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Unicellular diazotrophic cyanobacteria contribute significantly to the photosynthetic productivity of the ocean and the fixation of molecular nitrogen, with photosynthesis occurring during the day and nitrogen fixation during the night. In species like Crocosphaera watsonii WH8501, the decline in photosynthetic activity in the night is accompanied by the disassembly of oxygen-evolving photosystem II (PSII) complexes. Moreover, in the second half of the night phase, a small amount of rogue D1 (rD1), which is related to the standard form of the D1 subunit found in oxygen-evolving PSII, but of unknown function, accumulates but is quickly degraded at the start of the light phase. We show here that the removal of rD1 is independent of the rD1 transcript level, thylakoid redox state and trans-thylakoid pH but requires light and active protein synthesis. We also found that the maximal level of rD1 positively correlates with the maximal level of chlorophyll (Chl) biosynthesis precursors and enzymes, which suggests a possible role for rogue PSII (rPSII) in the activation of Chl biosynthesis just before or upon the onset of light, when new photosystems are synthesized. By studying strains of Synechocystis PCC 6803 expressing Crocosphaera rD1, we found that the accumulation of rD1 is controlled by the light-dependent synthesis of the standard D1 protein, which triggers the fast FtsH2-dependent degradation of rD1. Affinity purification of FLAG-tagged rD1 unequivocally demonstrated the incorporation of rD1 into a non-oxygen-evolving PSII complex, which we term rPSII. The complex lacks the extrinsic proteins stabilizing the oxygen-evolving Mn4CaO5 cluster but contains the Psb27 and Psb28-1 assembly factors.

Crocosphaera watsonii is as a key nitrogen (N) supplier in marine ecosystems, and it has been estimated to contribute up to half of oceanic N 2 fixation. Conversely, a recent study reported that Crocosphaera can assimilate combined N and proposed that unicellular diazotrophs can be competitors with non-N 2 fixing phytoplankton for combined N.

Graphical abstract, The photoautotrophic, unicellular N2-fixer, Cyanothece, is a model organism that has been widely used to study photosynthesis regulation, the structure of photosystems, and the temporal segregation of carbon (C) and nitrogen (N) fixation in light and dark phases of the diel cycle. Here, we present a simple quantitative model and experimental data that together, suggest external dissolved inorganic carbon (DIC) concentration as a major limiting factor for Cyanothece growth, due to its high C-storage requirement. Using experimental data from a parallel laboratory study as a basis, we show that after the onset of the light period, DIC was rapidly consumed by photosynthesis, leading to a sharp drop in the rate of photosynthesis and C accumulation. In N2-fixing cultures, high rates of photosynthesis in the morning enabled rapid conversion of DIC to intracellular C storage, hastening DIC consumption to levels that limited further uptake. The N2-fixing condition allows only a small fraction of fixed C for cellular growth since a large fraction was reserved in storage to fuel night-time N2 fixation. Our model provides a framework for resolving DIC limitation in aquatic ecosystem simulations, where DIC as a growth-limiting factor has rarely been considered, and importantly emphasizes the effect of intracellular C allocation on growth rate that varies depending on the growth environment.

Graphical abstract, Nitrogen-fixing organisms are of importance to the environment, providing bioavailable nitrogen to the biosphere. Quantitative models have been used to complement the laboratory experiments and in situ measurements, where such evaluations are difficult or costly. Here, we review the current state of the quantitative modeling of nitrogen-fixing organisms and ways to enhance the bridge between theoretical and empirical studies.

Crocosphaera watsonii(hereafterCrocosphaera) is a key nitrogen (N) fixer in the ocean, but its ability to consume combined N sources is still unclear. Usingin situmicrocosm incubations with an ecological model, we show thatCrocosphaerahas high competitive capability both under low and moderately high combined N concentrations. In field incubations,Crocosphaeraaccounted for the highest consumption of ammonium and nitrate, followed by pico-eukaryotes. The model analysis shows that cells have a high ammonium uptake rate (∼7 mol N (mol N)-1d-1at the maximum), which allows them to compete against pico-eukaryotes and non-diazotrophic cyanobacteria when combined N is sufficiently available. Even when combined N is depleted, their capability of nitrogen fixation allows higher growth rates compared to potential competitors. These results suggest the high fitness ofCrocosphaerain combined N limiting, oligotrophic oceans, and thus heightens its potential significance in its ecosystem and in biogeochemical cycling.

Prochlorococcus and Synechococcus are the two dominant picocyanobacteria in the low-nutrient surface waters of the subtropical ocean, but the basis for their coexistence in these biomes is still unclear. Here we combine in situ microcosm experiments and an ecological model to show that this coexistence can arise from specialization in the uptake of distinct nitrogen (N) substrates. In field incubations, the response of both Prochlorococcus and Synechococcus to nanomolar N amendments demonstrates N limitation of growth in both populations, but Prochlorococcus showed a higher affinity to ammonium whereas Synechococcus was more adapted to nitrate uptake. A simple ecological model demonstrates that the differential nutrient affinity of these species can explain their coexistence. Phylogenetic analysis of the presence of nitrate reductase and nitrite reductase further support the higher nitrate affinity of Synechococcus compared to Prochlorococcus. Our study suggests that the evolution of differential nutrient affinities is an important mechanism for sustaining coexistence of species under resource competition.

Effect of global change variables on the structure and photosynthesis of phytoplankton communities was evaluated in three different sites of the Patagonian coast of Argentina: enclosed bay (Puerto Madryn, PM), estuarine (Playa Unión, PU), and open waters (Isla Escondida, IE). We exposed samples to two contrasting scenarios: Present (nutrients at in situ levels) vs. Future (with lowered pH and higher nutrients inputs), and determined growth and photosynthetic responses after 2 days of acclimation. Under the Future condition phytoplankton growth was higher in the estuarine site compared to those in PM and IE. This effect was the most pronounced on large diatoms. While the increase of photosynthetic activity was not always observed in the Future scenario, the lower photosynthetic electron requirement for carbon fixation (Φe,C = ETR/PmB) in this scenario compared to the Present, suggests a more effective energy utilization. Long-term experiments were also conducted to assess the responses along a 4 days acclimation period in PU. Diatoms benefited from the Future conditions and had significantly higher growth rates than in the Present. In addition, Φe,C was lower after the acclimation period in the Future scenario, compared to the Present. Our results suggest that the availability, frequency and amount of nutrients play a key role when evaluating the effects of global change on natural phytoplankton communities. The observed changes in diatom growth under the Future scenario in PU and IE and photosynthesis may have implications in the local trophodynamics by bottom up control. Fil: Masuda, Takako. Czech Academy of Sciences; República Checa Fil: Prášil, Ondrej. Czech Academy of Sciences; República Checa Fil: Villafañe, Virginia Estela. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico; Argentina Fil: Valiñas, Macarena Soledad. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico; Argentina Fil: Inomura, Keisuke. University of Rhode Island; Estados Unidos Fil: Helbling, Eduardo Walter. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico; Argentina

Marine diazotrophs are a diverse group with key roles in biogeochemical fluxes linked to primary productivity. The unicellular, diazotrophic cyanobacterium Cyanothece is widely found in coastal, subtropical oceans. We analyze the consequences of diazotrophy on growth efficiency, compared to NO3–-supported growth in Cyanothece, to understand how cells cope with N2-fixation when they also have to face carbon limitation, which may transiently affect populations in coastal environments or during blooms of phytoplankton communities. When grown in obligate diazotrophy, cells face the double burden of a more ATP-demanding N-acquisition mode and additional metabolic losses imposed by the transient storage of reducing potential as carbohydrate, compared to a hypothetical N2 assimilation directly driven by photosynthetic electron transport. Further, this energetic burden imposed by N2-fixation could not be alleviated, despite the high irradiance level within the cultures, because photosynthesis was limited by the availability of dissolved inorganic carbon (DIC), and possibly by a constrained capacity for carbon storage. DIC limitation exacerbates the costs on growth imposed by nitrogen fixation. Therefore, the competitive efficiency of diazotrophs could be hindered in areas with insufficient renewal of dissolved gases and/or with intense phytoplankton biomass that both decrease available light energy and draw the DIC level down.

Nitrogen fixing plankton provide nitrogen to fuel marine ecosystems and biogeochemical cycles but the factors that constrain their growth and habitat remain poorly understood. Here we investigate the importance of metabolic specialization in unicellular diazotroph populations, using laboratory experiments and model simulations. In clonal cultures of Crocosphaera watsonii and Cyanothece sp. spiked with 15N2, cellular 15N enrichment developed a bimodal distribution within colonies, indicating that N2 fixation was confined to a subpopulation. In a model of population metabolism, heterogeneous nitrogen (N2) fixation rates substantially reduce the respiration rate required to protect nitrogenase from O2. The energy savings from metabolic specialization is highest at slow growth rates, allowing populations to survive in deeper waters where light is low but nutrients are high. Our results suggest that heterogeneous N2 fixation in colonies of unicellular diazotrophs confers an energetic advantage that expands the ecological niche and may have facilitated the evolution of multicellular diazotrophs., Takako Masuda et al. show that individual cells in clonal populations of Crocosphaera watsonii and Cyanothece sp exhibit varied nitrogen fixation rates. This heterogeneity within the population decreases the energetic cost of respiration and expands the viable habitats for these unicellular diazotrophs.

Diatom&ndash, diazotroph associations (DDAs) are symbioses where trichome-forming cyanobacteria support the host diatom with fixed nitrogen through dinitrogen (N2) fixation. It is inferred that the growth of the trichomes is also supported by the host, but the support mechanism has not been fully quantified. Here, we develop a coarse-grained, cellular model of the symbiosis between Hemiaulus and Richelia (one of the major DDAs), which shows that carbon (C) transfer from the diatom enables a faster growth and N2 fixation rate by the trichomes. The model predicts that the rate of N2 fixation is 5.5 times that of the hypothetical case without nitrogen (N) transfer to the host diatom. The model estimates that 25% of fixed C from the host diatom is transferred to the symbiotic trichomes to support the high rate of N2 fixation. In turn, 82% of N fixed by the trichomes ends up in the host. Modeled C fixation from the vegetative cells in the trichomes supports only one-third of their total C needs. Even if we ignore the C cost for N2 fixation and for N transfer to the host, the total C cost of the trichomes is higher than the C supply by their own photosynthesis. Having more trichomes in a single host diatom decreases the demand for N2 fixation per trichome and thus decreases their cost of C. However, even with five trichomes, which is about the highest observed for Hemiaulus and Richelia symbiosis, the model still predicts a significant C transfer from the diatom host. These results help quantitatively explain the observed high rates of growth and N2 fixation in symbiotic trichomes relative to other aquatic diazotrophs.

Crocosphaera is one of the major N2-fixing microorganisms in the open ocean. On a global scale, the process of N2 fixation is important in balancing the N budget, but the factors governing the rate of N2 fixation remain poorly resolved. Here, we combine a mechanistic model and both previous and present laboratory studies of Crocosphaera to quantify how chemical factors such as C, N, Fe, and O2 and physical factors such as temperature and light affect N2 fixation. Our study shows that Crocosphaera combines multiple mechanisms to reduce intracellular O2 to protect the O2-sensitive N2-fixing enzyme. Our model, however, indicates that these protections are insufficient at low temperature due to reduced respiration and the rate of N2 fixation becomes severely limited. This provides a physiological explanation for why the geographic distribution of Crocosphaera is confined to the warm low-latitude ocean., Crocosphaera is a major dinitrogen (N2)-fixing microorganism, providing bioavailable nitrogen (N) to marine ecosystems. The N2-fixing enzyme nitrogenase is deactivated by oxygen (O2), which is abundant in marine environments. Using a cellular scale model of Crocosphaera sp. and laboratory data, we quantify the role of three O2 management strategies by Crocosphaera sp.: size adjustment, reduced O2 diffusivity, and respiratory protection. Our model predicts that Crocosphaera cells increase their size under high O2. Using transmission electron microscopy, we show that starch granules and thylakoid membranes are located near the cytoplasmic membranes, forming a barrier for O2. The model indicates a critical role for respiration in protecting the rate of N2 fixation. Moreover, the rise in respiration rates and the decline in ambient O2 with temperature strengthen this mechanism in warmer water, providing a physiological rationale for the observed niche of Crocosphaera at temperatures exceeding 20°C. Our new measurements of the sensitivity to light intensity show that the rate of N2 fixation reaches saturation at a lower light intensity (∼100 μmol m−2 s−1) than photosynthesis and that both are similarly inhibited by light intensities of >500 μmol m−2 s−1. This suggests an explanation for the maximum population of Crocosphaera occurring slightly below the ocean surface. IMPORTANCE Crocosphaera is one of the major N2-fixing microorganisms in the open ocean. On a global scale, the process of N2 fixation is important in balancing the N budget, but the factors governing the rate of N2 fixation remain poorly resolved. Here, we combine a mechanistic model and both previous and present laboratory studies of Crocosphaera to quantify how chemical factors such as C, N, Fe, and O2 and physical factors such as temperature and light affect N2 fixation. Our study shows that Crocosphaera combines multiple mechanisms to reduce intracellular O2 to protect the O2-sensitive N2-fixing enzyme. Our model, however, indicates that these protections are insufficient at low temperature due to reduced respiration and the rate of N2 fixation becomes severely limited. This provides a physiological explanation for why the geographic distribution of Crocosphaera is confined to the warm low-latitude ocean.

Crocosphaera is a major dinitrogen (N2)-fixing microorganism, providing bioavailable nitrogen (N) to marine ecosystems. The N2-fixing enzyme nitrogenase is deactivated by oxygen (O2), which is abundant in marine environments. Using a cellular scale model of Crocosphaera sp. and laboratory data, we quantify the role of three O2 management strategies by Crocosphaera sp.: size adjustment, reduced O2 diffusivity, and respiratory protection. Our model predicts that Crocosphaera cells increase their size under high O2. Using transmission electron microscopy, we show that starch granules and thylakoid membranes are located near the cytoplasmic membranes, forming a barrier for O2. The model indicates a critical role for respiration in protecting the rate of N2 fixation. Moreover, the rise in respiration rates and the decline in ambient O2 with temperature strengthen this mechanism in warmer water, providing a physiological rationale for the observed niche of Crocosphaera at temperatures exceeding 20°C. Our new measurements of the sensitivity to light intensity show that the rate of N2 fixation reaches saturation at a lower light intensity (∼100 μmol m−2 s−1) than photosynthesis and that both are similarly inhibited by light intensities of >500 μmol m−2 s−1. This suggests an explanation for the maximum population of Crocosphaera occurring slightly below the ocean surface. IMPORTANCECrocosphaera is one of the major N2-fixing microorganisms in the open ocean. On a global scale, the process of N2 fixation is important in balancing the N budget, but the factors governing the rate of N2 fixation remain poorly resolved. Here, we combine a mechanistic model and both previous and present laboratory studies of Crocosphaera to quantify how chemical factors such as C, N, Fe, and O2 and physical factors such as temperature and light affect N2 fixation. Our study shows that Crocosphaera combines multiple mechanisms to reduce intracellular O2 to protect the O2-sensitive N2-fixing enzyme. Our model, however, indicates that these protections are insufficient at low temperature due to reduced respiration and the rate of N2 fixation becomes severely limited. This provides a physiological explanation for why the geographic distribution of Crocosphaera is confined to the warm low-latitude ocean.

The oceanic unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501 exhibits large diel changes in abundance of both Photosystem II (PSII) and Photosystem I (PSI). To understand the mechanisms underlying these dynamics, we assessed photosynthetic parameters, photosystem abundance and composition, and chlorophyll-protein biosynthesis over a diel cycle. Our data show that the decline in PSII activity and abundance observed during the dark period was related to a light-induced modification of PSII, which, in combination with the suppressed synthesis of membrane proteins, resulted in monomerization and gradual disassembly of a large portion of PSII core complexes. In the remaining population of assembled PSII monomeric complexes, we detected the non-functional version of the D1 protein, rD1, which was absent in PSII during the light phase. During the dark period, we also observed a significant decoupling of phycobilisomes from PSII and a decline in the chlorophyll a quota, which matched the complete loss of functional PSIIs and a substantial decrease in PSI abundance. However, the remaining PSI complexes maintained their photochemical activity. Thus, during the nocturnal period of nitrogen fixation C. watsonii operates a suite of regulatory mechanisms for efficient utilization/recycling of cellular resources and protection of the nitrogenase enzyme.

Unicellular nitrogen fixer Crocosphaera contributes substantially to nitrogen fixation in oligotrophic subtropical gyres. They fix nitrogen even when significant amounts of ammonium are available. This has been puzzling since fixing nitrogen is energetically inefficient compared with using available ammonium. Here we show that by fixing nitrogen, Crocosphaera can increase their population and expand their niche despite the presence of ammonium. We have developed a simple but mechanistic model of Crocosphaera based on their growth in steady state culture. The model shows that the growth of Crocosphaera can become nitrogen limited despite their capability to fix nitrogen. When they fix nitrogen, the population increases by up to 78% relative to the case without nitrogen fixation. When we simulate a simple ecological situation where Crocosphaera exists with non-nitrogen-fixing phytoplankton, the relative abundance of Crocosphaera increases with nitrogen fixation, while the population of non-nitrogen-fixing phytoplankton decreases since a larger fraction of fixed nitrogen is consumed by Crocosphaera. Our study quantitatively supports the benefit of nitrogen fixation despite the high electron/energy costs, even when an energetically efficient alternative is available. It demonstrates a competitive aspect of Crocosphaera, permitting them to be regionally significant nitrogen fixers.

Marine ecosystem services provide various benefits to people. In order to receive those benefits sustainably, conservation of marine environment is an important measure, and how to motivate people to marine conservation would be one of the keys to secure sustainable receipt of marine ecosystem services. This study explores perception of marine ecosystem services by residents of remote islands, namely Taketomi Town in Japan and how the perception would influence their behavioural intentions for marine conservation. A questionnaire survey was administered to the residents, and factor analysis and Structural Equation Model were applied to analyse data from 344 respondents. The results show that respondents perceive marine ecosystem services in four categories, namely “Benefits closely related to daily lives”, “Benefits from supporting services”, “Benefits from regulating services”, and “Benefits irrelevant to daily lives”. Among the four categories, “Benefits from regulating services” is the most influential to enhance behavioural intentions for marine conservation. The perception of marine ecosystem services by respondents of Taketomi Town and their influence on behavioural intentions for marine conservation are different from the results of previous studies administered to residents in the main island Honshu, Japan. This shows possibility that perception of marine ecosystem services and motivation for behavioural intention for marine conservation would relate to their connectednesspossibility to the sea.

The oceanic unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501 exhibits large diel changes in abundance of both Photosystem II (PSII) and Photosystem I (PSI). To understand the mechanisms underlying these dynamics, we assessed photosynthetic parameters, photosystem abundance and composition, and chlorophyll-protein biosynthesis over a diel cycle. Our data show that the decline in PSII activity and abundance observed during the dark period was related to a light-induced modification of PSII, which, in combination with the suppressed synthesis of membrane proteins, resulted in monomerization and gradual disassembly of a large portion of PSII core complexes. In the remaining population of assembled PSII monomeric complexes, we detected the non-functional version of the D1 protein, rD1, which was absent in PSII during the light phase. During the dark period, we also observed a significant decoupling of phycobilisomes from PSII and a decline in the chlorophyll a quota, which matched the complete loss of functional PSIIs and a substantial decrease in PSI abundance. However, the remaining PSI complexes maintained their photochemical activity. Thus, during the nocturnal period of nitrogen fixation C. watsonii operates a suite of regulatory mechanisms for efficient utilization/recycling of cellular resources and protection of the nitrogenase enzyme.

The relative importance of new nitrogen sources for primary production was investigated in well stratified subtropical waters in the vicinity of the shelf edge of the East China Sea by concurrent determinations of upward turbulent nitrate flux across the pycnocline, nitrate assimilation, N2 fixation and primary production. On the coastal side of the Kuroshio jet close to the shelf edge, strong vertical diffusivity Kρ was observed. The half-day mean Kρ at the top nitracline was 3.9×10−5 m2 s−1. Consequently, a higher turbulent upward nitrate flux of 445 µmol N m−2 d−1 across the nitracline was observed than that of 82 µmol N m−2 d−1 off the shelf near the Kuroshio jet and 146 µmol N m−2 d−1 on the slope at the outer side of the Kuroshio jet. This high upward flux supported the highest rates of primary production (27.3 mmol C m−2 d−1) and nitrate assimilation (764 µmol N m−2 d−1), and the contribution of the upward nitrate flux to new nitrogen was 5.7 times higher than that of N2 fixation. In contrast, N2 fixation was a major new nitrogen source at stations off the shelf, where the upward nitrate flux was less evident due to less vertical diffusivity and weaker vertical nitrate gradients than near the shelf break in the Kuroshio jet. These observations demonstrate that new nitrogen sources were highly variable according to relative locations at the shelf edge and Kuroshio jet in the East China Sea and the adjacent waters during summer.

Eutrophication of the world’s marine environment has been exacerbated due to discharge of a large volume of wastewater from rapidly growing cities. Between 2008 and 2009, a serious red tide appeared in the Gulf that brought about a temporary shutdown of water desalination plants in Oman and the UAE. In this study, a harmful dinoflagellate Cochlodinium polykrikoides known to be the cause of many red tides, including the one in the Gulf between 2008 and 2009, was cultured in the laboratory to investigate its impacts on a coagulation-microfiltration/ultrafiltration (MF/UF)-reverse osmosis (RO) desalination process. The culture medium for C. polykrikoides contained high amounts of dissolved organic matter that possessed fluorophores for fulvic-like, humic-like, and protein-like organic matter that can be detected by excitation-emission matrix (EEM). Although EEM peaks were diminished by coagulation-MF/UF, the reduction in dissolved organic carbon concentrations was not significant by MF and was small...

Phytoplankton dynamics in the lower euphotic zone were observed by tracking a subsurface water released at 20-m depth from Takumi, an artificial upwelling device. Takumi continually discharged seawater pumped up from a depth of 205 m: this water was mixed with 5-m depth water to adjust the density to that of 20-m depth water of Sagami Bay, Japan. The discharged water was pulse-labeled at Takumi with uranine and tracked for 63.9 h with a drifting buoy equipped with a drogue at 20-m depth. We present a simple model to estimate in situ phytoplankton net growth rates from temporal changes in phytoplankton abundance in the discharged water with correction for the influence of water exchange between the discharged water and neighboring layers. Lagrangian observation showed active growth of pico- and nanophytoplankton, especially cryptophytes and Synechococcus (Cyanobacteria), in the subsurface layer. In contrast, diatoms grew little in spite of micromolar concentrations of nutrients. The active growth of pico- and nanophytoplankton was in good agreement with shipboard serial dilution culture experiments. The low growth activity of diatoms was suggested to be related to low light availability in the subsurface layer.

Background The local anesthetic properties of tetrodotoxin, a potent naturally occurring sodium channel blocker, have been recently reexamined. It was found that sciatic nerve block duration could be greatly increased and systemic toxicity greatly decreased if epinephrine was injected with tetrodotoxin. The mechanism that underlies epinephrine-mediated prolongation of tetrodotoxin nerve blocks is not known, but indirect evidence suggests that epinephrine may slow the clearance of tetrodotoxin from the site of injection. The authors hypothesized that tetrodotoxin causes vasodilatation at its injection site, which accelerates its systemic uptake, and that this vasodilatation is attenuated by coinjected epinephrine. Methods The radiolabeled microsphere technique was used to measure tissue blood flow in anesthetized rats before and after perisciatic injection of tetrodotoxin alone and in combination with epinephrine. Results Tetrodotoxin, in a dose of 0.1 ml of a 60 microM solution, significantly increased blood flow in perisciatic muscle at the injected side compared with simultaneous contralateral control and ipsilateral preinjection baseline. Tetrodotoxin did not increase blood flow in the sciatic nerve. Coinjection of epinephrine with tetrodotoxin prevented tetrodotoxin-induced increases in perisciatic muscle blood flow over time and did not alter sciatic nerve blood flow. Arterial blood pressure was maintained with this dose of tetrodotoxin, although brain blood flow decreased. Coinjection of epinephrine with tetrodotoxin prevented decreases in brain blood flow. Higher doses of tetrodotoxin produced hypotension. Conclusions Vasoconstriction in the perisciatic muscles by epinephrine may contribute to the prolongation of tetrodotoxin-induced sciatic nerve blocks and the reduction of systemic toxicity of tetrodotoxin.

Background Stretch (mechanical stress)-induced membrane depolarization of smooth muscle may contribute to basal vascular tone and myogenic control. Propofol induces vasodilation and inhibits myogenic control. Hypotonic swelling was used as a model of mechanical stress. The authors investigated the effects of propofol and 5-nitro-2-(3-phenylpropylamino)benzoic acid, a chloride channel and nonselective cation channel blocker, on hypotonicity-induced membrane depolarization in cultured human coronary artery smooth muscle cells. Methods A voltage-sensitive fluorescent dye, bis-(1,3-diethylthiobarbiturate)trimethine oxonol, was used to assess relative changes in membrane potential semiquantitatively. The cells were continuously perfused with Earle's balanced salt solution containing 200 nM bis-(1,3-diethylthiobarbiturate)trimethine oxonol and exposed sequentially to isotonic and hypotonic medium. In a second series of experiments, the cells were exposed to hypotonic media in the presence and absence of 5-nitro-2-(3-phenylpropylamino)benzoic acid or propofol. Results The relative fluorescence values at 10, 20, and 30% hypotonicity were 147 +/- 29, 214 +/- 74, and 335 +/- 102% of baseline, respectively. The changes were all significantly different from the isotonic time control group. In the presence of 200 microM 5-nitro-2-(3-phenylpropylamino)benzoic acid or 0.1, 1, 10, or 100 microg/ml propofol, the relative fluorescence values at 30% hypotonicity were 87 +/- 17, 194 +/- 27, 160 +/- 18, 130 +/- 18, and 84 +/- 15%, respectively. These changes were significantly less than the 30% for the hypotonic control (246 +/- 23%). Conclusion These results suggest that volume-sensitive chloride channels and nonselective cation channels may participate in hypotonicity-induced membrane depolarization and that propofol inhibits hypotonicity-induced membrane depolarization in coronary artery smooth muscle.

Volume-sensitive chloride channels (VSCC) play an important role in regulation of cell volume and electrical activity. Activation of vascular smooth muscle VSCC causes smooth muscle depolarization and contraction. We investigated the effects of propofol on VSCC in cultured human coronary artery smooth muscle cells by using the chloride-sensitive dye 6-methoxy-N-ethylquinolinium (MEQ). To activate VSCC, cells were superfused for 2 min with hypotonic gluconate solutions and then potassium thiocyanate solution. The percentage reduction in MEQ fluorescence during 60 s in the presence of potassium thiocyanate was measured and used as an index of VSCC activity. 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a well characterized chloride channel blocker, and propofol were dissolved in hypotonic gluconate solution to test their effect on VSCC activity. The reduction in fluorescence was inversely related to osmolality, indicating that activation of VSCC is osmolality dependent. Hypotonic gluconate solution (210 mOsm/kg H(2)O) reduced fluorescence by 38.9% +/- 2.6% of the baseline value. The reduction in fluorescence was dose-dependently inhibited by NPPB. Propofol at 0.3, 1, 3, 10, 30, and 100 micro g/mL significantly inhibited the reduction in fluorescence to 23.6% +/- 4.8%, 19.7% +/- 7.4%, 18.2% +/- 3.5%, 17.6% +/- 5.0%, 15.8% +/- 3.1%, and 10.3% +/- 3.9% of the baseline value, respectively. Our results indicate that propofol inhibits VSCC in a dose-dependent manner in human coronary artery smooth muscle cells.

Two types of new anion-exchange stationary phases were readily prepared by the reaction of porous particles of chloromethylated styrene–divinylbenzene copolymer with various amines. The stationary phases Mn, where n is the length of alkyl chain, were obtained by the reaction with N,N-dimethylalkylamines. The reaction with N,N,N′,N′-tetramethyldiaminoalkanes also afforded the stationary phases Dn with a terminal functional group, where n is the length of methylene bridge. The HPLC separations of monosaccharides, disaccharides and oligosaccharides were successfully performed on these stationary phases using electrochemical detection with a Ni–Ti alloy working electrode in alkaline eluents. The effects of chemical structures of stationary phases on the separation of carbohydrates are described.

A three-layer membrane for optical pH chemical sensors has been developed consisting of layers of polyester support, chitosan-indicator and polyvinly alcohol-titanium dioxide for reflection. The chitosan membrane was more stable and easier for immobilizing of benzopurprine 4B or fluorescein isothiocyanate than a cellulose membrane on which polyvinyl alcohol with titanium dioxide and calcium carbonate was coated and heated at 140°C in nitrogen atmosphere followed by dissolving of calcium carbonate to form a porous water-resistant membrane for reflection of light. The sensor membrane shows a pH-induced color change than makes it capable of measuring pH in the 3-7 range.

To investigate the separations of all aldopentoses (ribose, arabinose, xylose and lyxose) and aldohexoses (glucose, galactose, allose, altrose, mannose, gulose, idose and talose) on the D₆ stationary phase prepared by the reaction of chloromethylated styrene-divinylbenzene copolymer and N,N,N',N'-tetramethyl-1,6-diaminohexane, we examined the effect of varying the concentration of the NaOH eluent on the elution orders. Separations of these aldoses were achieved using a 20 mM NaOH eluent. The elution behaviors of the aldoses were probably due to not only the individual pK(a) values, but also the chemical structures of the cyclic aldoses.

High-performance liquid chromatographic separation of monosaccharides and disaccharides was successfully carried out with novel anion-exchange stationary phases prepared from (CH3)2N-(CH2)n-N(CH3)2 and porous particles of chloromethylated styrene-divinylbenzene copolymer coupled with a pulsed amperometric detector.

Fifteen commercial samples of citrus fruits grown in Japan were analyzed for their sugar contents by high-performance anion-exchange chromatography (HPAEC) and electrochemical detection (ED) coupled with a stationary phase D10 column prepared using chloromethylated styrene-divinylbenzene copolymer and N,N,N',N'-tetramethyldiaminodecane. Myo-inositol, glucose, fructose, and sucrose in all of these various citrus fruits could be successfully separated within 25 min using 0.5 M NaOH eluent at a flow-rate of 0.4 mL/min. Myo-inositol, as a better nutritional source, was found in all the citrus fruits grown in Japan (0.7 +/- 0.04-2.1 +/- 0.2 g/L). The sugar contents of twelve citrus fruits grown in other countries were also determined.

New anion-exchange stationary phases On (n=1, 2 and 3) with a dimethylamino terminal functional group, where n is the number of oxyethylene units [–(CH2CH2O)n–], were prepared by the reaction of chloromethylated porous styrene–divinylbenzene copolymer beads and amines [(CH3)2N–(CH2CH2O)nCH2CH2–N(CH3)2]. HPLC separations of monosaccharides (sorbitol, fucose, glucosamine, mannose, glucose, galactose, fructose, allose and altrose) and disaccharides (trehalose, lactose, cellobiose and maltose) were performed successfully on these stationary phases. The ether group of the stationary phases On was found to affect the separation of carbohydrates.