Subsurface chlorophyll maximum (SCM) location and extension in the water column as governed by a density interface in the strongly stratified Kattegat estuary

The aims of the study were to analyse the relations between the physics of a water column and the location of the subsurface chlorophyll maximum (SCM) peaks in a strongly stratified estuary. Could extension and depth location of the SCM be explained by the physical conditions in terms of water column stratification and density interface? Questions were addressed by obtaining data on water column density (CTD), chlorophyll a (Chl a), nutrients, (F _v/F _m), σ_PSII and K _d(PAR) at 15 positions along a 575 km transect in the Kattegat estuary. Results showed that the estuary was strongly stratified with mixed surface and bottom layers intercepted by a layer where density increased with depth. The SCM occurred only in this density interface, and widths of SCM and density interface were highly correlated. The surface waters were nearly depleted of inorganic nitrogen, phosphate and silicate though with significant higher concentrations in the waters below the interface. The Chl a concentration was comparatively higher in the SCM peak as well as maximum quantum efficiency (F _v/F _m) and functional cross sectional area (σ_PSII). The SCM was maintained at very low light levels and by a diapycnal nitrogen flux, with a stratified water column and nutrient depleted surface waters as predecessors. It was concluded that the depth location and vertical extension of the SCM in the estuary were closely linked to the physical structure of the water column in terms of density interface and stratification.     

Chlorophyll content of Plešné Lake phytoplankton cells studied with image analysis

Using image analysis, chlorophyll autofluorescence was measured in single cells of green alga Monoraphidium dybowskii and in filaments of cyanobacteria (Pseudanabaena sp. and Limnothrix sp.) in the vertical profile of small acidified mountain lake Ple?né jezero (Ple?né Lake) from May to November of 2003. Cell chlorophyll autofluorescence was converted to cell chlorophyll content using a conversion factor determined by comparing the total autofluorescence of phytoplankton in a microscope field with spectrophotometrically determined total chlorophyll concentration; the conversion factor did not differ between epilimnion (0.5 m depth) and hypolimnion (9 m depth). Vertical patterns of chlorophyll concentration and of cellular chlorophyll content depended on water column mixing: during the period of stable thermal stratification, a metalimnetic peak in total chlorophyll concentration was present and cellular chlorophyll contents in the metalimnion and hypolimnion were notably elevated compared to the surface. Monotonous vertical profiles of both total chlorophyll concentration and cell chlorophyll content were typical for the period of water column overturn. During the stratification period, hypolimnetic Monoraphidium cell chlorophyll content was on average twice as high (maximum difference 2.7-fold) compared to surface values (of 3.2–12.9 fg µm^?3), while in filamentous cyanobacteria (surface cell chlorophyll content of 2.2–13.3 fg µm^?3), the difference was much higher — six-fold on average, with an 11.6-fold maximum value. The values measured with image analysis in 2003 were compared to unpublished values of total phytoplankton biomass-specific chlorophyll concentrations obtained using manual phytoplankton biomass determination and spectrophotometric chlorophyll measurement in 1998 at the same locality. Good agreement was found in seasonal patterns and vertical profiles of chlorophyll between both seasons.     

Nutrient cycling and productivity in a desert saline lake: observations from a dry,low-productivity year

Seasonal variability of nutrients and productivity were examined in Pyramid Lake, a hyposaline, N-deficient, terminal desert lake, during a dry period. River inflow and N-fixation during 1990 were minimal allowing internal nutrient cycling to be more closely studied. Nutrient cycling was strongly affected by seasonal thermal stratification that was typical for a warm monomictic lake. Concentrations of nitrate, phosphate, and silicate in surface waters were highest during winter mixing and decreased rapidly in the spring due to a diatom bloom. Maximum average chlorophyll concentration in surface waters was 2.7 ± 1.2 µg 1^–1 and occurred in April while surface nutrients were being depleted. In contrast to chlorophyll, maximum particulate carbon in surface waters occurred in July–August when areal productivity was highest (367–398 mg C m^–2 day^–1). Concurrent with spring nutrient depletion in surface waters was increasing N-deficiency in the plankton. After the spring bloom dissipated in May, particulate matter (POM) became increasingly N-deficient reaching maximum elemental C : N of > 18 during summer-fall. Profiles of the C : N ratio of POM were nearly constant with depth for individual sampling dates suggesting that the residence time of POM in the water column was short (< 1 month). While surface waters were nutrient depleted during summer stratification, nutrient concentrations of bottom waters progressively increased, presumably through the oxidation of POM sinking to the bottom (103 m). Converting the rate of oxygen depletion in bottom waters to carbon equivalents of POM suggests that 42 % of mean annual phytoplankton production in overlying waters during 1990 was mineralized in bottom waters.     

The resolution and biochemical characterization of subcomplexes of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC II)

LHC II isolated from carnation leaves has been solubilized and resolved by a newly developed, vertical-bed non-denaturing isoelectric focusing in polyacrylamide slab gels to yield three trimeric subcomplexes focusing at pH 4.52, 4.42 and 4.37 (designated a, b and c, respectively), comprising approximately 38%, 24% and 38% of the chlorophyll. The spectroscopic data demonstrated a close similarity among LHC II subcomplexes concerning their chlorophyll content and organization. The most alkaline and the most acidic subcomplex contained the 27 kDa polypeptide of LHC II while the intermediate pI fraction contained both LHC II polypeptides, i.e. 27 kDa and 26 kDa ones associated at 2:1 stoichiometry. The 27 kDa polypeptide could be resolved by denaturing isoelectrofocusing into 10 pI molecular isoforms covering 5.90–4.20 pH range. Three of the isoforms were found in the subcomplexes a and b and eight in the subcomplex c. The 26 kDa polypeptide comprised the unique pI molecular isoform focusing at pH 5.61.Abbreviations CBB G-250 Coomassie Brilliant Blue G-250 - chl chlorophyll - DM n-dodecyl--d-maltoside - EDTA ethylendiaminotetraacetic acid - IEF isoelectric focusing - LHC II the main light-harvesting chlorophyll a/b-protein complex of Photosystem II - LHCP II apoprotein of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II - NP-40 polyethyleneglycol-p-isooctylphenyl ether - pI isoelectric point - OG octyl--d-glucopyranoside - PS II Photosystem II - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TCA trichlorooacetic acid     

Die Wirkung von Phytochrom und Actinomycin D auf die Chlorophyll a-Synthese von Senfkeimlingen (Sinapis alba L.)

Zusammenfassung Eine Vorbestrahlung der etiolierten Senfkeimlinge mit 4 Std Dunkelrot bewirkt eine Steigerung der Chlorophyll a-Synthese im Weißlicht. — Andererseits wird die Chlorophyll a-Synthese bereits durch relativ niedrige Actinomycin D-Konzentrationen gehemmt oder verzögert. — Die hemmende Wirkung von Actinomycin D ist — ausgedrückt in Prozent Hemmung — mit und ohne Vorbelichtung genau dieselbe. Auch die übrigen Daten deuten darauf hin, daß Actinomycin D nicht auf die Protochlorophyllsynthese als solche wirkt, sondern vielmehr die Synthese bestimmter Strukturproteine in den Plastiden beeinträchtigt. —Die Daten der Arbeit werden genphysiologisch gedeutet. Der wesentliche Punkt dabei ist, daß aktive Gene (z.B. jene, welche die Enzyme der bekanntlich auch im Dunkeln ablaufenden Protochlorophyllsynthese codieren) relativ unempfindlich gegenüber Actinomycin D sind; potentiell aktive Gene hingegen (z.B. jene, die einige nur im Licht entstehende spezifische Strukturproteine der Plastiden codieren) scheinen sehr viel empfindlicher gegenüber Actinomycin D zu sein. Diese Schlüsse stehen im Einklang mit einer früher geäußerten Hypothese (Lange und Mohr, 1965) und mit den Daten von Schopfer (1966) über die Regulation der Ascorbatsynthese im Senfkeimling durch Phytochrom und Actinomycin D.

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The action of phytochrome and actinomycin D on chlorophyll a formation in mustard seedlings (Sinapis alba L.)

Summary In the mustard seedling chlorophyll a synthesis under white light is enhanced by a pretreatment with far-red which maintains a low but virtually stationary concentration of active phytochrome (=P₇₃₀) (Fig. 1) during the period of irradiation (4 hours). — On the other hand chlorophyll a synthesis is inhibited or delayed by relatively low concentrations of Actinomycin D(=Act) (Fig. 2)The inhibitory action of Act (on a percent basis) is exactly the same with and without a far-red pre-irradiation (Fig. 3). Act in relatively low doses (5 or 10 g/ml) greatly extends the lag-phase of chlorophyll synthesis; however, these doses do not influence the effect of the far-red pretreatment on the rate of chlorophyll synthesis when it finally takes place (Fig.4,5,6). The data presented in this paper indicate that Act does not inhibit protochlorophyll synthesis as such; we have rather to conclude that Act inhibits the de novo synthesis of some specific structural proteins which are prerequisites of chlorophyll accumulation and maintenance in the plastids (Table 1). Synthesis of these structural proteins seems to be under the control of phytochrome too.It is concluded that those genes which are already in function are relatively resistant to Act (e. g. those genes which are needed for protochlorophyll synthesis) whereas potentially active genes (e. g. those which code some specific structural proteins of the plastids) are very sensitive to Act. —A similar conclusion has been reached in an earlier paper in connection with phytochrome-induced antocyanin synthesis (Lange and Mohr, 1965). Our argumentation is further supported by Schopfer's data on control of ascorbate synthesis in the mustard seedling by phytochrome and Act (Schopfer, 1966).

     

Studies on the cation permeability of human red cell ghosts

Summary Net K movements in reconstituted human red cell ghosts and the resealing of ghosts to cations after osmotic hemolysis of red cells have been studied as functions of the free Ca ion concentration. The Ca-dependent specific increase in K permeability was shown to be mediated by a site close to the internal surface of the membrane with an apparent dissociation constant at pH 7.2 for Ca (K _D1) of 3–5×10^–7 m, for Sr of 7×10^–6 m. Ba and Mg did not increase the K-permeability of the membrane but inhibited the Ca-mediated permeability changes.K _D1 decreased in a nonlinear fashion when the pH was increased from 6.0 to 8.5. Two different pK values of this membrane site were found at pH 8.3 and 6.3. The Ca-activated net K efflux into a K-free medium was almost completely inhibited by an increase in intracellular Na from 4 to 70mm. Extracellular K antagonized this Na effect. Changes in the extracellular Na (0.1–140mm) or K(0.1–6mm) concentrations had little effect and did not changeK _D1. The Ca-stimulated recovery of a low cation permeability in ghost cells appeared to be mediated by a second membrane site which was accessible to divalent cations only during the process of hemolysis in media of low ionic strength. The apparent dissociation constant for Ca at this site (K _D2) varied between 6×10^–7 and 4×10^–6 m at pH 7.2. Mg, Sr, and Ba could replace Ca functionally. The selectivity sequence was Ca>Sr>Ba>Mg.K _D2 was independt on the pH value in the range between 6.0 and 8.0. Hill coefficients of 2 were observed for the interaction of Ca with both membrane sites suggesting that more than one Ca ion is bound per site. The Hill coefficients were affected neither by the ion composition nor by the pH values of the intra- and extracellular media. It is concluded that two different pathways for the permeation of cations across the membrane are controlled by membrane sites with high affinities for Ca: One specific for K, one unspecific with respect to cations. The K-specific channel has properties similar to the K channel in excitable tissues.     

The differential allosteric regulation of two chorismate-mutase isoenzymes of Nicotiana silvestris

The reaction catalyzed by chorismate mutase (EC 5.4.99.5) is a crucial step for biosynthesis of two aromatic amino acids as well as for the synthesis of phenylpropanoid compounds. The regulatory properties of two chorismate-mutase isoenzymes expressed in Nicotiana silvestris Speg. et Comes are consistent with their differential roles in pathway flow routes ending with l-phenylalanine and l-tyrosine on one hand (isoenzyme CM-1), and ending with secondary metabolites on the other hand (isoenzyme CM-2). Isoenzyme CM-1 was very sensitive to allosteric control by all three aromatic amino acids. At pH 6.1, l-tryptophan was a potent allosteric activator (K _a =1.5 M), while feedback inhibition was effected by l-tyrosine (K _i =15 M) or by l-phenylalanine (K_i=15 M). At pH 6.1, all three effectors acted competitively, influencing the apparent K _m for chorismate. All three allosteric effectors protected isoenzyme CM-1 at pH 6.1 from thermal inactivation at 52° C. l-Tryptophan abolished the weak positive cooperativity of substrate binding found with isoenzyme CM-1 only at low pH. At pH 7.2, the allosteric effects of l-tyrosine and l-tryptophan were only modestly different, in striking contrast to results obtained with l-phenylalanine. At pH 7.2 (i) the K _i for l-phenylalanine was elevated over 30-fold to 500 M, (ii) the kinetics of inhibition became non-competitive, and (iii) l-phenylalanine now failed to protect isoenzyme CM-1 against thermal inactivation. l-Phenylalanine may act at different binding sites depending upon the intracellular pH milieu. In-vitro data indicated that the relative ability of allosteric activation to dominate over allosteric inhibition increases markedly with both pH and temperature. The second isoenzyme, CM-2, was inhibited competitively by caffeic acid (K _i =0.2 mM). Aromatic amino acids failed to affect CM-2 activity over a broad range of pH and temperature. Inhibition curves obtained in the presence of caffeic acid were sigmoid, yielding an interaction coefficient (from Hill plots) of n=1.8.Abbreviation DAHP synthase 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase     

Distribution of marine viruses and their potential hosts in Prydz Bay and adjacent Southern Ocean,Antarctic

Viruses play a key role in all marine ecosystems, and yet little is known of their distribution in Antarctic waters, especially in bathypelagic waters (>1000 m). In this study, the abundance and distribution of viruses and their potential hosts from the surface to the bottom of Prydz Bay, Antarctic, was investigated using flow cytometry. Viruses and autotrophs were abundant in nearshore and continental shelf waters, while heterotrophic bacteria and picoeukaryotes were abundant in offshore waters. Virus and bacteria abundances generally decreased with increasing depth but increased slightly just above the seafloor. Within the water column, maximum virus numbers coincided with the maximum values of chlorophyll a (when greater than 0.1 μg l^?1), in the surface and subsurface (25 m). In the open ocean, however, virus abundance usually correlated with bacterial abundance at greater depths (50, 300 and 500 m) where the surface chlorophyll a concentration was lower than 0.1 μg l^?1. Viral abundance was correlated with the host cell abundance, and this was different in different pelagic zones (bacteria and autotrophs (i.e., chlorophyll a concentration) in the epipelagic waters, picoeukaryotes and bacteria in mesopelagic waters and bacteria in bathypelagic waters). Principle component analysis and Pearson correlation analysis indicated that there was a close relationship between virus abundance and chlorophyll a, bacteria and nutrients (NO₂ + NO₃, phosphate and silicate), and picoeukaryote abundance was mainly correlated with water depth and salinity.     

The fermentation process for l-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli

Summary A process for l-phenylalanine production was studied using a tyrosine auxotrophic regulatory mutant of Escherichia coli, resistant to both -2-thienyl-dl-alanine and p-fluoro-dl-phenylalanine. Fermentations were carried out in a 30-1 fermentor with intermittent feeding of glucose plus phosphate. The mutant accumulated l-phenylalanine in the fermentation broth up to 15 g/l at pH 7.0 and 33°C. Column chromatography on a strong cation exchanger was employed as the most effective step in the purification of l-phenyl-alanine from the broth. This step brought about 4-fold concentration of the product with 96% recovery.     

Hydrography and characteristics of the phytoplankton in shelf and oceanic waters off southeastern Brazil during winter (July/August 1982) and summer (February/March 1984)

The physical and chemical environment, and the phytoplankton primary production of southeastern Brazil were studied in relation to the general oceanographic structure during two research cruises (winter and summer). In each cruise, a total of 91 stations were occupied. Data were collected on the spatial distribution of nutrients, phytoplankton biomass and photosynthetic capacity over the coastal, shelf and oceanic areas off São Paulo, Paraná and Santa Catarina States.During wintertime, the mixing processes between tropical warm waters of the Brazil Current and subantarctic waters of the Malvinas Current formed strong environmental gradients. The drainings of Rio de La Plata and Lagoa dos Patos are transported northwards by coastal currents, enriching the shelf waters off Santa Catarina State with inorganic nutrients and consequently increasing the chlorophyll a to the highest concentrations (> 3.5 mg m ^–3) measured during the two cruises. In slope waters chlorophyll values were always low (0.05–0.45 mg m ^–3). The chlorophyll within the euphotic layer varied from 8.8–36.7 and 1.2–18.5 mg m^–2 during winter and summer, respectively.The surface photosynthetic rates during winter and summer cruises ranged respectively from 0.21–9.17 and 0.66–19.60 mgC/mgChl.a/h. The mean rates were higher in nearshore waters and decreased seaward.The thermal structure of the water column affected the vertical distribution of chlorophyll a and photosynthesis within the euphotic zone; During unstratified periods (winter) they were uniformly distributed but the occurrence of subsurface peaks of chlorophyll and strong photosynthetic inhibition of low light adapted cells in deeper layers are associated to the seasonal thermocline. Occasionally, upwelling of deep waters from shelf break enriched the deeper euphotic layers in offshore areas. Intensive upwelling was observed off Paranagua Bay (Parana State) and the mechanisms of its formation are discussed.     

Manganese cycling in an acidic Adirondack lake

There is considerable interest in the chemistry of Mn in acidic waters because of its role in the generation of acid neutralizing capacity during reduction processes, as an adsorbent in element cycling, and as a potential toxicant to aquatic organisms. Temporal and spatial variations in the concentration of Mn were evident in acidic Dart's Lake (1.0–2.3 mol l^–1), located in the Adirondack Region of New York. Seasonal changes in pH and dissolved oxygen concentration had subtle effects on the chemistry and transport of Mn. Despite oversaturation with respect to the solubility of manganite during periods of stratification, vertical deposition of Mn was minimal. The conservative nature of Mn appears to be due to the acidic conditions in Dart's Lake.     

Water quantity and quality as the factors driving the Serengeti ecosystem,Tanzania

Thirty nine years of rainfall data from 232 sites, 5 years of river discharge data from 3 rivers, 4 years of animal migration data and 4 years of water quality data at 60 sites were explored to quantify the role of water in the Serengeti ecosystem. Seasonal variations in rainfall are largely predictable; interannual fluctuations are huge and not predictable solely from the Southern Oscillation Index. The wildebeest and zebras start their annual migration at the end of the wet season well before surface water runs out, however these waters are very saline (salinity 5–17 psu). The timing of the migration appears predictable from a salinity model. Salinity is also important for the vegetation because high salinity coincides with the transition between wooded savanna and grassland. This transition has moved markedly southward in the last 30 years, this change may be due to decadal changes in annual rainfall. Most rivers are commonly ponded, with ponds having a flushing rate of 1 month in the wet season and zero flushing in the dry season. These ponds form the only source of water for wildlife for several months a year. The water quality varies spatially and temporally. pH values vary between 5.9 and 10 and are correlated with salinity. Surface waters are heavily eutrophicated from animal dung. As a result, the dissolved oxygen concentration near the surface fluctuates widely between 1 and 200% of saturation. Direct solar heating is restricted to the top few cm because of low visibility. A strong thermal stratification in temperature (2 °C/m) results and inhibits aeration. Bottom waters can be anoxic and are aerated only when hippopotamus stir the water. Poor water quality may affect wildlife health and production.     

Preparation of intact chloroplasts by chemically induced lysis of the green alga Dunaliella marina

A method for the isolation in high yield of intact chloroplasts from the unicellular green alga Dunaliella marina (Volvocales) is described. This procedure uses chemically induced lysis of cells with the polycationic macromolecules, DEAE-dextran (M=500,000) or poly-D,l-lysine (M=30,000-70,000). Reaction conditions were optimized with respect to obtaining a high yield of intact chloroplasts, after isopycnic centrifugation in a linear sucrose density gradient, by varying the concentration of polycation and the temperature and pH of incubation. Broken chloroplasts devoid of the stromal marker enzymes fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, but containing mitochondrial (fumarase) and microbody (catalase) contamination, were banded at a bouyant density of 1.18 g cm^-3. Intact chloroplasts, as indicated by their retention of alkaline fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, were found in 30% yield (chlorophyll in intact cells, 100%) at an equilibrium density of 1.24 g cm^-3. Contamination by cytoplasmic material (pyruvate kinase), mitochondria, and microbodies was less than 8% each.Abbreviations Chl chlorophyll - DEAE-dextran diethylaminoethyl-dextran - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - FBPase fructose-1,6-bisphosphate phosphatase, EC 3.1.3.11 - G6P-DH glucose 6-phosphate dehydrogenase, EC 1.1.1.49 - HEPES N-2-hydroxyethylpiperazine-N-ethanesulphonic acid - MES 2-(N-morpholino)ethanesulphonic acid - RuBP carboxylase D-ribulose-1,5-bisphosphate carboxylase or 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39     

Action of phenazine methyl sulfate,inhibitors, and uncouplers on the light-induced proton transport by cells ofRhodospirillum rubrum

Summary Suspensions of log phase cells ofRhodospirillum rubrum at pH 5.5 show a light-induced decrease in the pH of the medium which is reversed during the subsequent dark period. The velocity and magnitude of the pH change were the same whether the cells were bubbled with air, CO₂-free air or N₂ during experimentation. The pH response is temperature dependent. Phenazine methyl sulfate (PMS) at concentrations above 0.05mm stimulates the light-induced pH change. PMS at 1mm gives a 2-fold increase in the initial rate upon illumination and a 1.5-fold increase in the total change in pH after 2 min of illumination. The inhibition of the proton transport by 10 g/ml antimycin A or 20 m 2-n-heptyl-4-hydroxyquinoline-N-oxide can be partially relieved by PMS. However, inhibition of the light-induced proton transport with 0.5mm 2,4-dinitrophenol or 3 m carbonylcyanide-m-chlorophenylhydrazone (CCCP) cannot be overcome by addition of PMS. Valinomycin, at a concentration of 3 m, caused a slight stimulation of the light-induced proton transport in the presence of 200mm KCl. The inhibition of proton transport by 3 m CCCP was partially relieved with 3 m valinomycin in the presence of 200mm KCl, but the antibiotic was without effect when the cells were suspended in 200mm NaCl. The results are discussed in terms of current theories of the action of PMS, antimycin A, valinomycin, and uncouplers on the light-induced electron flow and photophosphorylation inR. rubrum.     

Proton fluxes associated with erythrocyte membrane anion exchange

Summary Transient extracellular pH changes accompany the exchange of chloride for sulfate across the erythrocyte membrane. The direction of the extracellular pH change during chloride efflux and sulfate influx depends on experimental conditions. When bicarbonate is present, the extracellular pH drops sharply at the outset of the anion exchange and tends to follow the partial ionic equilibrium described by Wilbrandt (W. Wilbrandt, 1942.Pfluegers Arch. 246:291). When bicarbonate is absent, however, the anion exchange causes the pH to rise, indicating that protons are cotransported with sulfate during chloridesulfate exchange. The pH rise can be reversed by the addition of HCO ₃ ^– (4 m) or 2,4-dinitrophenol (90 m). This demonstrates that the proton-sulfate cotransport can drive proton transport uphill. The stoichiometry of the transport is that one chloríde exchanges for one sulfate plus one proton. These results support the titratable carrier model proposed by Gunn (Gunn, R.B. 1972.In: Oxygen Affinity of Hemoglobin and Red Cell Acid-Base Status. M. Roth and P. Astrup, editors. p. 823. Munksgaard, Copenhagen) for erythrocyte membrane anion exchange.     

Role of vacuolar adenosine triphosphatase in the regulation of cytosolic pH in hepatocytes

The responses of the cytosolic pH of hepatocytes in suspension to agents affecting the activity of vacuolar adenosine triphosphatase (V-ATPase) and Na/H exchange have been studied. Changes of cytosolic pH were determined both with dual-wavelength excitation (500/440 nm) of the fluorescence of 2,7-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein and from the distribution of ¹⁴C-dimethyloxazolidinedione; both methods gave very similar results. Changes of vesicular pH were determined by comparing the fluorescence of fluorescein isothiocyanate-dextran and rhodamine B isothiocyanate-dextran taken up by endocytosis. Nitrate, which inhibits V-ATPase in isolated organelles, induced a concentration-dependent acidification of the cytosol and alkalinization of vesicles, with maximal effects at 25–37.5 mm in each case, indicating that V-ATPase contributes to removal of cytosolic protons. On continued exposure to nitrate, the acidification underwent an amiloride-inhibitable reversal. At the higher concentrations of NO ₃ ^– , both cytosolic acidification and vesicular alkalinization were reduced or absent. Bafilomycin A₁ caused alkalinization of vesicular pH; cytosolic acidification was not observed, possibly because of other ionic exchanges. Recovery of cytosolic pH from an acid load (2 min exposure to 5% CO₂) was sensitive to both 25 mm NO ₃ ^– and to ouabain. The pH dependence of the nitrate effect was tested with media of different pH; the activity was negligible at cytosolic pH 6.2 and rose to a maximum at cytosolic pH 7.3. Treatment of hepatocytes with 0.5–1.0 mm ouabain resulted in an initial alkalinization (0.5–2 min duration) of the cytosol, followed by a spontaneous reversal and, on occasion, further acidification. The alkalinization was blocked by 25 mm NO ₃ ^– , but not by 25 mm gluconate. The results suggest that the cytosolic alkalinization is caused by a stimulation of H⁺ uptake by V-ATPase activity. We conclude that V-ATPases make an important contribution to the regulation of the cytosolic pH of hepatocytes.This work was supported in part by National Institutes of Health B.R.S. Grant 507 RR05417 to Temple University.     

Methane and oxygen dynamics in a shallow floodplain lake: the significance of periodic stratification

Temperature, dissolved oxygen and dissolved methane profiles were measured during autumn and summer, in a shallow floodplain lake in south-eastern Australia to determine the effects of water-column stability on methane and oxygen dynamics. The water column was well mixed in autumn. Strong thermal stratification developed in the late afternoon in summer, with top-to-bottom temperature differences of up to 6 °C. Methane concentrations in surface waters varied over a daily cycle by an 18-fold range in summer, but only by a 2-fold range in autumn. The implication of short-term temporal variation is that static chambers deployed on the water surface for short times (less than a day) in summer will significantly underestimate the diffusive component of methane emissions across the water–atmosphere interface. There was a marked diel variation in dissolved oxygen concentrations in summer, with the highest oxygen values (commonly 5–8 mg l^–1) occurring in the surface waters in late afternoon; the bottom waters were then devoid of oxygen (< 0.2 mg l^–1). Because of high respiratory demands, even the surface water layers could be nearly anoxic by morning in summer. The concentration of dissolved oxygen in the surface waters was always less than the equilibrium value. When the water column became thermally stratified in summer, the dissolved oxygen and methane maxima were spatially separated, and planktonic methanotrophy would be limited to a moving zone, at variable depth, in the water column. In summer the whole-wetland rates of oxygen production and respiration, calculated from long-term (5 h) shifts in dissolved oxygen concentrations over a diel period, were approximately 6–10 and 3–6 mmol m^–3 h^–1, respectively. These values correspond to net and gross primary production rates of 0.7–1.2 and 1.0–1.9 g C m^–3 day^–1, respectively.     

Ethylenediamine uptake and metabolism in the cyanobacterium Anabaena variabilis

The cyanobacterium Anabaena variabilis showed a pH dependent uptake of ethylenediamine. No uptake of ethylenediamine was detected at pH 7.0. At higher pH values (e.g. pH 8.0 and pH 9.0) accumulation did occur and was attributed to diffusion of uncharged ethylenediamine in response to a pH gradient. A biphasic pattern of uptake was observed at these higher pH values. Treatment with l-methionine-d,l-sulphoximine (MSX) to inactivate glutamine synthetase (GS) inhibited the second slower phase of uptake without any significant alteration of the initial uptake. Therefore for sustained uptake, metabolism of ethylenediamine via GS was required. NH ₄ ⁺ did not alter the uptake of ethylenediamine. Ethylenediamine was converted in the second phase of uptake to an analogue of glutamine which could not be detected in uptake experiments at pH 7.0 or in uptake experiments at pH 9.0 following pretreatment of cells with MSX. Ethylenediamine treatment inhibited nitrogenase activity and this inhibition was greatest at high pH values.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1 piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - Tricine N-tris(hydroxymethyl) methylglycine     

Properties of α-l-iduronidase in cultured skin fibroblasts from α-l-iduronidase-deficient patients

Summary On DEAE cellulose column chromatography, -l-iduronidase in cultured skin fibroblasts was resolved into two distinct components, forms A and B. They had similar K_m values for 4-methylumbelliferyl--l-iduronide, but differed in pH optima and thermal stability. Form B was more heat-stable than form A.Residual -l-iduronidase activity in Hurler fibroblasts was heat-stable, while that in Scheie fibroblasts was heat-labile, and moreover, that in Hurler-Scheie compound fibroblasts lay intermediate between Hurler and Scheie syndromes. These findings demonstrated that Hurler syndrome, Scheie syndrome and Hurler-Scheie compound were enzymatically distinguishable.