway. Cell-surface-anchored Cthrc1 bound to Wnt proteins, Fzd proteins, and Ror2 and enhanced the interaction of Wnt proteins and Fzd/Ror2 by forming the Cthrc1-Wnt-Fzd/Ror2 complex. Consistent with this, Ror2 mutant mice also showed PCP-related abnormalities in the inner ear. These results suggest that Cthrc1 is a Wnt cofactor protein that selectively activates the Wnt/PCP pathway by stabilizing ligand-receptor interaction.

The remarkable regenerative ability of planarians is made possible by a system of pluripotent stem cells. Recent molecular biological and ultrastructural studies have revealed that planarian stem cells consist of heterogeneous populations, which can be classified into several subsets according to their differential expression of RNA binding protein genes. In this study, we focused on planarian musashi family genes. Musashi encodes an evolutionarily conserved RNA binding protein known to be expressed in neural lineage cells, including neural stem cells, in many animals. Here, we investigated whether planarian musashi-like genes can be used as markers for detecting neural fate-restricted cells. Three musashi family genes, DjmlgA, DjmlgB and DjmlgC (Dugesia japonica musashi-like gene A, B, C), and Djdmlg (Dugesia japonica DAZAP-like/musashi-like gene) were obtained by searching a planarian EST database and 5' RACE, and each was found to have two RNA recognition motifs. We analyzed the types of cells expressing DjmlgA, DjmlgB, DjmlgC and Djdmlg by in situ hybridization, RT-PCR and single-cell RT-PCR analysis. Although Djdmlg was expressed in X-ray-sensitive stem cells and various types of differentiated cells, expression of the other three musashi-like genes was restricted to neural cells, as we expected. Further detailed analyses yielded the unexpected finding that these three planarian musashi family genes were predominantly expressed in X-ray-resistant differentiated neurons, but not in X-ray-sensitive stem cells. RNAi experiments suggested that these planarian musashi family genes might be involved in neural cell differentiation after neural cell-fate commitment.

The effects of bacterial activity on the degradation of estrone (E1), estradiol (E2), estriol (E3) and ethinylestradiol (EE2) in nitrifying activated sludge (NAS) were studied with different substrates and organic loading rates (OLRs) and low temperature conditions. Heterotroph was shown to have utilized glucose prior to E1 for metabolism. The co-metabolism of ammonia oxidizing bacteria (AOB) dominated the degradation of E1, E2 and EE2 in NAS. The higher the organic loading, the higher the rate of organic matter transformation, with less ammonia oxidation and less degradation of E1, E2 and EE2. The degradation of E3 in NAS was shown to be largely due to heterotroph metabolism. On the basis of the difference of apparent activity between heterotroph and AOB at 4 degrees C, the process of estrogen degradation via the co-metabolism of AOB was able to be identified.

Temporal variations in sediment carbon content were tracked by sampling every 2 weeks for 6 to 33 months at 6 tidal flat stations with different carbon content levels (0.05–1.64%) in a single estuary. Three temporal series of current velocity at 5 cm above the sediment were also obtained. Non-cohesive and cohesive sediment stations differed in patterns of temporal variation in sediment carbon content, suggesting the difference in processes controlling sediment carbon content. In the stations of non-cohesive sandy sediment with relatively low carbon content (0.05–0.15%), sediment carbon content fluctuated within ranges specific to each station. In these stations, current velocity data suggested that frequency of sand resuspension washing out sediment carbon controls sediment carbon content level. In the stations of cohesive sediment with relatively high carbon content (0.77–1.64%), sediment carbon contents showed some unusual upward and downward peaks, recovered to usual levels specific to the stations, and was always kept higher than that in the non-cohesive sediment stations. We speculate that in the cohesive sediment stations, sand inputs may significantly control processes establishing the consolidated sediments with various carbon content levels. Spatial transitions from non-cohesive to cohesive sediments and from low to high sediment carbon contents likely occur as sand resuspension at spring tides become more irregular and less frequent. Based on these results, conceptual models describing physical processes controlling sediment carbon content at the studied stations were proposed.

To establish a simple and sensitive method to detect protein N-myristoylation, the usefulness of a newly developed cell-free protein synthesis system derived from insect cells for detecting protein N-myristoylation by in vitro metabolic labeling was examined. The results showed that in vitro translation of cDNA coding for N-myristoylated protein in the presence of [3H]myristic acid followed by SDS–PAGE and fluorography is a useful method for rapid detection of protein N-myristoylation. Differential labeling of N-myristoylated model proteins with [3H]leucine, [3H]myristic acid, and [35S]methionine revealed that the removal of the initiating Met during the N-myristoylation reaction could be detected using this system. Analysis of the N-myristoylation of a series of model proteins with mutated N-myristoylation motifs revealed that the amino acid sequence requirements for the N-myristoylation reaction in this system are quite similar to those observed in the rabbit reticulocyte lysate system. N-myristoylation of tBid (a posttranslationally N-myristoylated cytotoxic protein that could not be expressed in transfected cells) was successfully detected in this assay system. Thus, metabolic labeling in an insect cell-free protein synthesis system is an effective strategy to detect co- and posttranslational protein N-myristoylation irrespective of the cytotoxicity of the protein.

We have identified tyrosine-phosphorylated proteins on membrane from A-431 human epidermoid carcinoma cells by using detection with anti-phosphotyrosine antibody followed by PMF analysis. In there, on-membrane digestion for these protein spots was carried out on microscale region using chemical inkjet technology and the resulting tryptic digests were directly analyzed by MALDI-TOF MS. Proteins identified by a database search included phosphoproteins that are known to be markedly phosphorylated on tyrosine sites after the cells are treated with epidermal growth factor (EGF). This procedure is a rapid and easily handled approach that enables both detection and identification of phosphoproteins on a single blot membrane.

We determined the range of the tidal variations in nutrient flux across the sediment–water interface and elucidated mechanisms of the flux variation in two estuarine intertidal flats (one sand, one mud) in northeastern Japan. Nutrient flux was measured using in situ light and dark chambers, which were incubated for 2 h, 2–6 times per day. Results showed that nutrient concentration in overlying water varied by tide and was also affected by sewage-treated water inflow. The nutrient fluxes responded quickly to the tidal variation in overlying water chemistry and the range of the variation in flux was as large as the seasonal-scale variation reported in previous studies. In the sand flat, salinity increase likely enhanced benthos respiration and led to increases in both O 2 consumption and PO 4 3− regeneration under low illumination, while benthic microalgae were likely to actively generate O 2 , uptake PO 4 3− and suppress PO 4 3− release under high illumination (>900 μmol photons m −2 s −1 ). Also in the mud flat, PO 4 3− flux was related with O 2 flux, although the range of temporal variation in PO 4 3− flux was small. In both the flats, NH 4 + flux was always governed by NH 4 + concentration in the overlying water; either an increase in NH 4 + uptake or a decrease in NH 4 + release was observed as the NH 4 + concentration rose due to inflow of river water or input of sewage-treated water. Although NO 3 − tended to be released in both tidal flats when low NO 3 − concentration seawater dominated, their relationship was likely to be weakened under conditions of low oxygen consumption and suppressed denitrification. It is likely that tidal variation in nutrient flux is governed more by the nutrient concentration than other factors, such as benthic biological processes, particularly in the case where nutrient concentration in the overlying water is relatively high and with wide amplitude.

Embryonic stem (ES) cells have generated enormous interest due to their capacity to self-renew and the potential for growing many different cell types in vitro. Leukemia inhibitory factor (LIF), bone morphogenetic proteins, octamer-binding protein 3 or 4, and Nanog are important factors in the maintenance of pluripotency in mouse ES cells. However, the mechanisms by which these factors regulate the pluripotency remain poorly understood. To identify other proteins involved in this process, we did a proteomic analysis of mouse ES cells that were cultured in the presence or absence of LIF. More than 100 proteins were found to be involved specifically in either the differentiation process or the maintenance of undifferentiated state. Among these, chromatin-related proteins were identified as the major proteins in nuclear extracts of undifferentiated cells. Analysis with real-time RT-PCR revealed that enrichment of these proteins in pluripotent ES cells was regulated at the transcriptional levels. These results suggest that specific chromatin-related proteins may be involved in maintaining the unique properties of pluripotent ES cells.

Betacellulin (BTC) is a member of the epidermal growth factor family. It has two biological activities: mitogenic activity in fibroblasts and vascular smooth muscle cells, and differentiation activity for the differentiation of pancreatic acinar AR42J cells into insulin-secreting cells. The previous finding that recombinant BTC promotes the neogenesis of β-cells in a mouse model supports the possibility that BTC is a therapeutic protein. However, the mitogenic activity of BTC may not be needed for differentiation into β-cells and may cause a side effect in clinical use. We prepared several derivatives of BTC to segregate the two activities, to decrease the mitogenic activity, and to maintain the differentiation activity. We succeeded in obtaining BTC derivatives segregated by the two biological activities by preparing truncated-type derivatives. A derivative of BTC, BTC24–76, with a truncated N-terminal 23 amino acids and C-terminal 4 amino acids, was 2.5-fold more active in differentiation and had one-tenth of the mitogenic activity. The derivatives described in the present study should be helpful in future applications as therapeutic proteins and in basic research for discovery of a BTC-specific receptor.

In a search for novel genes expressed in human cancers, we identified one gene from an assembled expressed sequence tag database. Northern blot analysis revealed that the gene, termed alcan, was expressed in various types of human cancer cell lines and in the fetus, but not in normal tissues. The alcan gene is located on chromosome 6 and is encoded on a 246-amino-acid protein with weak homology to classical major histocompatibility complex class I. Its gene product, ALCAN, had hydrophobic amino acid clusters at both the N- and C-terminal regions and was predicted to be a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Flow cytometric analysis revealed that ALCAN was detected on the surface of human cancer cells and on alcan-transfected CHO-K1 cells. ALCAN was also secreted from these cells, suggesting that some portion of the molecules was secreted by enzymatic cleavage by, for example, phospholipases. Mutational analysis of ALCAN suggested that the GPI-anchored position was the Ser(216) residue. These findings indicate that ALCAN may be a potential target for cancer diagnosis or therapy.

We analyzed the tissue distribution of apelin mRNA in rats by a quantitative reverse transcription-polymerase chain reaction and that of immunoreactive apelin (ir-apelin) by an enzyme immunoassay (EIA) using a monoclonal antibody. The expression levels of apelin mRNA and ir-apelin seemed to be consistent among tissues: they were highly expressed in the lung and mammary gland. By the combination of gel filtration and EIA, we found that the molecular forms of apelin differ among respective tissues: apelin molecules with sizes close to apelin-36 (long forms) were major components in the lung, testis, and uterus, but both long and short (whose sizes were close to [

Neuromedin U is a bioactive peptide isolated originally from the porcine spinal cord. We recently identified neuromedin U as the cognate ligand for the orphan G protein-coupled receptor FM-3. In this study, we isolated cDNA coding for a novel G protein-coupled receptor, TGR-1, which was highly homologous with FM-3. We found that neuromedin U specifically and clearly elevated the extracellular acidification rates, arachidonic acid metabolite release, and intracellular Ca(2+) mobilization in Chinese hamster ovary cells expressing TGR-1. Radiolabeled neuromedin U specifically bound with high affinity to membrane fractions prepared from these cells. These results show that TGR-1, like FM-3, is a specific and functional receptor for neuromedin U. We analyzed TGR-1 mRNA tissue distribution in rats using quantitative reverse transcription-polymerase chain reaction and found it to considerably differ from that of FM-3 mRNA. TGR-1 mRNA was primarily expressed in the uterus, suggesting that TGR-1 mediates the contractile activity of neuromedin U in this tissue. The identification of specific and functional receptor subtypes for neuromedin U will facilitate the study of their physiological roles and the search for their specific agonists and antagonists.

We have recently identified apelin as the endogenous ligand for human APJ. In rats, the highest expression of APJ mRNA was detected in the lung, suggesting that APJ and its ligand play an important role in the pulmonary system. When apelin-36 and its pyroglutamylated C-terminal peptide, [

Prolactin-releasing peptide (PrRP) is a recently isolated hypothalamic peptide which is an endogenous ligand to an orphan receptor. We previously demonstrated that PrRP neurons are widely distributed throughout the rat brain and suggested that PrRP may have important functions in the central nervous system. To analyze the function of PrRP, we studied the effect of intracerebroventricular (i.c.v.) PrRP administration on c-Fos protein accumulation in the rat brain. The results clearly indicated that c-Fos protein accumulation was dramatically increased in the nuclei of corticotropin-releasing hormone (CRH)-positive parvocellular neurosecretory cells in the paraventricular nucleus (PVN). We also demonstrated synapse-like contact between PrRP neurons and CRH cell bodies in the PVN, which suggests that PrRP31 has some effect on CRH secretion. We therefore investigated the effect of i.c.v. administration of PrRP31 on the CRH-mediated increase in adrenocorticotropin (ACTH) levels, and found that plasma ACTH levels were indeed increased by i.c.v. PrRP31. In addition, animals pre-treated with intravenous alpha-helical CRH, a potent CRH antagonist, showed attenuated plasma ACTH responses after i.c.v. PrRP31 administration. These results strongly suggest that PrRP affects the hypothalamic-pituitary-adrenal axis.

Infarct volume and immunoreactivities for trkB and trkC in rat brain were compared at 24 h after 90 min of transient middle cerebral artery occlusion (MCAO) between animal groups with or without neurotrophin-3 (NT-3, 10 μg/250 g animal). Treatment of rat brain with topical application of NT-3 significantly reduced infarct volume ( P =0.02) and trkB and trkC inductions. These data suggest that NT-3 reduced the ischemic injury along with the reduction of trkB and trkC inductions.