Search

Continuous long-term monitoring is important for detecting ecological changes and understanding their causes, including anthropogenic impacts such as climate changes and eutrophication. Nonetheless, such long-term population studies have been rare, especially for sea urchins, which can affect community dynamics owing to their extensive herbivory and large population fluctuations. Here we present a long-term (from 1963 to 2014) dynamics of sea urchins in a fixed quadrat on a lower intertidal rocky flat in Hatakejima Island, southern Japan. We also conducted a complementary survey over the entire island approximately every five years from 1975 to 2013, and a 41-year assay for developmental abnormality of the sea urchin Heliocidaris crassispina using water adjacent to the island. The abundance of three commonest species in this area (H. crassispina, Echinostrephus molaris, and Echinometra spp.) and the richness of urchin species showed large variations, with high numbers in the 1960s and 1970s, followed by an abrupt decline in the late 1970s or early 1980s, and a gradual recovery subsequently. The species abundance and richness in the entire island survey showed good correlations with those in the quadrat census. Statistical analyses indicated that increasing water temperature and red tides were the major factors influencing the dynamics of abundance and species richness. Our studies reveal that anthropogenic environmental changes influence the long-term dynamics in abundance and richness of sea urchins.

Ocean acidification is widely recognised to have a negative impact on marine calcifying organisms by reducing calcifications, but controversy remains over whether such organisms could cope with ocean acidification within a range of phenotypic plasticity and/or adapt to future acidifying ocean. We performed a laboratory rearing experiment using clonal fragments of the common branching corals Montipora digitata and Porites cylindrica under control and acidified seawater (lower pH) conditions (approximately 400 and 900μatm pCO2, respectively) and evaluated the intraspecific variations in their responses to ocean acidification. Intra- and interspecific variations in calcification and photosynthetic efficiency were evident according to both pCO2 conditions and colony, indicating that responses to acidification may be individually variable at the colony level. Our results suggest that some corals may cope with ocean acidification within their present genotypic composition by adaptation through phenotypic plasticity, while others may be placed under selective pressures resulting in population alteration.

Coastal ecosystems can provide significantly high ecosystem services via various processes. Tropical and/or subtropical coastal ecosystems are constituted by reef-building corals, macroalgae, and seagrasses. The study was conducted in a subtropical coastal area, Okinawa Island, Japan, where macroalgal beds are distributed. Since few studies have focused on the macroalgal community compared to corals in the study area, the relationship between macroalgal (especially Sargassaceae species) distribution and environmental factors were evaluated. Environmental parameters used in the analysis were obtained by field observation, geographic analysis, satellite observation of water quality, and numerical simulation of wave height. Sampling was conducted mainly between May and August in 2018 and 2019, and 31 sampling stations were established along the coastline of Okinawa Island. Though hermatypic corals occurred in most of the stations, the distribution of Sargassaceae was spatially skewed from the southern and central part of the east coast. Canonical correspondence analysis (CCA) was applied to obtained environmental variables and presence–absence data on macroalgae (Sargassaceae), corals, and seagrasses. CCA results indicated that the geographical setting (openness) and wave exposure in winter are the primary factors determining the macroalgal distribution, whereas nutrient (dissolved inorganic nitrogen, phosphorous, and silicon) concentrations at each sampling occasion were not significant factors. Since coastal ecosystems are threatened by both local and global impacts, elucidation of the major mechanism limiting Sargassaceae distribution is significant for coastal ecosystem management, here we use Okinawa Island as an example.

Determining the success of eradication of an invasive species requires a way to decide when its risk of reoccurrence has become acceptably low. In Japan, the area populated by the Argentine ant, Linepithema humile (Mayr), is expanding, and eradication via chemical treatment is ongoing at various locations. One such program in Tokyo was apparently successful, because the ant population decreased to undetectable levels within a short time. However, construction of a population model for management purposes was difficult because the probability of detecting ants decreases rapidly as the population collapses. To predict the time when the ant was eradicated, we developed a multinomial-mixture model for chemical eradication based on monthly trapping data and the history of pesticide applications. We decided when to declare that eradication had been successful by considering both 'eradication' times, which we associated with eradication probabilities of 95% and 99%, and an optimal stopping time based on a 'minimum expected economic cost' that considered the possibility that surveys were stopped too soon. By applying these criteria, we retroactively declared that Argentine ants had been eradicated 38-42 months after the start of treatments (16-17 months after the last sighting).

We compared spatial variability in re- cruitment patterns between acroporid corals and the predatory crown-of-thorns starfish Acanthaster planci along the Onna village coast of Okinawa Island, Japan. Our objective was to understand population dynamics of both predator and prey. To quantify re- cruitment, we established artificial recruitment plates for acroporid corals and conducted quadrat surveys for juvenile A. planci at 7 sites. We also examined dis- tributional patterns of adult populations of both spe- cies and observed surface currents during larval dis- persal periods using GPS-equipped surface drifters. Acroporid recruits typically arrived in greater num- bers at more northern sites. Despite high variability, A. planci recruits displayed similar spatial patterns. Drifter trajectories suggest that these spatial recruit- ment patterns are probably driven by near-shore currents in the summer. Moreover, current data imply a complicated metapopulation relationship in this region, especially for A. planci, new recruits of which may mainly originate outside of the Onna area. Meta - population dynamics within the study area and among surrounding regions must be explained by the com- plex surface current circulation, because simpler ex- planations involving only the Kuroshio Current do not adequately describe the relationship between these populations.

Seaweed beds play a key role in providing essential habitats and energy to coastal areas, with enhancements in productivity and biodiversity and benefits to human societies. However, the spatial extent of seaweed beds around Japan has decreased due to coastal reclamation, water quality changes, rising water temperatures, and heavy grazing by herbivores. Using monthly mean sea surface temperature (SST) data from 1960 to 2099 and SST-based indices, we quantitatively evaluated the effects of warming seawater on the spatial extent of suitable versus unsuitable habitats for temperate seaweed Ecklonia cava, which is predominantly found in southern Japanese waters. SST data were generated using the most recent multiple climate projection models and emission scenarios (the Representative Concentration Pathways or RCPs) used in the Coupled Model Intercomparison Project phase 5 (CMIP5). In addition, grazing by Siganus fuscescens, an herbivorous fish, was evaluated under the four RCP simulations. Our results suggest that continued warming may drive a poleward shift in the distribution of E. cava, with large differences depending on the climate scenario. For the lowest emission scenario (RCP2.6), most existing E. cava populations would not be impacted by seawater warming directly but would be adversely affected by intensified year-round grazing. For the highest emission scenario (RCP8.5), previously suitable habitats throughout coastal Japan would become untenable for E. cava by the 2090s, due to both high-temperature stress and intensified grazing. Our projections highlight the importance of not only mitigating regional warming due to climate change, but also protecting E. cava from herbivores to conserve suitable habitats on the Japanese coast.

After the adoption of the Aichi Target, data accumulation and evaluation regarding biodiversity have progressed rapidly. The use of ecologically and biologically significant areas (EBSAs) criteria to evaluate important areas enables the identification of effective and prioritized areas for ecosystem management. This includes strategic environmental assessment and discussions aimed at establishing protected marine areas based on scientific data. This paper reviews previous and current ideas as well as the methods used, for the identification of EBSAs. In particular, the following issues are addressed: problems associated with different types of marine ecosystems in the Japanese Archipelago, such as seagrass and seaweed beds, coral reefs, offshore pelagic plankton, and deep-sea benthic ecosystems; and problems associated with the integration of multiple criteria that are not totally exclusive. Several candidate variables accounting for each of the 7 criteria used to identify ecologically important areas are presented. Data availability is the most important criterion that allowed for the comprehensive evaluation of different types of ecosystems in the same localities. In particular, for coastal ecosystems such as seagrass, seaweed beds, and coral reefs, it is possible to carry out broad spatial comparisons using variables representing most of these 7 criteria. Regarding methods for the quantitative evaluation of each criterion and their integration, application of these methods to kelp forest ecosystems in Hokkaido, Northern Japan is presented as a case study.

Determining the success of eradication of an invasive species requires a way to decide when its risk of reoccurrence has become acceptably low. In Japan, the area populated by the Argentine ant, Linepithema humile (Mayr), is expanding, and eradication via chemical treatment is ongoing at various locations. One such program in Tokyo was apparently successful, because the ant population decreased to undetectable levels within a short time. However, construction of a population model for management purposes was difficult because the probability of detecting ants decreases rapidly as the population collapses. To predict the time when the ant was eradicated, we developed a multinomial-mixture model for chemical eradication based on monthly trapping data and the history of pesticide applications. We decided when to declare that eradication had been successful by considering both ‘eradication’ times, which we associated with eradication probabilities of 95% and 99%, and an optimal stopping time based on a ‘minimum expected economic cost’ that considered the possibility that surveys were stopped too soon. By applying these criteria, we retroactively declared that Argentine ants had been eradicated 38–42 months after the start of treatments (16–17 months after the last sighting).

We evaluated acidification effects on two crustose coralline algal species common to Pacific coral reefs, Lithophyllum kotschyanum and Hydrolithon samoense. We used genetically homogeneous samples of both species to eliminate misidentification of species. The growth rates and percent calcification of the walls of the epithallial cells (thallus surface cells) of both species decreased with increasing pCO2. However, elevated pCO2 more strongly inhibited the growth of L. kotschyanum versus H. samoense. The trend of decreasing percent calcification of the cell wall did not differ between these species, although intercellular calcification of the epithallial cells in L. kotschyanum was apparently reduced at elevated pCO2, a result that might indicate that there are differences in the solubility or density of the calcite skeletons of these two species. These results can provide knowledge fundamental to future studies of the physiological and genetic mechanisms that underlie the response of crustose coralline algae to environmental stresses.

Background The coral genus Acropora contains more than 150 species with very high morphological diversity. This high diversity may have been caused by repeated hybridization via mass spawning. However, we have little information whether hybrids are formed in these corals. Identifying morphological differences between hybrids and their parental species would provide an opportunity to find wild hybrids in the field and to understand how colony shapes of Acropora have become highly diversified throughout evolutionary history. In the two morphologically distinctive coral species Acropora florida and A. intermedia in the Indo-Pacific, their gametes show high rates of bi-directional intercrossing in vitro, and thus these two species are ideal species to investigate the morphological traits of the hybrids. Methods We examined morphological characters of F1 hybrids from A. florida to A. intermedia, which were produced from in vitro crossing experiments. To compare morphological differences, we grew juveniles and mature colonies of reciprocal F1 hybrids (FLOint: A. florida eggs × A. intermedia sperm, and INTflo: A. intermedia eggs × A. florida sperm) and of the parental species (purebreds of A. intermedia and A. florida). We analyzed skeletal morphology such as colony size, branch length, and branching number, and compared them with those of a putative F1 hybrid between A. florida and A. intermedia found in the field. We also confirmed the molecular phylogenetic position of F1 hybrids, parental species, and a putative F1 hybrid using the mitochondrial non-coding region. Results Our morphological analysis revealed that branching number of the F1 hybrids was intermediate relative to the parental species. Moreover, the FLOint hybrids were morphologically more closely related to the maternal species A. florida, and the INTflo hybrids were to A. intermedia. Molecular data showed that A. florida and A. intermedia were clearly divided into two clades, and that F1 hybrids grouped in the clade based on their maternal parent. A very similar pattern to the INTflo hybrids was obtained for the putative F1 hybrid in nature. Discussion Our results revealed that F1 hybrids between two Indo-Pacific species A. florida and A. intermedia had intermediate morphology relative to their parent species but reflected the maternal parent more. Similarity to maternal species in hybrids is opposite to the Caribbean Acropora species that had more paternal morphological characters in hybrids. These results further suggest that some genetic factor in eggs is likely to affect determination of colony shape in the Indo-Pacific. At present, we have considered colonies with intermediate morphs between different species to be intra-specific morphological variation, but they may be real F1 hybrids. Indeed, a putative F1 hybrid represented similar morphological and molecular features to the F1 hybrids, and thus it is plausible to be attributed as a “real” F1 hybrid in nature.

Human embryonic stem cells (hESCs) and induced pluripotent cells have the potential to provide an unlimited source of tissues for regenerative medicine. For this purpose, development of defined/xeno-free culture systems under feeder-free conditions is essential for the expansion of hESCs. Most defined/xeno-free media for the culture of hESCs contain basic fibroblast growth factor (bFGF). Therefore, bFGF is thought to have an almost essential role for the expansion of hESCs in an undifferentiated state. Here, we report identification of small molecules, some of which were neurotransmitter antagonists (trimipramine and ethopropazine), which promote long-term hESC self-renewal without bFGF in the medium. The hESCs maintained high expression levels of pluripotency markers, had a normal karyotype after 20 passages, and could differentiate into all three germ layers.

Seagrass meadows provide both habitats and a range of food sources for macrofaunal communities. These functions facilitate the coexistence of less mobile invertebrates (in comparison with mysids, such as amphipods) that are associated with seagrass leaves, and may also enhance the coexistence of highly mobile invertebrates such as mysid. We investigated the function of seagrass in supporting the coexistence of two mysid species, Neomysis awatschensis and N. mirabilis. These taxa are dominant in seagrass ecosystems of temperate coastal areas. We compared patterns of habitat use between the two species at mesoscales (among seagrass patches) and microscales (among seagrass leaves) by performing field surveys and laboratory experiments. The field survey results showed positive correlations in the abundance of the two mysid species, indicating that both species select similar habitats at the mesoscale level. In the laboratory experiments, the pattern of microhabitat selection (fundamental habitat) was similar for both species, even at increased densities and with the presence of an immobile habitat-competitor (the gastropod Barleeia angustata) on the leaves. However, this pattern changed significantly when a food source (epiphytic microalgae) was present on the leaves. This result indicates that (i) inter- and intraspecific interference competition does not affect microhabitat selection in these two mysids and (ii) both Neomysis species use similar habitats at the feeding stage. Although these two closely related mysids species may have similar requirements for microhabitat and food, the evidence that they did not act as competitors is attributable to unrestricted microhabitat and food (e.g., epiphytic algae) in the presence of seagrass vegetation.

Two closely related, Coffea arabica cultivars Tall Mokka and Typica, yield coffee with excellent flavor, but they differ distinctively in the size of aerial organs including the fruit and in the degree of branching. Using potato cross-species microarray hybridization, we identified prolyl oligopeptidase (CaPOP) gene(s) as differentially expressed between the shoot tips of ‘Tall Mokka’ and ‘Typica’. Isolation and sequencing of these POP genes identified three paralogs, CaPOP1, CaPOP2 and CaPOP3. All three genes were present in both cultivars, which suggest that differences in the expression of CaPOP are caused by factor(s) regulating their transcription. CaPOP1 differs in sequence from CaPOP2 primarily in having two large deletions in the promoter region. CaPOP3 is probably a recombination product of CaPOP1 and CaPOP2 and encodes a protein identical to CaPOP2. Coffee’s POP (CaPOP) genes are homologous to the arabidopsis gene At1g20380, encoding a post-proline cleaving enzyme that acts on substrates shorter than 30 amino acids. Ectopic expression of CaPOP1 under its native promoter in transgenic arabidopsis resulted in more secondary branches than in the wild type. This is the first study to successfully isolate CaPOP genes and characterize their expression in the developing tissues of coffee. This study also reports a novel role for prolyl oligopeptidase in control of lateral shoot branching.

Metabolic experiments were conducted for each Thai native (TN) and Anglo Nubian–TN 50% (AN × TN) bucks, 26.5–38 kg BW and 35–52 months of age, by assigning the following concentrate diets, which had four levels of CP concentrations, T1: 14%, T2: 21%, T3: 28% and T4: 35%, on a DM basis. The animals were fed the concentrates at a rate of 1% of their BW and had ad libitum access to Paspalum plicatulum hay. As the CP level increased, digestibility of CP, N excretion in urine and N retention increased ( P P 0.75 day) versus 36.0 g/(kg BW 0.75 day)), thus N intake of AN × TN was higher than that of TN ( P 0.75 day) versus 0.90 g/(kg BW 0.75 day)). Digestibility of CP and blood urea N concentrations of TN were higher than those of AN × TN ( P 0.75 day) versus 0.11 g/(kg BW 0.75 day)) and T4 (0.25 g/(kg BW 0.75 day) versus 0.17 g/(kg BW 0.75 day)). Digestibility of NDF and ADF, and TDN of TN were higher than those of AN × TN ( P 0.75 day), and no significant difference of DE among CP levels nor breeds of bucks was observed. Thai native was superior to AN × TN in digestibility of N and fiber fractions at the maintenance level of DE, which might have overcome inferiority of TN to AN × TN in amount of N and energy intake caused by the lower DMI. It is likely that TN goats are well adapted to fodder shortage condition due to their efficient utilization of nutrients.

Similar to pelagic larval dispersal, it has been suggested that dispersal of post-larval benthic organisms and species with direct development plays an important role in population persis- tence. However, effective dispersal distances required for the maintenance of local populations have been poorly characterized. This study elucidated distance effects on patterns and processes in disper- sal of the amphipod Incisocalliope symbioticus, which is associated exclusively with the gorgonian octocoral Melithaea flabellifera. Dispersal patterns over an annual cycle were examined by the place- ment of defaunated gorgonians at 3 distance levels (

We have developed a novel 'electronic barcode' system that uses radio frequency identification (RFID) tags, cell phones, and portable computers to link phenotypic, environmental, and genomic data. We describe a secure, inexpensive system to record and retrieve data from plant samples. It utilizes RFID tags, computers, PDAs, and cell phones to link, record, and retrieve positional, and functional genomic data. Our results suggest that RFID tags can be used in functional genomic screens to record information that is involved in plant development or disease.

A case of extracerebellar lipomatous primitive neuroectodermal tumor (PNET) with glioblastoma multiforme (GBM) areas is reported. A 44-year-old woman who had been on antipsychotic agents for schizophrenia complained of hemiparesis and drowsiness. She deteriorated progressively and died 3 months later. The autopsy revealed a huge, ill-defined tumor located from right basal ganglia to brain stem. Microscopically, the tumor consisted of three distinct components: clusters of small primitive cells consistent with PNET, mature lipoma-like islands, and a GBM-like component. Neuronal differentiation in PNET areas was confirmed by the presence of Homer Wright rosette, synaptophysin-positive fibrillary background, and ultrastructural demonstration of neuritic processes. Lipoma-like areas composed of lipidized cells containing large lipid droplets were intimately intermingled and closely related with PNET areas. Furthermore, GBM areas were, although predominantly located in the brain stem, often blended with the previous two components. This component was characterized by glial fibrillary acid protein immunoreactivity of atypical tumor cells and the presence of necrosis and endothelial proliferation. PNET areas with lipomatous differentiation in the present tumor may suggest the morphological and histogenetic similarity to liponeurocytoma, although the neuronal element in the former was anaplastic. The association with a GBM component makes the present tumor a unique, and, to our knowledge, previously unrecognized lesion.

Neoxanthin, a precursor of the plant hormone abscisic acid, is an allenic xanthophyll recognized as the last product of carotenoid synthesis in green plants. A cDNA for neoxanthin synthase (NSY) was isolated from tomato using a molecular approach based on the mechanistic and structural similarities of NSY to two other closely related carotenogenic enzymes, lycopene cyclase (LCY) and capsanthin-capsorubin synthase (CCS). The identified tomato NSY cDNA (T.NSY) encodes a 56-kDa plastid-targeted protein that when expressed in Escherichia coli, catalyzes the conversion of violaxanthin to neoxanthin. In tobacco leaves that transiently express T.NSY, an increase in neoxanthin content with a concomitant decrease in violaxanthin is observed. NSY is structurally similar to LCY and CCS. However, in Cyanobacteria, the generally accepted progenitor of plastids, both CCS and NSY are absent while LCY is present. LCY catalyzes a simplified version of the reaction catalyzed by NSY and CCS suggesting that these two enzymes were remodeled from LCY during higher plant evolution to create new forms of oxidized carotenoids.

Rapid production of protein-based tumor-specific vaccines for the treatment of malignancies is possible with the plant-based transient expression system described here. We created a modified tobamoviral vector that encodes the idiotype-specific single-chain Fv fragment (scFv) of the immunoglobulin from the 38C13 mouse B cell lymphoma. InfectedNicotiana benthamianaplants contain high levels of secreted scFv protein in the extracellular compartment. This material reacts with an anti-idiotype antibody by Western blotting, ELISA, and affinity chromatography, suggesting that the plant-produced 38C13 scFv protein is properly folded in solution. Mice vaccinated with the affinity-purified 38C13 scFv generate >10 μg/ml anti-idiotype immunoglobulins. These mice were protected from challenge by a lethal dose of the syngeneic 38C13 tumor, similar to mice immunized with the native 38C13 IgM-keyhole limpet hemocyanin conjugate vaccine. This rapid production system for generating tumor-specific protein vaccines may provide a viable strategy for the treatment of non-Hodgkin’s lymphoma.

The biosynthesis of leaf carotenoids in Nicotiana benthamiana was altered by forced re-routing of the pathway to the synthesis of capsanthin, a non-native chromoplast-specific xanthophyll, using an RNA viral vector containing capsanthin-capsorubin synthase (Ccs) cDNA. The cDNA encoding Ccs was placed under the transcriptional control of a tobamovirus subgenomic promoter. Leaves from transfected plants expressing Ccs developed an orange phenotype and accumulated high levels of capsanthin (up to 36% of total carotenoids). This phenomenon was associated with thylakoid membrane distortion and reduction of grana stacking. In contrast to the situation prevailing in chromoplasts, capsanthin was not esterified and its increased level was balanced by a concomitant decrease of the major leaf xanthophylls, suggesting an autoregulatory control of chloroplast carotenoid composition. Capsanthin was exclusively recruited into the trimeric and monomeric light-harvesting complexes of photosystem II (PSII) and shown to significantly contribute to the light-harvesting capacity. On a chlorophyll basis, the concentrations of PSI and PSII reaction centres were not modified. This demonstration that higher plant antenna complexes can accommodate non-native carotenoids provides compelling evidence for functional remodelling of photosynthetic membranes toward a better photoreactivity by rational design of the incorporated carotenoid structures.

A chimeric enzyme, engineered from two rice α-amylase isozymes, Amy1A and Amy3D, showed unique characteristics in soluble-starch and maltoheptaose hydrolysis. Effects of pH on soluble-starch hydrolysis and the thermostability of the chimeric enzyme were similar to those of the isozyme Amy3D. The previous study revealed that Amy1A shows high activity in soluble-starch hydrolysis and low activity in oligosaccharide degradation, while Amy3D shows low activity in soluble-starch hydrolysis and high activity in oligosaccharide degradation. The chimeric enzyme showed high activities in both soluble-starch hydrolysis and oligosaccharide degradation. These results suggest that protein modules of highly homologous enzymes are interchangeable, and that a novel enzyme with unique characteristics can be obtained by creating a chimeric enzyme.

This paper describes the flora of habitat-forming seaweeds (fucoids and temperate kelps) at 7673 sites of the Japanese coast encompassing its warm to cold temperate zone, recorded from 1887 to 2014. The data set includes 86 species (21,168 presence and 20,845 absence records), compiled from 355 literature sources, most of which were written in Japanese and published as grey literature in local journals or individual reports. Scientific names were consolidated under currently-accepted nomenclature based on Algaebase ( http://www.algaebase.org ). The data set compiled the seaweed flora at each study site each year, the geographical location and the scientific names. Additionally, three supporting data sets were created respectively including name of each site, synonyms of the seaweeds, and the corresponding literature list. This rich collection of data can be used to study the biogeography and long-term changes of particular species and the diversity of habitat-forming seaweeds of the Japanese coast.

The carotenoid biosynthetic pathway in higher plants was manipulated by using an RNA viral vector. A cDNA encoding phytoene synthase and a partial cDNA encoding phytoene desaturase (PDS) were placed under the transcriptional control of a tobamovirus subgenomic promoter. One to two weeks after inoculation, systemically infected Nicotiana benthamiana plants were analyzed for phytoene. Leaves from transfected plants expressing phytoene synthase developed a bright orange phenotype and accumulated high levels of phytoene. Cytoplasmic inhibition of plant gene expression by viral RNA was demonstrated with an antisense RNA transcript to a partial PDS cDNA derived from tomato. The leaves of the plants transfected with the antisense PDS sequence developed a white phenotype and also accumulated high levels of phytoene. A partial cDNA to the corresponding N. benthamiana PDS gene was isolated and found to share significant homology with the tomato antisense PDS transcript. This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway. In addition, our results indicate that an antisense transcript generated in the cytoplasm of a plant cell can turn off endogenous gene expression.

We determined the levels of angiotensin I (ANG I), angiotensin II (ANG II), and the heptapeptide angiotensin(1-7) [ANG(1-7)] in the blood and brain of female Hannover Sprague-Dawley (SD) and transgenic hypertensive rats [mRen-2]27 by radioimmunoassay and high performance liquid chromatography. Hypertension was accompanied by higher plasma concentrations of ANG II, no statistical changes in ANG(1-7), and no differences in plasma ANG I levels. In the hypothalamus of transgenic rats, concentrations of ANG II and ANG(1-7) averaged 827% and 168% above values in SD rats (p < 0.005) whereas both ANG I and ANG II increased in the medulla oblongata. The data showed that the established phase of hypertension in rats harboring the mouse Ren-2 gene is associated with overexpression of the renin-angiotensin system in brain regions participating in the endocrine regulation of blood pressure.

We engineered cDNA of tobacco mosaic tobamovirus (TMV) into Agrobacterium tumefaciens for inoculation of plant cells. The resulting bacterial strains were used to transfect tobacco (Nicotiana tabacum cv. Xanthi and Xanthi/nc) with wild type and a defective virus. Lesion formation on Xanthi/nc tobacco was used to measure the timing and efficiency of transfection. Infections mediated by Agrobacterium produced lesions an average of two days later than infections produced by inoculation with virions. The addition of approximately 80 bp of non-viral sequences to the 5'-end of TMV transcripts abolished transfection. Transcripts with non-viral sequences at the 3'-end initiated infections, while precise transcript termination with a synthetic ribozyme sequence increased transfection frequencies two-fold. Culture conditions reported to induce genes of the vir region of the Agrobacterium Ti plasmid also increased the transfection frequency approximately two-fold. Therefore, in addition to the pararetroviruses and geminiviruses previously described, 'agroinoculation' may be used to infect plants with plus-sense RNA viruses.

A recombinant Saccharomyces cerevisiae, expressing and secreting rice alpha-amylase, converts starch to ethanol. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of the promoter from a newly described Pichia pastoris alcohol oxidase genomic clone. The nucleotide sequences of ZZA1 and other methanol-regulated promoters were analyzed. A highly conserved sequence (TTG-N3-GCTTCCAA-N5-TGGT) was found in the 5' flanking regions of alcohol oxidase, methanol oxidase, and dihydroxyacetone synthase genes in Pichia pastoris, Hansenula polymorpha, and Candida boidinii S2. The yeast strain containing the ZZA1-OS103 fusion secreted biologically active enzyme into the culture media while fermenting soluble starch.

We studied the effects of chronic oral inhibition of angiotensin-converting enzyme on the baroreflex control of renal sympathetic nerve activity (RSNA) and heart rate (HR) in 14-wk-old conscious spontaneously hypertensive rats (SHR; n = 12) and age-matched Wistar-Kyoto rats (WKY; n = 11). Rats were treated with lisinopril (10 mg.kg-1.day-1 in tap water) or vehicle for 7 days. We evaluated the baroreflex control of efferent RSNA and HR in awake rats 2 days after implantation of electrodes around the renal sympathetic nerves. The relation between mean arterial pressure (MAP) and either RSNA or HR was analyzed by a logistic function curve during intravenous infusions of phenylephrine and nitroglycerin. The maximum gain of the curve was considered as the sensitivity of the baroreflex. MAP in lisinopril-treated SHR averaged 93 +/- 3 mmHg, a value lower than that obtained in vehicle-treated SHR (147 +/- 5 mmHg) but not in WKY (96 +/- 4 mmHg). In vehicle-treated SHR baroreflex sensitivity (-4.3 +/- 0.5% change nerve activity/mmHg) was significantly (P less than 0.005) smaller than that of WKY (-15.8 +/- 1.5%/mmHg). Seven days of oral treatment of lisinopril caused significant improvement of the baroreflex sensitivity in SHR (-10.5 +/- 0.7%/mmHg, P less than 0.01). The maximum gain of MAP and HR relation of lisinopril-treated SHR was also larger (P less than 0.05) than that of vehicle-treated SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Two cDNA clones, pOS103 and pOS137, were isolated which code for distinct alpha-amylase isozymes in germinating rice seeds. Sequence analysis indicated that the clones encode polypeptides of approximately 48 kDa, both of which possess a signal peptide involved in directing secretion of the protein. Comparison of the two rice alpha-amylase amino acid sequence showed that they are 76% similar to each other, while showing 85% to 90% similarity with other cereal alpha-amylases. A comparison of eleven cereal alpha-amylases also revealed three new conserved regions (I', II', and IV') not previously identified in the animal, bacterial, and fungal alpha-amylases. Regions I' and IV' are sites for intron splicing while region II' is probably involved in calcium binding. One of the rice alpha-amylase cDNAs, pOS103, encodes a protein that has two potential N-glycosylation sites, one in the signal peptide and the other in the mature portion of the protein. The cDNA clone, pOS137, encodes an alpha-amylase with a single glycosylation site in the signal peptide, suggesting that the mature OS137 isozyme is not glycosylated. Analysis of the expression of these genes in germinating rice seeds indicated that mRNA corresponding to pOS103 and pOS137 could be detected throughout a 48 h period of seed imbibition. RNA levels, however, were dramatically stimulated by treatment of embryoless half-seeds with exogenous GA3. Our results demonstrate that at least two forms of alpha-amylase are expressed in germinating rice seeds and that the expression of these genes is regulated by the phytohormone GA3.

We examined the hypothesis that the positive inotropic effect of angiotensin I (Ang I) may be retained in the presence of angiotensin converting enzyme inhibitors so that it may have a direct beneficial effect on the heart. Accordingly, isolated perfused hearts (Langendorff preparation) of 300-day-old cardiomyopathic hamsters (a model of spontaneous cardiomyopathy) and age-matched normal hamsters (controls) were infused with Ang I in the presence of captopril; propranolol was added to the perfusing medium to block catecholamine-mediated effects of angiotensins on the heart. Left ventricular developed pressure and the rate of increase in left ventricular developed pressure increased significantly (p less than 0.001) in both the cardiomyopathic and the normal hamster heart despite concomitant reduction in myocardial flow rate favoring a direct inotropic effect of Ang I in both normal and myopathic hearts; these changes were significantly higher by almost threefold in the cardiomyopathic than in the normal hamsters (p less than 0.01) and were blocked by the angiotensin II (Ang II) antagonist [Sar1,Thr8]Ang II. Comparing dose-left ventricular contractility response curves for Ang I and Ang II, ED50 for responses was identical in both normal and myopathic hearts, whereas peak responses to Ang II were double those to Ang I in normal hearts but were almost identical in the myopathic hearts. Binding of [125I]Ang II in six cardiomyopathic and four normal hamster hearts was of high affinity, but there was no evidence for Ang I-saturable high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

We report the high level expression and secretion of rice α-amylase isozyme by Saccharomyces cerevisiae . Transcription of this gene was under control of the yeast enolase promoter. The synthesized protein had an approximate molecular size of 45 kDa and a pI of approx 4.7 to 5.0. The rice α-amylase signal peptide was recognized and efficiently processed by yeast and the active, glycosylated enzyme was secreted into the culture media. This enzyme was purified to homogeneity by affinity chromotography and its enzymatic properties were characterized. The K m and V max were found to be similar to those of α-amylase from other organisms. The high level of secretion observed in these studies may be due to the unique features of the rice signal peptide and/or to the glycosylation of the recombinant enzyme.

Small marine invertebrates often use sessile organisms as microhabitats, which can provide food sources and/or serve as refugees from predators. Because of the availability of external food items such as epibionts and detritus in the marine environment, these invertebrates may not depend on the sessile organisms as sole food sources. In this study, I hypothesized that habitat specialization by a marine invertebrate is determined by factors other than food. Results of field surveys off the coast of the Izu Peninsula, on the eastern coast of Japan, showed that, with few exceptions, the distribution of the amphipod Incisocalliope symbioticus was restricted to the octocoral Melithaea flabellifera. When presented with several habitat options, I. symbioticus selected M. flabellifera most frequently, although some individuals chose the octocoral Acabaria japonica. The selection was proximately determined by water-borne cues from M. flabellifera that appear to be unrelated to the octocoral as a food source, since the amphipod preferred detritus to the octocoral. As a chemical refuge, M. flabellifera had an allelopathic effect that deterred fish predation on the exposed epifauna. With regard to octocoral habitat in the study area, I. symbioticus may be restricted to M. flabellifera because this was the only abundant octocoral consistently occurring in shallow water (or =10 m), where predation is intensive. The relationship between I. symbioticus and M. flabellifera was commensal and was ultimately driven by the value of M. flabellifera as a chemical refuge from predation, rather than its food value. This study supports the idea that protection from predators, rather than food utilization, can promote ecological specialization.

Tobamoviral vectors have been developed for the heterologous expression of glycoproteins in plants. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of a tobamovirus subgenomic promoter in a RNA viral vector. One to two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated glycosylated alpha-amylase to levels of at least 5% total soluble protein. The 46kDa recombinant enzyme was purified, and its structural and biological properties were analyzed. Post-translational modifications of the secreted protein were compared to rice alpha-amylase expressed in amylolytic strains of Pichia pastoris and Saccharomyces cerevisiae. Endo-H analysis revealed that the alpha-amylase was moderately glycosylated in transfected plants and hyperglycosylated in yeast.

The rnfABCDE genes in Rhodobacter capsulatus are essential for diazotrophic growth under illuminated conditions. They locate near fdxN gene whose product is a major electron donor to nitrogenase. Since disruption of rnf genes conferred metronidazole resistance under pseudo-diazotrophic conditions and two of the rnf products, RnfB and RnfC, are supposed to hold iron-sulfur clusters, the genes are expected to encode some ferredoxin reductase. Because most of rnf products are highly hydrophobic it is hypothesized that rnf products constitute a membrane protein complex. In this study, we have 1) experimentally investigated their presence at chromatophore membranes, 2) made a prediction of enzymatic function and 3) collected data that the rnf products also serve for nitrogen fixation in darkness and that their homologs present in non-diazotrophs.

Copper-containing monoamine oxidase (EC 1.4.3.6) from Escherichia coli is a periplasmic enzyme containing topa quinone in addition to divalent copper as a cofactor. The amino acid sequence of E. coli monoamine oxidase was compared to several cloned amine oxidase genes and five well-conserved histidine residues were found. Site-directed mutagenesis studies were performed to determine which histidine residue serves as the binding Ligand to the copper. Enzyme activity, absorption spectrum, and atomic absorption spectrophotometry of the mutant enzymes indicated that histidines 470, 554, and 556 are the copper binding ligands. The absorption spectra of phenylhydrazine derivatives suggested that copper is necessary for topa quinone formation.

Periodic surveys of the subtidal epifaunal community on the gorgonian Melithaea flabellifera were conducted over a five year period at the Izu Peninsula, southern Japan. The occurrence patterns of epifaunal species on M. flabellifera were examined. The epifauna consisted of 16 species representing five phyla. The amphipod Incisocalliope symbioticus dominated (usually >80%) and occurred throughout the study.

Miyazaki, T. et al.. Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nat. Commun. 3:1236 doi: 10.1038/ncomms2231 (2012)., Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the potential to provide an infinite source of tissues for regenerative medicine. Although defined xeno-free media have been developed, culture conditions for reliable propagation of hESCs still require considerable improvement. Here we show that recombinant E8 fragments of laminin isoforms (LM-E8s), which are the minimum fragments conferring integrin-binding activity, promote greater adhesion of hESCs and hiPSCs than do Matrigel and intact laminin isoforms. Furthermore, LM-E8s sustain long-term self-renewal of hESCs and hiPSCs in defined xeno-free media with dissociated cell passaging. We successfully maintained three hESC and two hiPSC lines on LM-E8s in three defined media for 10 passages. hESCs maintained high level expression of pluripotency markers, had a normal karyotype after 30 passages and could differentiate into all three germ layers. This culture system allows robust proliferation of hESCs and hiPSCs for therapeutic applications.

alpha-Trichosanthin, a eukaryotic ribosome-inactivating protein from Trichosanthes kirilowii, inhibits the replication of the human immunodeficiency virus (HIV) in vitro. The alpha-trichosanthin gene was placed under the transcriptional control of a tobamovirus subgenomic promoter in a plant RNA viral vector. Two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated alpha-trichosanthin to levels of at least 2% of total soluble protein. The recombinant alpha-trichosanthin was purified and its structural and biological properties were analyzed. The 23-amino acid signal peptide was recognized by N. benthamiana and the processed enzyme caused a concentration-dependent inhibition of protein synthesis in vitro. The high level of heterologous gene expression observed in these studies is due to the unique features of the RNA viral-based transfection system.

Sheep require both sulfur and selenium for normal wool growth and to protect against diseases caused by free radical damage. This work describes the seasonal changes in the concentrations of sulfur, both as glutathione and inorganic sulfur, and selenium, in pregnant ewes, and the effects thereon of a multi-element supplement containing sulfate, selenium, and vitamins A and E. Merino ewes (420) were divided into 4 equal treatment groups, control (C), vitamins (V), minerals (M), and vitamins plus minerals (V+M), in a 22 factorial design. The control group was offered a loose salt lick NaCl at 175 g/head· day and the mineral group an equivalent amount of a multi-element mineral mix (Siromin). The vitamin groups were offered either salt or the mineral mix with added vitamins E and A. Treatments started 3 weeks prior to mating in November and lasted until 1 week prior to lambing in May. Sheep were sampled until weaning in September. Regardless of treatment, the concentrations of glutathione in blood and liver declined by 60% and 40%, respectively, when sheep were grazing dry pasture during summer. Sulfate-sulfur concentration in plasma of ewes in the C and V groups declined by 80% in the same period to

Four hundred and twenty Merino ewes were divided into four treatment groups: control (NaCl at 175 g head-1 week-1 ), vitamins (salt containing 0.24 g/kg retinol plus 12 g/kg �-tocopherol), minerals (a multi-element mineral mix (Siromin ) at 175 g head-1 week-1) and vitamins plus minerals (mineral mix plus vitamins) in a two by two factorial design. Treatments started in November prior to mating and finished in May 1 week prior to lambing. Measurements of vitamin status and animal productivity were made on ewes and foetal and weanling lambs. Compared with salt, the mineral mix increased ewe body weight by up to 3% ( P < 0.05), annual wool growth by 4.4% ( P < 0.001) and birth and weaning weights of single lambs by 7.3% ( P < 0.01) and 3.7% (P < 0.05) respectively. Since ewes had been given selenium and cobalt bullets at weaning, the results suggest either that the bullets do not offer protection for the lifetime of the ewe and that additional bulleting is warranted, or that other minerals are limiting production. As pastures dried off, the concentrations of �-tocopherol and G-carotene fell and this was reflected in a decline in the concentrations of �-tocopherol and retinol in liver. However, the liver stores at the beginning of summer were sufficient to prevent the appearance of clinical signs of vitamin deficiency. Vitamin supplementation significantly increased the concentration of vitamins in the plasma and liver of ewes ( P < 0.001)) but it had no significant effect on ewe reproductive performance or on ewe or lamb body weights or wool growth. Vitamin supplements increased the concentrations of retinol and �-tocopherol in the liver of foetal lambs, but even with supplements the levels remained well below normal adult reference values.

Tyrosine phenol-lyase of Escherichia intermedia is inactivated by treatment with diethylpyrocarbonate at pH 6.0 AND 4 degrees. Spectrophotometric studies show that the inactivation is stoichiometric, with a modification of 2 histidyl residues per molecule of the enzyme. Finding that this inactivation is largely reversed by treatment with hydroxylamine indicates that the inactivation is mainly due to modification of the histidyl residues. No changes in the sulfhydryl content or in the aromatic amino acids are observed as a result of this modification. The modified tyrosine phenol-lyase retains most of its ability to form a nearly normal complex with its coenzyme, pyridoxal phosphate. This has been shown by studies of its absorption, by the determination of pyridoxal phosphate, and by reduction of the holoenzyme with tritiated sodium borohydride. The modified enzyme also appears to form a Schiff base intermediate with L-alanine. The modified holoenzyme fails to catalyze the exchange of the alpha-hydrogen of L-alanine with tritium from tritiated water. This is consistent with a catalytic role for modified histidyl residues at the active site of the enzyme; this role is the removal of the alpha-hydrogen of substrates.