Longitudinal Spatial Patterns of Bacterial Production and Respiration in a Large River–Estuary: Implications for Ecosystem Carbon Consumption

Rivers and estuaries transport organic carbon (C) from terrestrial and freshwater ecosystems to the marine environment. During this transit, bacteria actively utilize and transform organic C, but few studies have measured detailed spatial variation in rates of bacterial respiration (BR) and production (BP). We measured BP at 39 stations and BR at 12 stations at monthly intervals along a 200-km reach of the tidal Hudson River. We observed strong repeatable spatial patterns for both BP and BR, with rates declining in the downstream direction. Bacterial Production had much greater dynamic range of spatial variation than BR. We used the detailed seasonal and spatial data on BP and BR to measure the total C demand of bacteria at several scales. We calculated volumetric and areal rates for 12 sections of the Hudson, as well as the total C utilization. Volumetric BR averaged 20 g-C-m^–3 y^–1, but it was highest in the most upstream section at 30 g C m^–3 y^–1. Areal rates averaged over the entire river were 174 g C m^–2 y^–1, but they were 318 g C m^–2 y^–1 in the deepest section of the river, indicating the importance of morphometric variation. Total bacterial C demand increased downriver with increasing total volume. Overall, bacteria in the freshwater section of the river consumed approximately 18–25.5 × 10⁹ g C y^–1, about 20% of the total organic C load.     

Effectiveness of SC2053 as a chemical hybridizing agent for winter wheat

The use of chemical hybridizing agents (CHA) allows production of hybrid wheat seeds. We evaluated the effectiveness of a new CHA (SC2053) to induce male sterility on winter wheat in controlled growth conditions. CHA effectiveness was measured with the application of 4 doses (0–400–700–1000 g.ha^–1) at 7 stages. These stages were defined by the length of the main stem spike (1–4–7–11–15–20–40 mm). At heading, individual ears were isolated with a greaseproof paper bag. The seeds formed were counted on treated and control ears. The spikes' sterility was calculated three weeks after flowering. The sterility of the main stem's spike reached 95% to 100% for application of 700 g.ha^–1 and 1000 g.ha^–1 for main stem spike length of 7 mm to 20 mm. The effects of ear tillering (5 tillers per plant) on CHA effectiveness were also investigated. We observed a significant delay of ear development between the main stem and tillers so that complete sterilities were not reached for each dose. Since tillering in field conditions rarely exceeds 3 ears per plant, CHA effectiveness was studied on plants bearing 3 ears. The mean sterility of the first 3 ears was close to 100% for applications with 700 g.ha^–1 and 1000 g.ha^–1 at stages from 11 mm to 20 mm of main stem spike length.     

Metalloproteinase with factor X activating and fibrinogenolytic activities from Vipera berus berus venom

We have previously shown that Vipera berus berus venom contains several factor X activating enzymes. In the present study we have investigated one of them. The enzyme was separated from venom by gel filtration on Sephadex G-100 superfine and chromatography on agarose HPS-7 and phenyl-agarose. The enzyme is a glycosylated metalloproteinase containing hexoses, hexosamines and neuraminic acid. The purified factor X activating enzyme consists of two equal chains (59 kDa). The specificity studies have shown that enzyme is nonspecific factor X activating proteinase hydrolysing also proteins such as azocasein, gelatin and fibrinogen. The enzyme hydrolyses oxidized insulin B-chain at the positions Ala¹⁴–Leu¹⁵ and Tyr¹⁶–Leu¹⁷ but it is inactive on fibrin, plasminogen and prothrombin. We used 8–10 amino acid residues containing peptides, which reproduce the sequence around the cleavage sites in factor X, factor IX and fibrinogen, as potential substrates for enzyme. Cleavage products of peptide hydrolysis were determined by MALDI-TOF MS. The peptide Asn–Asn–Leu–Thr–Arg–Ile–Val–Gly–Gly—factor X fragment was cleaved by enzyme at positions Leu³–Thr⁴ and Arg⁵–Ile⁶. The fibrinogen peptide fragment Glu–Tyr–His–Thr–Glu–Lys–Leu–Val–Thr–Ser was hydrolysed at position Lys⁶–Leu⁷.     

Identification of the cardiac sarcolemmal Na+−Ca2+ exchanger using monoclonal antibodies

Summary We have previously partially purified the sarcolemmal Na⁺–Ca²⁺ exchange protein and produced rabbit polyclonal antibodies to the exchanger (Philipson, K. D., Longoni, S., Ward, R. 1988.Biochim. Biophys.Acta 945:298–306). We now describe the generation of three stable murine hybridoma lines which secrete monoclonal antibodies (MAb's) to the exchanger. These MAb's immunoprecipitate 50–75% of solubilized Na⁺–Ca²⁺ exchange activity. The MAb's appear to be reactive with native conformation-dependent expitopes on the Na⁺–Ca²⁺ exchanger since they do not react on immunoblots. An indirect method was used to identify Na⁺–Ca²⁺ exchange proteins. A column containing Na⁺–Ca²⁺ exchanger immobilized by MAb's was used to affinity purify the rabbit polyclonal antibody. The affinity-purified polyclonal antibody reacted with proteinsof, apparent molecular weights of 70, 120, and 160 kDa on immunoblots of sarcolemma. The data provide strong support for our prevous association of Na⁺–Ca²⁺ exchange with these proteins.     

Chloride-activated water permeability in the frog corneal epithelium

We have previously reported that the isolated frog corneal epithelium (a Cl^–-secreting epithelium) has a large diffusional water permeability (P_dw 1.8×10^–4 cm/s). We now report that the presence of Cl^– in the apical-side bathing solution increases the diffusional water flux, J_dw (in both directions) by 63% from 11.3 to 18.4 l min^–1 · cm^–2 with 60 mm [Cl] exerting the maximum effect. The presence of Cl^– in the basolateral-side bathing solution had no effect on the water flux. In Cl^–-free solutions amphotericin B increased J_dw by 29% but only by 3% in Cl^–-rich apical-side bathing solution, suggesting that in Cl^–-rich apical side bathing solution, the apical barrier is no longer rate limiting. Apical Br^– (75 mm) also increased J_dw by 68%. The effect of Cl^– on J_dw was observed within 1 min after its addition to the apicalside bathing solution. HgCl₂ (0.5 mm) reduced the Cl^–-increased P_dw by 31%. The osmotic permeability (P_f) was also measured under an osmotic gradient yielding values of 0.34 and 2.88 (x 10^–3 cm/s) in Cl^–-free and Cl^–-rich apical-side bathing solutions respectively. It seems that apical Cl^–, or Cl^– secretion into the apical bath could activate normally present but inactive water channels. In the absence of Cl^–, water permeability of the apical membrane seems to be limited to the permeability of the lipid bilayer.This work was supported by National Eye Institute grants EY-00160 and EY-01867.     

Chaff piles of harvester ant (Messor spp.) nests in a desert ecosystem

Summary The plant species composition of the chaff piles of three species of harvester ant (Messor spp.) and the contribution of the chaff to the organic pool were studied from August 1985 to July 1987. There were distinct differences in the plant species composition of the chaff of the three species. We attribute this to the different diets of the three species, which reflect the relative sizes of their individuals and their foraging strategies. The amount of chaff accumulated varies greatly between the three species (Messor rugossus: 127–196 g · ha^–1 · y^–1;Messor ebeninus: 2823–4437 g · ha^–1 · y^–1;Messor arenarius: 2165–2535 g · ha^–1 · y^–1), although the number of nests per hectare is virtually the same. We found that the amount of chaff is related to the rate of activity and the size of the individuals of each of the three ant species. The total chaff accumulated during the study period was 19.2 kg · ha^–1, which is an important contribution to the organic matter in the soil in the Negev desert ecosystem.     

Hydrological and nutrient budgets of freshwater and estuarine wetlands of Taylor Slough in Southern Everglades, Florida (U.S.A.)

Hydrological restoration of the Southern Everglades will result in increased freshwater flow to the freshwater and estuarine wetlands bordering Florida Bay. We evaluated the contribution of surface freshwater runoff versus atmospheric deposition and ground water on the water and nutrient budgets of these wetlands. These estimates were used to assess the importance of hydrologic inputs and losses relative to sediment burial, denitrification, and nitrogen fixation. We calculated seasonal inputs and outputs of water, total phosphorus (TP) and total nitrogen (TN) from surface water, precipitation, and evapotranspiration in the Taylor Slough/C-111 basin wetlands for 1.5 years. Atmospheric deposition was the dominant source of water and TP for these oligotrophic, phosphorus-limited wetlands. Surface water was the major TN source of during the wet season, but on an annual basis was equal to the atmospheric TN deposition. We calculated a net annual import of 31.4 mg m^–2 yr^–1 P and 694 mg m^–2 yr^–1N into the wetland from hydrologic sources. Hydrologic import of P was within range of estimates of sediment P burial (33–70 mg m^–2 yr^–1 P), while sediment burial of N (1890–4027 mg m^–2 yr^–1 N) greatly exceeded estimated hydrologic N import. High nitrogen fixation rates or an underestimation of groundwater N flux may explain the discrepancy between estimates of hydrologic N import and sediment N burial rates.     

Settling flux and sinking velocity of Seston in Lago di Mergozzo (Northern Italy) and influence of microbial activity on the decomposition of entrapped organic material

Settling flux and velocity of seston, Particulate Organic Carbon (POC) and chlorophyll a were measured at three depths during 8 seven-day exposure periods in Lago di Mergozzo (Northern Italy). Sedimentation rates of seston varied from 250 to 1200 mg m^–2 d^–1 with a prevalence of the inorganic fraction (130–900 mg m^–2 d^–1) over the organic (160–320 mg m^–2 d^–1).The percentage of organic fraction inside the traps was always lower than outside. The comparison of preserved and unpreserved traps showed no significant difference in both organic matter content and bacterial numbers. We inferred from this result that bacterial activity in the traps did not cause a measurable POC loss during the seven day exposures. Therefore, the higher settling velocity of the inorganic particles was responsible for the higher percentage of this fraction in the traps. The settling velocity of sestonic particles increased, during the stratification period, with increasing depth and reached a maximum value of 2.5 m d^–1.     

Feeding rates and energy deficits of juvenile and adult Japanese monkeys in a cool temperate area with snow coverage

Japanese monkeys, Macaca fuscata, living in a cool temperate forest experienced energy crises in winter. We measured feeding times and feeding rates (mass of foods eaten per unit time of feeding) in six different-sized, age–sex classes (1.2–12.6 kg body mass) in autumn and winter. One-, 2- and 3~4-year-olds spent 34–35% and 44–46% of the day feeding in autumn and winter, respectively. Monkeys less than 0 years old spent less time feeding (16–28%) than average in winter and autumn; adult females spent less (41%) in winter; and adult males spent less (25%) in autumn. All age–sex classes ate mainly fruits in autumn and the heavier classes fed more on tree bark than buds in winter. The feeding rate for fruits (2.3–53.5 g min^–1) was nine to 12 times faster than those for buds (1.0– 4.8 g min^–1) and bark (0.5–4.4 g min^–1), and energy content did not differ among fruits (22.1 kJ g^–1 dry mass), buds (19.9 kJ g^–1 dry mass) and bark (23.2 kJ g^–1 dry mass). Energy base feeding rates increased with body mass where the body mass exponent for buds (0.29) was smaller than those for bark (0.64) and fruits (0.63), which might be attributable to the unit size of food items and mass dependency of masticatory apparatus. Our monkeys obtained two to five times more energy in autumn (1567–1150 kJ day^–1) than in winter (604–3020 kJ day^–1). Adult females obtained 60% of expected energy expenditure and other classes obtained 77–88% of that in winter.     

Forest nitrogen sinks in large eastern U.S. watersheds: estimates from forest inventory and an ecosystem model

The eastern U.S. receives elevated rates of Ndeposition compared to preindustrial times, yetrelatively little of this N is exported indrainage waters. Net uptake of N into forestbiomass and soils could account for asubstantial portion of the difference between Ndeposition and solution exports. We quantifiedforest N sinks in biomass accumulation andharvest export for 16 large river basins in theeastern U.S. with two separate approaches: (1)using growth data from the USDA ForestService's Forest Inventory and Analysis (FIA)program, and (2) using a model of forestnitrogen cycling (PnET-CN) linked to FIAinformation on forest age-class structure. Themodel was also used to quantify N sinks in soiland dead wood, and nitrate losses below therooting zone. Both methods agreed that netgrowth rates were highest in the relativelyyoung forests on the Schuylkill watershed, andlowest in the cool forests of northern Maine. Across the 16 watersheds, wood export removedan average of 2.7 kg N ha^–1 yr^–1(range: 1–5 kg N ha^–1 yr^–1), andstanding stocks increased by 4.0 kg N ha^–1yr^–1 (–3 to 8 kg N ha^–1 yr^–1). Together, these sinks for N in woody biomassamounted to a mean of 6.7 kg N ha^–1yr^–1 (2–9 kg N ha^–1 yr^–1), or73% (15–115%) of atmospheric N deposition. Modeled rates of net N sinks in dead wood andsoil were small; soils were only a significantnet sink for N during simulations ofreforestation of degraded agricultural sites. Predicted losses of nitrate depended on thecombined effects of N deposition, and bothshort- and long-term effects of disturbance. Linking the model with forest inventoryinformation on age-class structure provided auseful step toward incorporating realisticpatterns of forest disturbance status acrossthe landscape.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.     

Methane and nitrous oxide exchange in differently fertilised grassland in southern Germany

We examined the effect of fertilisation (200 kg cattle slurry-N ha^–1 year^–1) on the exchange of N₂O and CH₄ in the soil–plant system of meadow agroecosystems in southern Germany. From 1996 to 1998, we regularly determined the gas fluxes (closed chamber method) and associated environmental parameters. N₂O and CH₄ fluxes were not significantly affected by fertilisation. N₂O fluxes at the unfertilised and fertilised plots were small, generally between 50 and –20 g N m^–2 h^–1. We identified some incidents of N₂O uptake. CH₄-C fluxes ranged from 1.3 to –0.2 mg m^–2 h^–1 and were not significantly different from 0 at both plots. We budgeted an annual net emission of 15.5 and 29.6 mg m^–2 N₂O-N and an annual CH₄-C net emission of 184.2 and 122.7 mg m^–2 at the unfertilised and fertilised plots, respectively. Apparently, rapid N mineralization and uptake in the densely rooted topsoil prevents N losses and the inhibition of CH₄ oxidation.     

Benithic-pelagic coupling of nutrient metabolism along an estuarine eutrophication gradient

The shallow, brackish (11–18% salinity) Roskilde Fjord represents a eutrophication gradient with annual averages of chlorophyll, ranging from 3 to 25 mg chl a m^–3. Nutrient loadings in 1985 were 11.3–62.4 g N m^–2 yr^–1 and 0.4–7.3 g P m^–2 yr^–1. A simple one-layer advection-diffusion model was used to calculate mass balances for 7 boxes in the fjord. Net loss rates varied from –32.2 to 17.9 g P m^–2 yr^–1 and from –3.3 to 66.8 g N m^–2, corresponding to 74% of the external P-loading and 88% of the external N-loading to the entire estuary.Gross sedimentation rates measured by sediment traps were between 7 and 52 g p m^–2 yr^–1 and 50 and 426 g N M^–2 yr^–1, respectively. Exchangeable sediment phosphorus varied in annual average between 2.0 and 4.8 g P m^–2 and exchangeable sediment nitrogen varied from 1.9 to 33.1 g N m–1. Amplitudes in the exchangeable pools followed sedimentation peaks with delays corresponding to settling rates of 0.3 m d^–1. Short term nutrient exchange experiments performed in the laboratory with simultaneous measurements of sediment oxygen uptake showed a release pattern following the oxygen uptake, the changes in the exchangeable pools and the sedimentation peaks.The close benthic-pelagic coupling also exists for the denitrification with maxima during spring of 5 to 20 mmol N m^–2 d–1. Denitrification during the nitrogen-limited summer period suggests dependence on nitrification. Comparisons with denitrification from other shallow estuaries indicate a maximum for denitrification in estuaries of about 250 µmol N m^–2 h^–2 achieved at loading rates of about 25–125 g N m^–2 yr^–1.     

An hypothesis for the interpretation of the contractile response of vascular smooth muscle at the cellular level

The long preservation and recovery of functional (contractile) properties in cultured aortic smooth muscle cells, even after replating or deep-frozen storage and the measurement of their responses are now technically settled issues. We could thus study extensively the responses of single cultured cells from rat thoracic aorta. Responses were elicited by the addition of KCl 40 mmol/L without or with a calcium blocker PN 200-100 (10^–6 mol/L); angiostein II (10^–11–10^–6 mol/L) without or with antagonist (losartan 10^–5 mol/L); or serotonin (10^–9–10^–4 mol/L) without or with antagonist (naftidrofuryl 10^–5 mol/L). Results thus obtained enabled us to propose a new hypothesis for the interpretation of the contractile responses of an elastic vascular smooth muscle. The different maximal effects of different agonists result mainly from the different proportions of cells they can mobilize; the agonist concentration-contraction relationship is mainly due to the increase of the proportion of cells involved up to a maximal value typical of the agonist used. An antagonist primarily reduce the proportion of cells an agonist can mobilize. Some of the consequences of this hypothesis are briefly outlined.     

Determination of β-glucosidase aggregating factor (BGAF) binding and polymerization regions on the maize β-glucosidase isozyme Glu1

β-Glucosidases (Glu1 and Glu2) in maize specifically interact with a lectin called β-glucosidase aggregating factor (BGAF). We have shown that the N-terminal (Glu⁵⁰–Val¹⁴⁵) and the C-terminal (Phe⁴⁶⁶–Ala⁵¹²) regions of maize Glu1 are involved in binding to BGAF. Sequence comparison between sorghum β-glucosidases (dhurrinases, which do not bind to BGAF) and maize β-glucosidases, and the 3D-structure of Glu1 suggested that the BGAF-binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF-binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile⁷²–Thr⁸²) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser¹–Thr²⁹, together with C-terminal region Phe⁴⁶⁶–Ala⁵¹², affects the size of Glu1–BGAF complexes. The dissociation constants (K_d) of chimeric β-glucosidase–BGAF interactions also demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile⁷²–Thr⁸² on Glu1 for BGAF binding, we constructed a chimeric sorghum β-glucosidase, Dhr2 (C-11, Dhr2 whose Val⁷²–Glu⁸² region was replaced with the Ile⁷²–Thr⁸² region of Glu1). C-11 binds to BGAF, indicating that the Ile⁷²–Thr⁸² region is indeed a major interaction site on Glu1 involved in BGAF binding.     

Change in water quality during the passage through a tropical montane rain forest in Ecuador

We studied five 20-m transects onthe lower slope under tropical lower montanerain forest at 1900–2200 m above sea level. We collectedsamples of soil and of weekly rainfall,throughfall, litter leachate, and stream waterbetween 14 March 1998 and 30 April 1999 anddetermined the concentrations of Al, totalorganic C (TOC), Ca, Cl^–, Cu, K, Mg, Mn,NH₄ ⁺-N, NO₃ ^–-N, total N (TN), Na, P, S, and Zn. The soils were shallowInceptisols; pH ranged 4.4–6.3 in the Ohorizons and 3.9–5.3 in the A horizons, totalCa (6.3–19.3 mg kg^–1) and Mgconcentrations (1.4–5.4) in the O horizon weresignificantly different between the transects.Annual rainfall was 2193 mm; throughfall variedbetween 43 and 91% of rainfall, cloud waterinputs were 3.3 mm a^–1 except forone transect (203). The volume-weighted mean pHwas 5.3 in rainfall and 6.1–6.7 in throughfall.The median of the pH of litter leachate andstream water was 4.8–6.8 and 6.8, respectively.The concentrations of Ca and Mg in litterleachate and throughfall correlatedsignificantly with those in the soil (r =0.76–0.95). Element concentrations inthroughfall were larger than in rainfallbecause of leaching from the leaves (Al, TOC,Ca, K, Mg), particulate dry deposition (TOC,Cu, Cl^–, NH₄ ⁺-N), and gaseousdry deposition (NO₃ ^–-N, total N, S).Net throughfall (= throughfall-rainfalldeposition) was positive for most elementsexcept for Mn, Na, and Zn. High-flow eventswere associated with elevated Al, TOC, Cu, Mn,and Zn concentrations.     

Localization of epitopes for antibodies that differentially label sodium channels in skeletal muscle surface and T-tubular membranes

Summary We previously characterized two monoclonal antibodies, A/B2 and L/D3, that bind to the amino-terminus of the sodium channel but produce distinct immunocytochemical patterns in innervated adult skeletal muscle. Because these findings suggested the presence of several channel isoforms, we sought to define the epitopes for each antibody. Five peptides encompassing the amino-terminal 126 residues of the adult skeletal muscle sodium channel were synthesized and tested by radioimmunoassay against each antibody. Both monoclonals bound only to a peptide comprising residues 1–30 (I^1–30). A nested set of peptides within this region was then synthesized and used to compete for antibody binding to II^1–30. L/D3 binding was quantitatively inhibited by oligopeptides 1–30, 7–30, 13–30, and 19–30 but not 25–30, while binding of A/B2 was blocked only by the intact I^1–30 peptide. This data implies that the epitope for L/D3 lies within residues 19–25 while the epitope for A/B2 is contained within residues 1–6. These tentative epitope localizations were confirmed using both proteolytic cleavage of I^1–30 and immunoreactivity of a peptide corresponding to residues 1–12 with A/B2 but not L/D3. Therefore, epitopes for each monoclonal antibody are present in the SkM-1 sequence and are in close proximity in the amino-terminus of the protein. Their characteristic immunocytochemical labeling patterns may reflect differing accessibility of the epitopes in various membrane environments.We wish to thank Dr. John Lambris for helpful discussions. We also thank Ms. Candace Mello and Mr. James Hills for their expert technical assistance. This work was supported in part by NIH Grant NS 18013 (RLB) and by a grant from the W.W. Smith Charitable Trust (SAC). SAC is a Scholar of the Pfizer Scholar's Program for New Faculty.     

A scrutiny of the biochemical pathways from Ang II to Ang-(3–4) in renal basolateral membranes

In a previous paper we demonstrated that Ang-(3–4) counteracts inhibition of the Ca²⁺-ATPase by Ang II in the basolateral membranes of kidney proximal tubules cells (BLM). We have now investigated the enzymatic routs by which Ang II is converted to Ang-(3–4). Membrane-bound angiotensin converting enzyme, aminopeptidases and neprilysin were identified using fluorescent substrates. HPLC showed that Plummer's inhibitor but not Z–pro–prolinal blocks Ang II metabolism, suggesting that carboxypeptidase N catalyzes the conversion Ang II→ Ang-(1–7). Different combinations of bestatin, thiorphan, Plummer's inhibitor, Ang II and Ang-(1–5), and use of short proteolysis times, indicate that Ang-(1–7)→ Ang-(1–5)→ Ang-(1–4)→ Ang-(3–4) is a major route. When Ang III was combined with the same inhibitors, the following pathway was demonstrated: Ang III→ Ang IV→ Ang-(3–4). Ca²⁺-ATPase assays with different Ang II concentrations and different peptidase inhibitors confirm the existence of these pathways in BLM and show that a prolyl-carboxypeptidase may be an alternative catalyst for converting Ang II to Ang-(1–7). Overall, we demonstrated that BLM have all the peptidase machinery required to produce Ang-(3–4) in the vicinity of the Ca²⁺-ATPase, enabling a local RAS axis to effect rapid modulation of active Ca²⁺ fluxes.     

Removal of bacteria and nutrient dynamics within the coral reef framework of Curaçao (Netherlands Antilles)

The authors studied removal rates of bacteria and the regeneration of inorganic nutrients in coral reef cavities in the reef slope of Curaçao, Netherlands Antilles. We found that in cavities the hard substratum surface area (=ca 68% of cavity surface area) is 65% covered with sessile filter feeders. The cryptic cavity surface area exceeds the projected surface area of the reef by 1.5–8 times. Consequently, the organisms living in these cryptic habitats have potentially a large impact on pico- and nano-plankton densities and are important in reef water nutrient dynamics. We closed cavities (±70 l volume, 15 m depth) in seven experiments to study changes in bacterial densities and dissolved inorganic nutrients (DIN, DIP, and silicate) over time. Water samples were taken from the middle of the cavity at 5-min intervals, for 30 min, and analyzed for heterotrophic bacterial abundance and nutrient concentrations. After closure, bacterial abundance dropped rapidly. Of the initial bacterial concentration in the cavities, 50–60% had disappeared after 30 min, an average disappearance rate of 1.43×10⁴ bacteria ml^–1 min^–1 (0.62 mg C l^–1 d^–1; or 30.1 mg C m^–2 cavity surface area d^–1). NO_x concentrations increased significantly during the time of closure. Efflux rates varied between 1.02–9.77 mmol m^–2 cavity surface area d^–1. NH₄⁺ and PO₄^3– concentrations were variable and did not show a consistent change over time in the experiments. Comparison of bacterial organic nitrogen disappearance rates and DIN (NO_x+NH₄⁺) release rates suggests that on average only 30–40% of additional sources of N besides bacteria were required to balance the nitrogen budget. This highlights the importance of heterotrophic bacterioplankton as food for cryptic filter feeders on coral reefs. Silicate concentrations significantly decreased after closure with 0.50 mmol m^–2 cavity surface area d^–1, suggesting the net deposition of SiO₄^2– in spicules of cryptic filter feeding sponges. We conclude that coral reef cavities are a major sink for heterotrophic bacteria, a sink for dissolved silicon (DSi), and a source for NO_x. That reef cavities are a source for NO_x suggests strong remineralization and nitrification in cavities with a potential role for sponge-symbiotic microbial nitrification.Communicated by K.S. Sealey     

Properties of the chloride-ATPase fromLimonium salt glands: Activation by,and binding to,specific sugars

Summary Attempts to separate membrane fractions enriched in Cl^–-ATPase activity fromLimonium leaf microsomes were hampered because, it seemed, the microsomal membranes were aggregated in clumps. We found hemagglutination activity, specific for N-acetylgalactosamine and to a lesser extent galactose, in the soluble phase of the homogenate, and we were able to prevent membrane aggregation by adding galactose to the microsomes. We discovered that the Cl^–-ATPase activity of the microsomes was increased by galactose and to an even greater extent by N-acetylgalactosamine. We report that the Cl^–-ATPase binds to galactosamine-sepharose, from which it can be eluted in 0.1m galactose, i.e., the enzyme is associated with a saccharide-binding site similar to that of the hemagglutinins. This procedure results in a 100-fold enrichment of the Cl^–-ATPase activity and represents a new way of purifying a membrane-bound enzyme from a heterogeneous membrane preparation in one step. Enzyme isolated by affinity chromatography of Triton-solubilized membranes was essentially free of other ATPase and accounted for a substantial proportion (sometimes all) of the Cl^–-ATPase of the microsomes. This purified preparation of the enzyme shows N-acetylgalactosamine-specific hemagglutination activity. However, we can show that the Cl^–-ATPase and the hemagglutinins are different entities. Thus, material isolated in the same way from salt-free plants showed hemagglutination but not Cl^–-ATPase activity. Also, the hemagglutinins, but not the Cl^–-ATPase, will bind to galactosaminesepharose in the absence of ATP.This is the first report of enzyme activity associated with a carbohydrate receptorspecific protein. Possible roles for saccharide-binding in the control, assembly, and orientation of the chloride-pump are discussed.