Characteristics of ouabain binding to isolated trout hepatocytes

Summary Na⁺, K⁺ exchanges were studied in isolated hepatocytes of the rainbow trout, Salmo gairdneri. Ouabain at 10^–4 M produced maximal inhibition (95%) of K⁺ uptake and enhanced intracellular Na⁺ accumulation, showing that active fluxes account for a very large proportion of Na⁺ and K⁺ exchanges. Inhibition of the Na–K pump by ouabain was significant at low concentrations (10^–8 M). When external K⁺ concentration was reduced from 7 mM to 0.5 mM, half maximum inhibition (IC₅₀) of K⁺ uptake was obtained at a 22-fold lower concentration of ouabain confirming that ouabain and potassium compete at the same pump site. Time-course analysis of [³H]ouabain binding indicated a two-component kinetics: one component saturable and dependent on K⁺ concentration in the medium, the other linear and independent of external K⁺. The ouabain binding site number, determined by Scatchard plots, remained constant (ca. 2.5·10⁵ per cell) and independent of the external K⁺ concentration (7, 0.5 or 0 mM), while the dissociation constant (K_D) decreased from 4.2 M to 7.3 nM when K⁺ was removed from the Hank's medium. These ouabain binding sites are characterized by an exceptionally low turnover rate (400 min^–1), as estimated from ouabain-sensitive K⁺ flux, in comparison to those described in other cell types of higher vertebrates. At each external K⁺ concentration studied, the inhibition of K⁺ uptake and ouabain binding measured as a function of ouabain concentration indicated a strict correlation between the degree of K pump inhibition and the amount of bound glycoside.     

A Simple Method for Mass-Production of Liposomes,in Particular Large Liposomes,Suitable for Delivery of Free Amino Acids to Filter Feeding Zooplankton

In marine fish larviculture the live feed organisms are often enriched in order to enhance their nutritional value. One of the challenges is to enhance the phospholipids (PL) content, and another is to enhance the content of specific water soluble nutrients, like free amino acids (FAA). There are a few studies where this has been achieved by the use of liposomes. The aim of this study was to develop a simple method for mass-production of liposomes within a size range of 1–5 μm a size range suitable to feed live food organisms. Furthermore, the liposomes should have a high FAA concentration and be stable under conditions typical for short-time enrichment of live feed organisms. The method used in the present study is based on a combination of a reverse-phase evaporation method for preparing liposomes and re-hydration of freeze-dried, empty liposomes. The liposomal membrane was made of soy phosphatydilcholine and was loaded with a highly concentrated free amino acids solution. Most of the liposomes produced were 2–8 μm in diameter and the FAA encapsulation efficiency was 42.6%. Two experiments simulating 2 hr of live food enrichment were used to evaluate the liposomes. The results showed the liposome did not disintegrate or aggregate when suspended in seawater and that only 9% of the FAA content of the liposomes was lost after 2 hr suspension. The developed method was easy and reliable, producing tens of grams of liposomes per batch.     

Preconditioning of leaves by solar radiation and anoxia affects microbial colonisation and rate of leaf mass loss in an intermittent stream

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Increased quantitative proteome coverage with (13)C/(12)C-based, acid-cleavable isotope-coded affinity tag reagent and modified data acquisition scheme

Quantitative protein profiling using the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS) enables the pair-wise comparison of protein expression levels in biological samples. A new version of the ICAT reagent with an acid-cleavable bond, which allows removal of the biotin moiety prior to MS and which utilizes (13)C substitution for (12)C in the heavy-ICAT reagent rather than (2)H (for (1)H) as in the original reagent, was investigated. We developed and validated an MS data acquisition strategy using this new reagent that results in an increased number of protein identifications per experiment, without losing the accuracy of protein quantification. This was achieved by following a single survey (precursor) ion scan and serial collision induced dissociations (CIDs) of four different precursor ions observed in the prior survey scan. This strategy is common to many high-performance liquid chromatography-electrospray ionization (HPLC-ESI)-MS shotgun proteomic strategies, but heretofore not to ICAT experiments. This advance is possible because the new ICAT reagent uses (13)C as the "heavy" element rather than (2)H, thus, eliminating the slight delay in retention time of ICAT-labeled "light" peptides on a C18-based HPLC separation that occurs with (2)H and (1)H. Analyses using this new scheme of an ICAT-labeled trypsin-digested six protein mixture as well as a tryptic digest of a total yeast lysate, indicated that about two times more proteins were identified in a single analysis, and that there was no loss in accuracy of quantification.     

Nitrogen Export from Forested Watersheds in the Oregon Coast Range: The Role of N<Subscript>2</Subscript>-fixing Red Alder

Variations in plant community composition across the landscape can influence nutrient retention and loss at the watershed scale. A striking example of plant species importance is the influence of N₂-fixing red alder (Alnus rubra) on nutrient cycling in the forests of the Pacific Northwest. To understand the influence of red alder on watershed nutrient export, we studied the chemistry of 26 small watershed streams within the Salmon River basin of the Oregon Coast Range. Nitrate and dissolved organic nitrogen (DON) concentrations were positively related to broadleaf cover (dominated by red alder: 94% of basal area), particularly when near-coastal sites were excluded (r ² = 0.65 and 0.68 for nitrate-N and DON, respectively). Nitrate and DON concentrations were more strongly related to broadleaf cover within entire watersheds than broadleaf cover within the riparian area alone, which indicates that leaching from upland alder stands plays an important role in watershed nitrogen (N) export. Nitrate dominated over DON in hydrologic export (92% of total dissolved N), and nitrate and DON concentrations were strongly correlated. Annual N export was highly variable among watersheds (2.4–30.8 kg N ha^–1 y^–1), described by a multiple linear regression combining broadleaf and mixed broadleaf–conifer cover (r² = 0.74). Base cation concentrations were positively related to nitrate concentrations, which suggests that nitrate leaching increases cation losses. Our findings provide evidence for strong control of ecosystem function by a single plant species, where leaching from N saturated red alder stands is a major control on N export from these coastal watersheds.     

Regulation of excitation energy distribution in Photosystem-II fragments by magnesium ions

Fluorescence characteristics of Photosystem-II subchloroplasts (TSF-II and TSF-IIa) fractionated by Triton X-100 treatment were studied in relation to cation-induced regulation of excitation-energy distribution within subchloroplast fragments. Absorption spectra and fluorescence-emission spectra at 77 K showed that TSF-II contains the light-harvesting chlorophyll-protein complex in addition to the reaction-center complex, which is present alone in TSF-IIa.Mg²⁺ increased the ratio of F_695nm to F_685nm in the fluorescence-emission spectrum of TSF-II particles at 77 K, but had no effect on TSF-IIa particles. Mg²⁺ also induced a quenching of chlorophyll fluorescence at room temperature in TSF-II, an effect that was insensitive to the presence of DCMU. The DCMU-insensitive fluorescence quenching was not observed in the TSF-IIa preparation. These results suggest an existence of cation-induced regulation of excitation-energy transfer in TSF-II preparations. Presence of antenna chlorophyll molecules alone does not seem to be sufficient for observing energytransfer regulation by cations in Photosystem-II preparations.     

Antibodies Specific for α-N-Acetyl-β-Endorphins: Radioimmunoassays and Detection of Acetylated β-Endorphins in Pituitary Extracts

Abstract: Antibodies specific for α-N-acetyl-β-endorphins have been prepared by injecting into rabbits either α-N-acetyl-β-endorphin(1-31) or [α-N-acetyl, ε-acetyl-Lys⁹]-β-endorphin(1-9) linked by carbodiimide to bovine thyroglobulin. Both antisera were used to develop specific radioimmunoassays for α-N-acetyl-β-endorphins. The radioimmunoassays were used to measure α-N-acetylated β-endorphins in extracts of pituitary regions from different species. By comparison of the amounts of total β-endorphin and α-N-acetyl-β-endorphin immunoreactivity, a relative ratio of β-endorphin acetylation was obtained. The relative acetylation of β-endorphin was highest in rat posterior-intermediate lobe extracts (>90%). Beef and monkey intermediate lobes had a lower degree of acetylation (53 and 31%, respectively). Anterior lobe extracts from all three species contained low amounts of acetylated β-endorphin. Human pituitary extracts did not contain acetylated β-endorphins. By the use of cation exchange and high performance liquid chromatography, six different acetylated derivatives and fragments of β-endorphin were resolved in extracts of rat posterior-intermediate pituitaries. Two of these peptides corresponded to α-N-acetyl-β-endorphin(1-31) and -(1-27). One acetylated β-endorphin fragment had the same size as α-N-acetyl-β-endorphin(1-27) but was eluted earlier from the cation exchange column. This peptide had full cross-reactivity with antibodies directed against the middle and amino-terminal parts of β-endorphin. Compared with α-N-acetyl-β-endorphin(1-27), it had much less cross-reactivity with antibodies directed against the COOH-terminal part of β-endorphin, suggesting that it was a COOH-terminally modified derivative of β-endorphin(1-27). The remaining N-acetylated β-endorphin derivatives were eluted even earlier from the cation exchange column. The majority of these fragments were slightly larger in size than y-endorphin, i.e., β-endorphin(1-17), but smaller than β-endorphin(1-27). They had full cross-reactivity in an amino-terminally directed β-endorphin radioimmunoassay and a greatly diminished cross-reactivity with antibodies to the middle region of β-endorphin.     

A grape berry (Vitis vinifera L.) cation/proton antiporter is associated with berry ripening

We have cloned and characterized VvNHX1, a gene encoding a vacuolar cation/H(+) antiporter from Vitis vinifera cv. Cabernet Sauvignon. VvNHX1 belongs to the vacuolar NHX protein family and showed high similarity to other known vacuolar antiporters. The expression of VvNHX1 partially complements the salt- and hygromycin-sensitive phenotypes of an ena1-4 nhx1 yeast strain. Immunoblots of vacuoles of yeast expressing a VvNHX1, together with the expression of a VvNHX1-GFP (green fluorescent protein) chimera demonstrated that VvNHX1 localized to the vacuoles. VvNHX1 displayed low affinity K(+)/H(+) and Na(+)/H(+) exchange activities (12.8 and 40.2 mM, respectively). The high levels of expression of VvNHX1 during the véraison and post-véraison stages would indicate that the increase in vacuolar K(+) accumulation, mediated by VvNHX1, is needed for vacuolar expansion. This process, together with the rapid accumulation of reducing sugars, would drive water uptake to the berry and the concomitant berry size increase, typical of the post-véraison stage of growth.     

Effects of soil frost on nitrogen net mineralization, soil solution chemistry and seepage losses in a temperate forest soil

Freezing and thawing may alter element turnover and solute fluxes in soils by changing physical and biological soil properties. We simulated soil frost in replicated snow removal plots in a mountainous Norway spruce stand in the Fichtelgebirge area, Germany, and investigated N net mineralization, solute concentrations and fluxes of dissolved organic carbon (DOC) and of mineral ions (NH₄⁺, NO₃⁻, Na⁺, K⁺, Ca²⁺, Mg²⁺). At the snow removal plots the minimum soil temperature was −5 °C at 5 cm depth, while the control plots were covered by snow and experienced no soil frost. The soil frost lasted for about 3 months and penetrated the soil to about 15 cm depth. In the 3 months after thawing, the in situ N net mineralization in the forest floor and upper mineral soil was not affected by soil frost. In late summer, NO₃⁻ concentrations increased in forest floor percolates and soil solutions at 20 cm soil depth in the snow removal plots relative to the control. The increase lasted for about 2–4 months at a time of low seepage water fluxes. Soil frost did not affect DOC concentrations and radiocarbon signatures of DOC. No specific frost effect was observed for K⁺, Ca²⁺ and Mg²⁺ in soil solutions, however, the Na⁺ concentrations in the upper mineral soil increased. In the 12 months following snowmelt, the solute fluxes of N, DOC, and mineral ions were not influenced by the previous soil frost at any depth. Our experiment did not support the hypothesis that moderate soil frost triggers solute losses of N, DOC, and mineral ions from temperate forest soils.