An In Vitro Examination Of Surface Tension Reduction By Pulmonary Surfactant In The Presence And Absence Of Inhibitory Agents

The surface activity of Lipid Extract Surfactant (LES), a clinical preparation used for the treatment of Neonatal Respiratory Distress Syndrome, and the effects of addition of surfactant-associated protein A (SP-A) and palmitic acid were assayed in vitro in the presence and absence of challenge by inhibitory agents. Measurements of surface activity were made with the pulsating bubble surfactometer and the captive bubble technique. The addition of SP-A to LES enhances the surface activity of the preparation, especially at low surfactant concentrations. SP-A accelerates the adsorption of surfactant lipids from the aqueous subphase to the air-liquid interface and causes enrichment of the monolayer film in dipalmitoylphosphatidylcholine.;Blood proteins are potent inhibitors of LES. The addition of SP-A completely reverses the inhibition by fibrinogen, albumin and alpha-globulin. As little as 0.5% (w/w of the surfactant lipid concentration) of SP-A causes reversal of inhibition. This effect is absolutely dependent upon the presence of calcium in the assay mixture. The addition of SP-A to another clinical preparation, Survanta, does not result in enhancement of surface activity or reversal of blood protein inhibition.;The addition of palmitic acid to LES enhances the surface activity, and accelerates adsorption. This effect requires relatively high concentrations of palmitic acid ({dollar}\sim{dollar}8% w/w). Addition of palmitic acid causes a partial reversal of inhibition by blood proteins. Lysophosphatidylcholine (lyso-PC) also inhibits LES. This inhibition is reversed by the addition of palmitic acid, but not SP-A.;The presence of small amounts of lyso-PC in preparations of LES sensitizes the surfactant to inhibition by fibrinogen. This effect is observed regardless of whether the lyso-PC is endogenous, exogenous, or endogenously generated in vitro.;The results of these experiments demonstrate the importance of SP-A in the surface activity of surfactant and the interactions of palmitic acid and lyso-PC with LES. The addition of SP-A and/or palmitic acid should be considered for some applications of surfactant replacement therapy

Shifting Patterns of Nitrogen Excretion and Amino Acid Catabolism Capacity during the Life Cycle of the Sea Lamprey (\u3cem\u3ePetromyzon mariunus\u3c/em\u3e)

The jawless fish, the sea lamprey (Petromyzon marinus), spends part of its life as a burrow-dwelling, suspension-feeding larva (ammocoete) before undergoing a metamorphosis into a free swimming, parasitic juvenile that feeds on the blood of fishes. We predicted that animals in this juvenile, parasitic stage have a great capacity for catabolizing amino acids when large quantities of protein-rich blood are ingested. The sixfold to 20-fold greater ammonia excretion rates (JAmm) in postmetamorphic (nonfeeding) and parasitic lampreys compared with ammocoetes suggested that basal rates of amino acid catabolism increased following metamorphosis. This was likely due to a greater basal amino acid catabolizing capacity in which there was a sixfold higher hepatic glutamate dehydrogenase (GDH) activity in parasitic lampreys compared with ammocoetes. Immunoblotting also revealed that GDH quantity was 10-fold and threefold greater in parasitic lampreys than in ammocoetes and upstream migrant lampreys, respectively. Higher hepatic alanine and aspartate aminotransferase activities in the parasitic lampreys also suggested an enhanced amino acid catabolizing capacity in this life stage. In contrast to parasitic lampreys, the twofold larger free amino acid pool in the muscle of upstream migrant lampreys confirmed that this period of natural starvation is accompanied by a prominent proteolysis. Carbamoyl phosphate synthetase III was detected at low levels in the liver of parasitic and upstream migrant lampreys, but there was no evidence of extrahepatic (muscle, intestine) urea production via the ornithine urea cycle. However, detection of arginase activity and high concentrations of arginine in the liver at all life stages examined infers that arginine hydrolysis is an important source of urea. We conclude that metamorphosis is accompanied by a metabolic reorganization that increases the capacity of parasitic sea lampreys to catabolize intermittently large amino acid loads arising from the ingestion of protein rich blood from their prey/hosts. The subsequent generation of energy-rich carbon skeletons can then be oxidized or retained for glycogen and fatty acid synthesis, which are essential fuels for the upstream migratory and spawning phases of the sea lamprey’s life cycle

Quantitative and Functional Characterization of the Hyper-Conserved Protein of Prochlorococcus and Marine Synechococcus

A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs). While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP) of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein\u27s binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly

Quantitative and Functional Characterization of the Hyper-Conserved Protein of Prochlorococcus and Marine Synechococcus

A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs). While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP) of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein\u27s binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly

A Key Marine Diazotroph in a Changing Ocean: The Interacting Effects of Temperature, CO2 and Light on the Growth of Trichodesmium erythraeum IMS101

Trichodesmium is a globally important marine diazotroph that accounts for approximately 60-80% of marine biological N2 fixation and as such plays a key role in marine N and C cycles. We undertook a comprehensive assessment of how the growth rate of Trichodesmium erythraeum IMS101 was directly affected by the combined interactions of temperature, pCO2 and light intensity. Our key findings were: low pCO2 affected the lower temperature tolerance limit (Tmin) but had no effect on the optimum temperature (Topt) at which growth was maximal or the maximum temperature tolerance limit (Tmax); low pCO2 had a greater effect on the thermal niche width than low-light; the effect of pCO2 on growth rate was more pronounced at suboptimal temperatures than at supraoptimal temperatures; temperature and light had a stronger effect on the photosynthetic efficiency (Fv/Fm) than did CO2; and at Topt, the maximum growth rate increased with increasing CO2, but the initial slope of the growth-irradiance curve was not affected by CO2. In the context of environmental change, our results suggest that the (i) nutrient replete growth rate of Trichodesmium IMS101 would have been severely limited by low pCO2 at the last glacial maximum (LGM), (ii) future increases in pCO2 will increase growth rates in areas where temperature ranges between Tmin to Topt, but will have negligible effect at temperatures between Topt and Tmax, (iii) areal increase of warm surface waters (> 18°C) has allowed the geographic range to increase significantly from the LGM to present and that the range will continue to expand to higher latitudes with continued warming, but (iv) continued global warming may exclude Trichodesmium spp. from some tropical regions by 2100 where temperature exceeds Topt

Determining Diversity of Freshwater Fungi on Decaying Leaves: Comparison of Traditional and Molecular Approaches

Traditional microscope-based estimates of species richness of aquatic hyphomycetes depend upon the ability of the species in the community to sporulate. Molecular techniques which detect DNA from all stages of the life cycle could potentially circumvent the problems associated with traditional methods. Leaf disks from red maple, alder, linden, beech, and oak as well as birch wood sticks were submerged in a stream in southeastern Canada for 7, 14, and 28 days. Fungal biomass, estimated by the amount of ergosterol present, increased with time on all substrates. Alder, linden, and maple leaves were colonized earlier and accumulated the highest fungal biomass. Counts and identifications of released conidia suggested that fungal species richness increased, while community evenness decreased, with time (up to 11 species on day 28). Conidia of Articulospora tetracladia dominated. Modifications of two molecular methods—denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis—suggested that both species richness and community evenness decreased with time. The dominant ribotype matched that of A. tetracladia. Species richness estimates based on DGGE were consistently higher than those based on T-RFLP analysis and exceeded those based on spore identification on days 7 and 14. Since traditional and molecular techniques assess different aspects of the fungal organism, both are essential for a balanced view of fungal succession on leaves decaying in streams

Combined effects of simulated acidification and hypoxia on the harmful dinoflagellate Amphidinium carterae

Hypoxia and acidification frequently co-occur in coastal marine ecosystems, and will likely become more intense and persis- tent with anthropogenic climate change. Although the separate effects of these stressors have previously been described, their combined effects on marine phytoplankton are currently unknown. In this novel study, multi-stressor incubation experiments using the harmful dinoflagellate, Amphidinium carterae, examined the effects of acidification and hypoxia both individually and in combination. Long-term (7 days) and short-term (6 h) experiments under controlled carbon dioxide (CO2) and oxygen (O2) conditions examined the interactive effects of the stressors and the physiological mechanisms driving their interaction. In the long-term experiment, synergistically negative effects were observed for A. carterae growth, photosynthesis, carbon fixation, nitrate uptake, and photosynthetic efficiency (Fv/Fm) under combined high CO2 (low pH) and low O2 conditions. In the short-term experiment, delayed recovery of photosystem II (PSII) reaction centers was observed following photoinhibi- tion, suggesting that high CO2 and low O2 conditions negatively affect photosynthesis in A. carterae even after relatively short exposures. Although high CO2, low O2 conditions should decrease photorespiration and favor carbon fixation by the key photosynthetic enzyme ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO), these findings demonstrate that the affinity of RuBisCO for CO2 relative to O2 alone does not predict phytoplankton responses to CO2 and O2 conditions in vivo, complicating predictions of phytoplankton community responses to hypoxia and acidification. Results of these experi- ments suggest that the combination of low pH and O2 concentrations may negatively impact the growth of some harmful dinoflagellates in coastal marine ecosystems

Quantitative Targeted Proteomics and Electrochromic Shift for Measuring Photosystem Content of Marine Phytoplankton

Abundance and stoichiometry data for the photosystems, the intersystem electron transport complexes and the Calvin cycle enzymes are rich in information about light and nutrient acclimation. Quantifying these complexes is essential for understanding limitations on and capacities for photosynthesis. Targeted quantitative immunodetections of conserved subunits (eg. PsbA for PSII; PsaC for PSI) are becoming an established method for absolute measurement of these complexes. An advantage of protein measurements is that they can be done with non-living flash-frozen samples and processed post-field. A pitfall of physical versus functional measures is that in some scenarios, such as during photoinhibition of photosystem II (PSII), physical and functional measures give different values, but such disparities are often meaningful, informing targeted studies of regulation, repair and enzyme kinetics. Electrochromic Shift (ECS) is an alternative, fast and noninvasive method which can be exploited to determine functional PSI:PSII ratios in living cells. The basis for ECS is that pigments in the photosynthetic membrane exhibit a shift in their absorption spectra when the electric component of the proton motive force is generated across the membrane in the light. Cross-validation of methods by independent measures builds confidence in results from both approaches and can be useful for ground truthing of underway or high-throughput optical measurements or functional measurements from bioassays. We present comparative data from immunoquantitation and ECS for an array of diatom taxa. The physical data fall within established ranges. The basis for similarities and disparities in the photosystem stoichiometries between the methods are discussed

The RUBISCO to Photosystem II Ratio Limits the Maximum Photosynthetic Rate in Picocyanobacteria

Marine Synechococcus and Prochlorococcus are picocyanobacteria predominating in subtropical, oligotrophic marine environments, a niche predicted to expand with climate change. When grown under common low light conditions Synechococcus WH 8102 and Prochlorococcus MED 4 show similar Cytochrome b6f and Photosystem I contents normalized to Photosystem II content, while Prochlorococcus MIT 9313 has twice the Cytochrome b6f content and four times the Photosystem I content of the other strains. Interestingly, the Prochlorococcus strains contain only one third to one half of the RUBISCO catalytic subunits compared to the marine Synechococcus strain. The maximum Photosystem II electron transport rates were similar for the two Prochlorococcus strains but higher for the marine Synechococcus strain. Photosystem II electron transport capacity is highly correlated to the molar ratio of RUBISCO active sites to Photosystem II but not to the ratio of cytochrome b6f to Photosystem II, nor to the ratio of Photosystem I: Photosystem II. Thus, the catalytic capacity for the rate-limiting step of carbon fixation, the ultimate electron sink, appears to limit electron transport rates. The high abundance of Cytochrome b6f and Photosystem I in MIT 9313, combined with the slower flow of electrons away from Photosystem II and the relatively low level of RUBISCO, are consistent with cyclic electron flow around Photosystem I in this strain