Seawater carbonate chemistry and biological processes of oysters Crassostrea virginica during experiments, 2010

Estuarine organisms are exposed to periodic strong fluctuations in seawater pH driven by biological carbon dioxide (CO2) production, which may in the future be further exacerbated by the ocean acidification associated with the global rise in CO2. Calcium carbonate-producing marine species such as mollusks are expected to be vulnerable to acidification of estuarine waters, since elevated CO2 concentration and lower pH lead to a decrease in the degree of saturation of water with respect to calcium carbonate, potentially affecting biomineralization. Our study demonstrates that the increase in CO2 partial pressure (pCO2) in seawater and associated decrease in pH within the environmentally relevant range for estuaries have negative effects on physiology, rates of shell deposition and mechanical properties of the shells of eastern oysters Crassostrea virginica (Gmelin). High CO2 levels (pH ~7.5, pCO2 ~3500 µatm) caused significant increases in juvenile mortality rates and inhibited both shell and soft-body growth compared to the control conditions (pH ~8.2, pCO2 ~380 µatm). Furthermore, elevated CO2 concentrations resulted in higher standard metabolic rates in oyster juveniles, likely due to the higher energy cost of homeostasis. The high CO2 conditions also led to changes in the ultrastructure and mechanical properties of shells, including increased thickness of the calcite laths within the hypostracum and reduced hardness and fracture toughness of the shells, indicating that elevated CO2 levels have negative effects on the biomineralization process. These data strongly suggest that the rise in CO2 can impact physiology and biomineralization in marine calcifiers such as eastern oysters, threatening their survival and potentially leading to profound ecological and economic impacts in estuarine ecosystems.

(Table 1) Microplankton biomass determined from ATP and by microscopy

Simultaneous determinations of the microplankton biomass by direct microscopy and by measuring concentration of adenosine triphosphate (ATP) in samples from different depths of the Eastern Pacific revealed considerable differences between values obtained. With the exception of 3 of 62 determinations, the biomass determined from ATP exceeded that measured by microscopy; the latter averaged about 10% of the former for the region as a whole. This sharp difference is largely due to underestimation of ultramicroscopic cells by direct microscopy and to substantial variations in the factor used to convert ATP concentrations to cell biomass.

Interactive effects of salinity and elevated CO2 levels on juvenile eastern oysters, Crassostrea virginica

Rising levels of atmospheric CO2 lead to acidification of the ocean and alter seawater carbonate chemistry, which can negatively impact calcifying organisms, including mollusks. In estuaries, exposure to elevated CO2 levels often co-occurs with other stressors, such as reduced salinity, which enhances the acidification trend, affects ion and acid-base regulation of estuarine calcifiers and modifies their response to ocean acidification. We studied the interactive effects of salinity and partial pressure of CO2 (PCO2) on biomineralization and energy homeostasis in juveniles of the eastern oyster, Crassostrea virginica, a common estuarine bivalve. Juveniles were exposed for 11 weeks to one of two environmentally relevant salinities (30 or 15 PSU) either at current atmospheric PCO2 (400 µatm, normocapnia) or PCO2 projected by moderate IPCC scenarios for the year 2100 (700-800 µatm, hypercapnia). Exposure of the juvenile oysters to elevated PCO2 and/or low salinity led to a significant increase in mortality, reduction of tissue energy stores (glycogen and lipid) and negative soft tissue growth, indicating energy deficiency. Interestingly, tissue ATP levels were not affected by exposure to changing salinity and PCO2, suggesting that juvenile oysters maintain their cellular energy status at the expense of lipid and glycogen stores. At the same time, no compensatory upregulation of carbonic anhydrase activity was found under the conditions of low salinity and high PCO2. Metabolic profiling using magnetic resonance spectroscopy revealed altered metabolite status following low salinity exposure; specifically, acetate levels were lower in hypercapnic than in normocapnic individuals at low salinity. Combined exposure to hypercapnia and low salinity negatively affected mechanical properties of shells of the juveniles, resulting in reduced hardness and fracture resistance. Thus, our data suggest that the combined effects of elevated PCO2 and fluctuating salinity may jeopardize the survival of eastern oysters because of weakening of their shells and increased energy consumption.

Trichodesmium's strategies to alleviate phosphorus limitation in the future acidified oceans

Global warming may exacerbate inorganic nutrient limitation, including phosphorus (P), in the surface-waters of tropical oceans that are home to extensive blooms of the marine diazotrophic cyanobacterium, Trichodesmium. We examined the combined effects of P limitation and pCO2, forecast under ocean acidification scenarios, on Trichodesmium erythraeum IMS101 cultures. We measured nitrogen acquisition, glutamine synthetase activity, C uptake rates, intracellular Adenosine Triphosphate (ATP) concentration and the pool sizes of related key proteins. Here, we present data supporting the idea that cellular energy re-allocation enables the higher growth and N2 fixation rates detected in Trichodesmium cultured under high pCO2. This is reflected in altered protein abundance and metabolic pools. Also modified are particulate organic carbon and nitrogen production rates, enzymatic activities, and cellular ATP concentrations. We suggest that adjusting these cellular pathways to changing environmental conditions enables Trichodesmium to compensate for low P availability and to thrive in acidified oceans. Moreover, elevated pCO2 could provide Trichodesmium with a competitive dominance that would extend its niche, particularly in P-limited regions of the tropical and subtropical oceans.

The effects of thermal and high-CO2 stresses on the metabolism and surrounding microenvironment of the coral Galaxea fascicularis

The effects of elevated temperature and high pCO2 on the metabolism of Galaxea fascicularis were studied with oxygen and pH microsensors. Photosynthesis and respiration rates were evaluated from the oxygen fluxes from and to the coral polyps. High-temperature alone lowered both photosynthetic and respiration rates. High pCO2 alone did not significantly affect either photosynthesis or respiration rates. Under a combination of high-temperature and high-CO2, the photosynthetic rate increased to values close to those of the controls. The same pH in the diffusion boundary layer was observed under light in both (400 and 750 ppm) CO2 treatments, but decreased significantly in the dark as a result of increased CO2. The ATP contents decreased with increasing temperature. The effects of temperature on the metabolism of corals were stronger than the effects of increased CO2. The effects of acidification were minimal without combined temperature stress. However, acidification combined with higher temperature may affect coral metabolism due to the amplification of diel variations in the microenvironment surrounding the coral and the decrease in ATP contents.

Response of Nodularia spumigena to pCO2 - Part 3: Turnover of phosphorus compounds

Diazotrophic cyanobacteria often form extensive summer blooms in the Baltic Sea driving their environment into phosphate limitation. One of the main species is the heterocystous cyanobacterium Nodularia spumigena. N. spumigena exhibits accelerated uptake of phosphate through the release of the exoenzyme alkaline phosphatase that also serves as an indicator of the hydrolysis of dissolved organic phosphorus (DOP). The present study investigated the utilization of DOP and its compounds (e.g. ATP) by N. spumigena during growth under varying CO2 concentrations, in order to estimate potential consequences of ocean acidification on the cell's supply with phosphorus. Cell growth, phosphorus pool fractions, and four DOP-compounds (ATP, DNA, RNA, and phospholipids) were determined in three set-ups with different CO2 concentrations (341, 399, and 508 µatm) during a 15-day batch experiment. The results showed rapid depletion of dissolved inorganic phosphorus (DIP) in all pCO2 treatments while DOP utilization increased with elevated pCO2, in parallel with the growth stimulation of N. spumigena. During the growth phase, DOP uptake was enhanced by a factor of 1.32 at 399 µatm and of 2.25 at 508 µatm compared to the lowest pCO2 concentration. Among the measured DOP compounds, none was found to accumulate preferentially during the incubation or in response to a specific pCO2 treatment. However, at the beginning 61.9 ± 4.3% of the DOP were not characterized but comprised the most highly utilized fraction. This is demonstrated by the decrement of this fraction to 27.4 ± 9.9% of total DOP during the growth phase, especially in response to the medium and high pCO2 treatment. Our results indicate a stimulated growth of diazotrophic cyanobacteria at increasing CO2 concentrations that is accompanied by increasing utilization of DOP as an alternative P source.

Surface protein phosphorylation by ecto-protein kinase is required for the maintenance of hippocampal long-term potentiation.

During the induction of long-term potentiation (LTP) in hippocampal slices adenosine triphosphate (ATP) is secreted into the synaptic cleft, and a 48 kDa/50 kDa protein duplex becomes phosphorylated by extracellular ATP. All the criteria required as evidence that these two proteins serve as principal substrates of ecto-protein kinase activity on the surface of hippocampal pyramidal neurons have been fulfilled. This phosphorylation activity was detected on the surface of pyramidal neurons assayed after synaptogenesis, but not in immature neurons nor in glial cells. Addition to the extracellular medium of a monoclonal antibody termed mAb 1.9, directed to the catalytic domain of protein kinase C (PKC), inhibited selectively this surface protein phosphorylation activity and blocked the stabilization of LTP induced by high frequency stimulation (HFS) in hippocampal slices. This antibody did not interfere with routine synaptic transmission nor prevent the initial enhancement of synaptic responses observed during the 1-5 min period immediately after the application of HFS (the induction phase of LTP). However, the initial increase in the slope of excitatory postsynaptic potentials, as well as the elevated amplitude of the population spike induced by HFS, both declined gradually and returned to prestimulus values within 30-40 min after HFS was applied in the presence of mAb 1.9. A control antibody that binds to PKC but does not inhibit its activity had no effect on LTP. The selective inhibitory effects observed with mAb 1.9 provide the first direct evidence of a causal role for ecto-PK in the maintenance of stable LTP, an event implicated in the process of learning and the formation of memory in the brain.

Swiveling-domain mechanism for enzymatic phosphotransfer between remote reaction sites.

The crystal structure of pyruvate phosphate dikinase, a histidyl multiphosphotransfer enzyme that synthesizes adenosine triphosphate, reveals a three-domain molecule in which the phosphohistidine domain is flanked by the nucleotide and the phosphoenolpyruvate/pyruvate domains, with the two substrate binding sites approximately 45 angstroms apart. The modes of substrate binding have been deduced by analogy to D-Ala-D-Ala ligase and to pyruvate kinase. Coupling between the two remote active sites is facilitated by two conformational states of the phosphohistidine domain. While the crystal structure represents the state of interaction with the nucleotide, the second state is achieved by swiveling around two flexible peptide linkers. This dramatic conformational transition brings the phosphocarrier residue in close proximity to phosphoenolpyruvate/pyruvate. The swiveling-domain paradigm provides an effective mechanism for communication in complex multidomain/multiactive site proteins.

Efeito da estimulação purinérgica sobre a produção de melatonina em macrófagos da linhagem RAW 264.7

A melatonina é um hormônio produzido de forma rítmica e no período de escuro pela glândula pineal bem como de forma não rítmica por diversos tecidos e células imunocompetentes. É sintetizada pela acetilação e metilação da serotonina pela ação das enzimas arilalquilamina N-acetiltransferase (AA-NAT) e acetilserotonina -O-metiltransferase (ASMT) que levam à formação de N-acetilserotonina (NAS) e melatonina (MEL), respectivamente. Nos últimos anos temos demonstrado que síntese de melatonina pela pineal pode ser negativamente modulada por mediadores inflamatórios e pelo ATP que atua como co-transmissor juntamente com a noradrenalina liberada no terminal nervoso simpático que a inerva. Perifericamente, contudo, estes mediadores inflamatórios apresentam um efeito contrário induzindo a produção de melatonina em células imunocompetentes. Estas observações levaram à criação da hipótese de um eixo imune-pineal. Esse trabalho teve como objetivo verificar o efeito do ATP sobre produção de melatonina em macrófagos da linhagem RAW 264.7 Os dados desse trabalho mostram que o ATP é capaz de induzir de maneira dose dependente a produção de melatonina em macrófagos através da modulação das enzimas AA-NAT e ASMT. Foi demostrado também que esse efeito é mediado pelo receptor P2X7 e que a melatonina produzida age autocrina e paracrinamente aumentando a fagocitose de particulas de zimosan. Com isso, podemos concluir que o ATP é um ativador endógeno do eixo imune-pineal

Investigating the role of glycine in astrocyte signalling using calcium imaging of rat primary cultures of cortical astrocytes

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Proton Conducting Devices and Materials

Thesis (Ph.D.)--University of Washington, 2016-06

Presynaptic Anchoring of PKA by AKAP7 is Required for Pattern Separation by Dentate Gyrus

Thesis (Ph.D.)--University of Washington, 2016-06

The V-ATPase in Paramecium:functional specialization by multiple gene isoforms

The vacuolar H(+)-ATPase (V-ATPase), a multisubunit, adenosine triphosphate (ATP)-driven proton pump, is essential for numerous cellular processes in all eukaryotes investigated so far. While structure and catalytic mechanism are similar to the evolutionarily related F-type ATPases, the V-ATPase's main function is to establish an electrochemical proton potential across membranes using ATP hydrolysis. The holoenzyme is formed by two subcomplexes, the transmembraneous V(0) and the cytoplasmic V(1) complexes. Sequencing of the whole genome of the ciliate Paramecium tetraurelia enabled the identification of virtually all the genes encoding V-ATPase subunits in this organism and the studying of the localization of the enzyme and roles in membrane trafficking and osmoregulation. Surprisingly, the number of V-ATPase genes in this free-living protozoan is strikingly higher than in any other species previously studied. Especially abundant are V(0)-a-subunits with as many as 17 encoding genes. This abundance creates the possibility of forming a large number of different V-ATPase holoenzymes by combination and has functional consequences by differential targeting to various organelles.