Synthesis and evaluation of affinity adsorbents for glycoproteins: an artificial lectin

A combination of rational design based on mimicking natural protein–carbohydrate interactions and solid-phase combinatorial chemistry has led to the identification of an affinity ligand which displays selectivity for the mannose moiety of glycoproteins. The ligand, denoted 18/18 and comprising a triazine scaffold bis-substituted with 5-aminoindan, has been synthesised in solution, characterised by TLC, ¹H-NMR and MS. When immobilised to amine-derivatised agarose at concentrations >24 μmol/g moist weight gel, ligand 18/18 selectively binds glucose oxidase. The adsorbed enzyme was quantitatively eluted with 0.5 M α- -methyl-mannoside and to a lesser extent with the equivalent glucoside. An investigation of the comparative retention times of saccharidic solutes showed that significant retardation was observed for α- -mannose, mannobiose and mannan, with little or no evidence for selective retention of other saccharides, with the exception of α- -fucose. Interestingly, α- -fucose and α- -mannose share an identical configuration of the hydroxyl groups on C-2, C-3 and C-4. Analysis of Scatchard plots from partition equilibrium studies on the interaction of glucose oxidase and the p-nitrophenyl-glycosides of -mannose, -glucose, -fucose and -galactose with immobilised 18/18 establish that the affinity constants (K_AX) for the enzyme, the glycosides of mannose, glucose and fucose, and the p-nitrophenyl-galactoside are 4.3×10⁵ M⁻¹, 1.9×10⁴ M⁻¹ and 1.2×10⁴ M⁻¹ respectively. ¹H-NMR studies on the interaction of α- -methyl-mannoside with ligand 18/18 in solution confirm the involvement of the hydroxyl group in the C-2 position. Molecular modelling suggests the formation of four hydrogen bonds between the hydroxyl groups at positions C-2, C-3 and C-4 of α- -methyl-mannoside and the bridging and ring nitrogen atoms of the triazine scaffold, with aromatic stacking of a second ligand against the carbohydrate face. The greater specificity of ligand 18/18 for mannose and glucose than for galactose parallels that exhibited by concanavalin A.     

Isolation and characterization of free glycans of the oligomannoside type from the extracellular medium of a plant cell suspension

The oligosaccharides Man₅GlcNAc and Man₃(Xyl)GlcNAc(Fuc)GlcNAc presumed to originate fromN-glycosyl proteins have been purified from an extracellular medium (concentration: 2–5 mg/l of 14 day cultures) of white campion (Silene alba) suspension culture. Their primary structures have been determined by¹H-400-MHz NMR spectroscopy and FAB-MS spectrometry. They are probably the result of an autophagic process including protein catabolism due to sucrose starvation. Additional identification of digalactosylglycerol (galactolipid breakdown) argues for this hypothesis.Abbreviations Fuc l-fucose - Man d-mannose - Xyl d-xylose - GlcNAc N-acetyl-d-glucosamine - Gal d-galactose - Glc d-glucose - FAB-MS fast atom bombardment mass spectrometry - NMR nuclear magnetic resonance     

Isolation of species-specific and stage-specific adhesion promoting component by disaggregation of intact sea urchin embryo cells

The species-specific and developmental stage-specific aggregation-enhancing supernatant isolated from intact sea urchin (Strongylocentrotus purpuratus) blastula cells incubated in Ca²⁺---Mg²⁺-free sea water is a hemagglutinin. This material agglutinated trypsinized, fixed human type O and B (inhibited by -galactose) erythrocytes, whereas control erythrocytes in Millipore-filtered sea water did not agglutinate. The blastula supernatant agglutinates both live and fixed S. purpuratus blastula cells. Fixed cells were chosen in these experiments so that a standardized, highly reproducible system could be produced by pooling batches of blastula cells. Dissociation supernatant (DS)-mediated agglutination of S. purpuratus blastula cells was blocked by -galactose and N-acetyl- -galactosamine by 10 min of incubation, but not by -glucose, -fucose, -mannose, -glucosamine, -mannosamine or N-acetyl- -mannosamine (all at 0.1 M concentration, the concentration chosen as a result of preliminary experiments). The results were consistently observed in scores of experiments and suggest that DS binds cells together via -galactose-like and N-acetyl- -galactosamine-like residues. We also found that aggregates of live blastula cells formed in the presence of DS gave rise, after 24 h incubation, to viable, swimming embryoids, suggesting that DS-mediated adhesion is physiologically meaningful.     

Histochemical study of Hurler's disease by the use of peroxidase-labelled lectins

Summary Peroxidase-labelled lectins specific for various carbohydrate residues were used as histochemical reagents in the investigation of Hurler's syndrome. Peanut lectin was used to detect terminald-galactose, wheatgerm lectin forN-acetyl-d-glucosamine, soybean lectin forN-acetyl-d-galactosamine,Tetragonolobus lotus lectin for -l-fucose andBandeiraea S. lectin for -d-galactose. It was found that Kupffer cells in the liver and splenic reticulo-endothelial cells contain acid mucopolysaccharides which bind lectins in paraffin sections after appropriate fixation. The pattern of lectin binding suggests that such cells contain significant amounts ofd-galactose,l-fucose,N-acetyl-d-galactosamine andN-acetyl-d-glucosamine. It is likely that the last named carbohydrate is present as a polymer. Neurones contain a different carbohydrate, rich in galactose and fucose but poor inN-acetyl-d-glucosamine. This compound is resistant to lipid extraction. Hepatocytes, as a rule, do not react with lectins, most likely because of loss of the more soluble mucopolysaccharides during fixation. The results are consistent with the biochemical data of Hurler's syndrome and indicate that lectins can be a useful tool for the investigation of the cytochemistry of storage disorders.     

Mannose-Specific Lectin Activity of Parasporal Proteins from a Lepidoptera-Specific <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain

Lectin activity, agglutinating sheep erythrocytes, was associated with parasporal inclusion proteins from a Lepidoptera-specific isolate of Bacillus thuringiensis serovar galleriae (H5ab). The activity was generated when parasporal inclusions were solubilized in an alkaline condition. Proteolytic processing was not required for generation of the lectin activity; the activity level was not affected by the presence/absence of the three proteases (trypsin, chymotrypsin, and proteinase K). SDS-PAGE analysis revealed that (1) alkali-solubilized parasporal inclusion proteins consisted of two major components of 130 kDa and 65 kDa, and (2) proteinase K treatment of alkali-solubilized proteins yielded a single major protein of 60 kDa. Lectin activity of our isolate was strongly inhibited by preincubation with D-mannose, but not with the six other monosaccharides: D-galactose, D-glucose, L-fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and N-acetylneuraminic acid. In contrast, D-mannose did not inhibit the in vivo larvicidal activity of the proteins against the silkworm, Bombyx mori. Received: 21 February 2002 / Accepted: 28 March 2002     

Fractionation of plant chromatin with immobilized RNA-polymerase.

A new heptasaccharide, lacto-N-fucoheptaose has been isolated from human milk. It contains D(+)-galactose, D(+)-glucose, L(?)-fucose and N-acetyl-D-(+)-glucosamine in a 3 : 1 : 1 : 2 ratio. The glucose residue is at the reducing end of the oligosaccharide. Data obtained by partial acid hydrolysis, permethylation and enzymic hydrolysis establish the structure of lacto-N-fucoheptaose as follows:

     

Halogenated 2,5-pyrrolidinediones: synthesis, bacterial mutagenicity in Ames tester strain TA-100 and semi-empirical molecular orbital calculations

The chloroimide 3,3-dichloro-4-(dichloromethylene)-2,5-pyrrolidinedione, a tetrachloroitaconimide, is the principal mutagen produced by chlorination of simulated poultry chiller water. It is the second most potent mutagenic disinfection by-product of chlorination ever reported. Six of seven new synthetic analogs of this compound are direct-acting mutagens in Ames tester strain TA-100. Computed energies of the lowest unoccupied molecular orbital (E_LUMO) and of the radical anion stability (ΔH_f^rad−ΔH_f) from MNDO-PM3 for the chloroimides show a quantitative correlation with the Ames TA-100 bacterial mutagenicity values. The molar mutagenicities of these direct acting mutagenic imides having an exocyclic double bond fit the same linear correlation (ln M_m vs. E_LUMO; ln M_m vs. ΔH_f^rad−ΔH_f) as the chlorinated 2(5H)-furanones, including the potent mutagen MX, 3-chloro-4-(dichloro-methyl)-5-hydroxy-2(5H)-furanone, a by-product of water chlorination and paper bleaching with chlorine. Mutagenicity data for related haloimides having endocyclic double bonds are also given. For the same number of chlorine atoms, the imides with endocyclic double bonds have significantly higher Ames mutagenicity compared to their structural analogs with exocyclic double bonds, but do not follow the same E_LUMO or ΔH_f^rad−ΔH_f correlation as the exocyclic chloroimides and the chlorinated 2(5H)-furanones.     

Validation of a high-performance liquid chromatographic method for the determination of ibuprofen enantiomers in plasma of broiler chickens

To characterise the pharmacokinetic properties of each enantiomer of ibuprofen in broiler chickens, a stereospecific HPLC method based on a α₁-acid glycoprotein bonded chiral stationary phase has been validated. S-(+)-naproxen was used as internal standard. Enantiomers of ibuprofen and S-(+)-naproxen were baseline separated using a mobile phase consisting of 0.1 M phosphate buffer pH=7 and 0.4% 2-propanol. The method is precise, specific, accurate and reproducible. Recoveries were higher than 80% and the limits of quantification for R-(−)- and S-(+)-ibuprofen were 1.16 and 1.37 μg ml⁻¹, respectively. The method seemed suitable for the pharmacokinetic studies of ibuprofen in chickens.     

Acceptor Specificity of Cyclodextrin Glycosyltransferase from Bacillus stearothermophilus and Synthesis of α-D-Glucosyl O-β-D-galactosyl-(1→4)-β-D-glucoside

Bacillus stearothermophilus CGTase had a wider acceptor specificity than Bacillus macerans CGTase did and produced large amounts of transfer products of various acceptors such as D-galactose, D-mannose, D-fructose, D- and L-arabinose, d- and L-fucose, L-rhamnose, D-glucosamine, and lactose, which were inefficient acceptors for B. macerans CGTase. The main component of the smallest transfer products of lactose was assumed to be α-D-glucosyl O-β-D-galactosyl-(l→4)-β-D-glucoside.     

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

Growth and functional responses of different cultivars of Lotus corniculatus to aluminum and low pH stress

Aluminum toxicity is an important stress factor in acid soils. Growth, respiration and permeability properties of root cells were studied in five cultivars of Lotus corniculatus subjected to aluminum (Al) or low pH stress. The cultivars showed significant differences in root elongation under stress conditions, which correlated with changes in membrane potential (E_M) of root cortical cells. A pH drop from 5.5 to 4.0 resulted in significant membrane depolarization and root growth inhibition. The strongest inhibition was observed in cv. São Gabriel (33.6%) and least in cv. UFRGS (25.8%). Application of an extremely high Al concentration (2 mM) stopped the root growth in cv. INIA Draco, while inhibition in cv. UFRGS reached only 75%.The E_M values of cortical cells of Lotus roots varied between −115 and −144 mV. Treatment with 250 μM of AlCl₃ (pH 4) resulted in rapid membrane depolarization. The extent of the membrane depolarization ranged between 51 mV (cv. UFGRS) and 16 mV (cv. INIA Draco). The membrane depolarization was followed by a loss of K⁺ from Al-treated roots (2 mM Al) and resulted in a decrease of the diffusion potential (E_D). The total amount of K⁺ in Al-treated roots dropped from 31.4 to 16.8 μmol g⁻¹ FW in sensitive cv. INIA Draco, or from 26.1 to 22.7 μmol g⁻¹ FW in tolerant cv. UFGRS. The rate of root respiration under control conditions as well as under Al treatment was higher in cv. INIA Draco than in cv. UFRGS. Al-induced inhibition of root respiration was 21–34% of the control.     

Distribution of the glycoconjugate oligosaccharides in the human placenta from pregnancies complicated by altered glycemia: lectin histochemistry

The aim of this study was to investigate the distribution of the oligosaccharides of the glycoconjugates in placentas from pregnancies complicated by different degree of altered glycaemia. Placentas from women with physiological pregnancies (group 1), with pregnancies complicated by minor degree of glucose intolerance (group 2) and with pregnancies complicated by gestational diabetes mellitus (GDM) treated with insulin (group 3) were collected. Ten lectins were used (ConA, WGA, PNA, SBA, DBA, LTA, UEA I, GSL II, MAL II and SNA) in combination with chemical and enzymatic treatments. The data showed a decrease of sialic acid linked α(2–6) to galactose/N-acetyl-d-galactosamine and an increase of N-acetyl-d-glucosamine in the placentas of the pathological groups, in particular the group 3, comparing to the group 1. A decrease of l-fucose (LTA) and d-galactose-(β1–3)-N-acetyl-d-galactosamine, and an increase and/or appearance of l-fucose (UEA I) and N-acetyl-d-galactosamine were observed in both the pathological groups, particularly in the group 2, with respect to the group 1. In GDM, and even in pregnancies with a simple alteration of maternal glycaemia, the changes in the distribution of oligosaccharides could be related to alteration of the structure and functionality of the placenta.     

Chemical and biological studies on the lipopolysaccharide (O-antigen) of Anacystis nidulans

The O-antigen (lipopolysaccharide) of Anacystis nidulans, strain KM, has been isolated from whole cells and from cell wall preparations by phenolwater extraction. The polysaccharide moiety consists of a D-mannose polymer accompanied by smaller amounts of 3- and 4-O-methyl-D-mannoses, D-galactose, D-glucose, L-fucose, D-glucosamine, mannosamine and 2-keto-3-deoxyoctonate. Aldoheptoses are lacking. The degraded polysaccharide is split from lipid A by acid hydrolysis (10% acetic acid, 100°C, 3 h) whereby 2-keto-3-deoxyoctonate is released in small amounts. Degraded polysaccharide forms only one major fraction by Sephadex G-50 gel-filtration. This fraction includes all the sugars mentioned above except L-fucose, which is released during the acetic acid degradation. Periodate studies and methylation analysis revealed that the poly-mannose chain consists of about 75% 13 linked and of 25% 14 linked D-mannose units.Lipid A of A. nidulans is phosphate-free. The main fatty acid, -hydroxypalmitic acid, is exclusively amide-bound, presumably to the amino group of D-glucosamine. Other fatty acids, found as minor constituents, are -hydroxymyristic, palmitic and stearic acids. Lipopolysaccharide of A. nidulans KM exhibits high anticomplementary activity in guineapig serum. It is about 800 times less toxic for adrenalectomized mice than endotoxin from Salmonella typhimurium.The isolated lipopolysaccharide reacts with rabbit antisera against living or heat-killed cells of A. nidulans in passive hemagglutination, when untreated or heated, but not when alkali-treated lipopolysaccharide is used for red blood cell sensibilization. It is concluded that lipopolysaccharide of A. nidulans KM is exposed on the surface of the cell.     

Quantitation of entacapone glucuronide in rat plasma by on-line coupled restricted access media column and liquid chromatography–tandem mass spectrometry

A column-switching liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method was developed for the direct analysis of entacapone glucuronide in plasma. The plasma samples (5 μl) were injected onto a C₁₈-alkyl-diol silica (ADS) column and the matrix compounds were washed to waste with a mixture of 20 mM ammonium acetate solution at pH 4.0–acetonitrile (97:3). The retained analyte fraction containing (E)- and (Z)-isomers of glucuronides of entacapone and tolcapone glucuronide (internal standard) was backflushed to the analytical C₁₈ column, with a mixture of 20 mM ammonium acetate–acetonitrile (85:15) for the final separation at pH 7.0. The eluate was directed to the mass spectrometer after splitting (1:100). The mass spectrometer was operated in the negative ion mode and the deprotonated molecules [M−H]⁻ were chosen as precursor ions for the analytes and internal standard. Collisionally induced dissociation of [M−H]⁻ in MS–MS resulted in loss of the neutral glucuronide moiety and in the appearance of intensive negatively charged aglycones [M−H−Glu]⁻, which were chosen as the product ions for single reaction monitoring. Quantitative studies showed a wide dynamic range (0.0025–100 μg/ml) with correlation coefficients better than 0.995. The method was repeatable within-day (relative standard deviation, RSD<7%) and between-day (RSD<14%) and the recovery (78–103%) was better than with the traditional, laborious pretreatment method. The use of tandem mass spectrometry permitted low limits of detection (1 ng/ml of entacapone glucuronide). The method was applied for the quantitation of (E)- and (Z)-isomers of entacapone glucuronide in plasma of rats used in absorption studies.     

Surface-enhanced Raman spectroscopy combined with atomic force microscopy for ultrasensitive detection of thrombin

Indole-3-acetic acid (IAA) amide conjugates play an important role in balancing levels of free IAA in plant cells. The GH3 family of proteins conjugates free IAA with various amino acids. For example, auxin levels modulate expression of the Oryza sativa (rice) GH3-8 protein, which acts to prevent IAA accumulation by coupling the hormone to aspartate. To examine the kinetic properties of the enzyme, we developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay system. Bacterially expressed OsGH3-8 was purified to homogeneity and used to establish the assay system. Monitoring of the reaction confirms the reaction product as IAA–Asp and demonstrates that production of the conjugate increases proportionally with both time and enzyme amount. Steady-state kinetic analysis using the LC–MS/MS-based assay yields the following parameters: V/E_t^IAA = 20.3 min⁻¹, K_m^IAA = 123 μM, V/E_t^ATP = 14.1 min⁻¹, K_m^ATP = 50 μM, V/E_t^Asp = 28.8 min⁻¹, K_m^Asp = 1580 μM. This is the first assignment of kinetic values for any IAA–amido synthetase from plants. Compared with previously described LC- and thin-layer chromatography (TLC)-based assays, this LC–MS/MS method provides a robust and sensitive means for performing direct kinetic studies on a range of IAA-conjugating enzymes.     

Approximate Michaelis-Menten kinetics displayed in a stochastic model of cell-mediated cytotoxicity

A nonlinear continuous-time Markov chain describing a two-step process of cytolytic cells binding to target and the subsequent lysis and release of label is shown to have kinetics which resemble standard enzyme-substrate kinetics. The Michaelis-Menten saturation function is found as a special case resulting when the target population is in excess. A comparison theorem for the pseudo-steady-state distribution Π is constructed to enable examination of that distribution whose expected value E and variance V satisfy - K_mE + (C_T − E)(T_T − E) + V = 0, where K_m is the Michaelis half-saturation constant and C_T and T_T are the initial populations of the two cell types. Using Π as an initial condition, the release of label process is examined. The main result is that the fraction of specific release, ƒ, has the approximate form when T_t is large, so that a nonlinear regression procedure is appropriate for the determination of the parameters.     

The effect of PPAR ligands to modulate glucose metabolism alters the incorporation of metabolic precursors into proteoglycans synthesized by human vascular smooth muscle cells

PPAR ligands are important effectors of energy metabolism and can modify proteoglycan synthesis by vascular smooth muscle cells (VSMCs). Describing the cell biology of these important clinical agents is important for understanding their full clinical potential, including toxicity. Troglitazone (10 microM) and fenofibrate (30 microM) treatment of VSMCs reduces ((35)S)-sulphate incorporation into proteoglycans due to a reduction of glycosaminoglycan (GAG) chain length. Conversely, under physiological glucose conditions (5.5 mM), the same treatment increases ((3)H)-glucosamine incorporation into GAGs. This apparent paradox is the consequence of an increase in the intracellular ((3)H)-galactosamine specific activity from 48.2 +/- 3.2 microCi/ micromol to 90.7 +/- 11.0 microCi/ micromol (P < 0.001) and 57.1 +/- 2.6 microCi/ micromol (P < 0.05) when VSMCs were treated with troglitazone and fenofibrate, respectively. The increased specific activity observed with troglitazone (10 microM) treatment correlates with a two-fold increase in glucose consumption, while fenofibrate (50 microM) treatment showed a modest (14.6%) increase in glucose consumption. We conclude that the sole use of glucosamine precursors to assess GAG biosynthesis results in misleading conclusions when assessing the effect of PPAR ligands on VSMC proteoglycan biosynthesis.     

Chloride conductance and mitochondria-rich cell density in isolated skin of Rana catesbeiana acclimated to various environments

The Cl⁻ conductance in isolated skin of frogs (Rana catesbeiana) acclimated to 30 mM solutions of NaCl, Na₂SO₄, MgCl₂ and distilled water (DW) was studied. Transepithelial potential difference (PD_trans), short-circuit current (I_SC) and total conductance (G_t) were measured under conditions such that there was Cl⁻ flux in the presence and absence of Na⁺ transport. The Cl⁻ content of the mucosal solution was acutely replaced with SO₄²⁻ or gluconate to evaluate the effect of removal of Cl⁻ conductance on electrophysiological parameters. Mitochondria-rich cell density (D_MRC) was also measured. Skins from frogs acclimated to NaCl and Na₂SO₄ showed the lowest and the highest D_MRC, respectively, but no difference could be found between the skins from frogs acclimated to DW and MgCl₂ indicating that D_MRC is not unconditionally dependent on environmental Cl⁻ in this species. Frogs acclimated to NaCl showed marked differences when compared to the other groups: the highest G_t, probably represented by a higher paracellular conductance; the lowest transepithelial electrical potential difference which remained invariant after replacement of mucosal Cl⁻ with SO₄²⁻ or replacement of mucosal Cl⁻ with gluconate and an inwardly oriented positive current in the absence of bilateral Na⁺.     

Time-activity budgets and energetics of Dipper Cinclus cinclus are dictated by temporal variability of river flow

The white-throated Dipper (Cinclus cinclus) is unique among passerine birds by its reliance on diving to achieve energy gain in fast-flowing waters. Consequently, it should have evolved behavioural adaptations allowing responding directly to runoff patterns (one of the assumptions of the Natural Flow Regime Paradigm—NRFP). In this study (October 1998–August 2001), we investigated how behavioural and energy use strategies in Dippers might vary under the natural flow regime of snowmelt-dominated streams in The Pyrénées (France) where natural flow regime is highly seasonal and predictable. We recorded time spent in each of 5 behavioural activities of ringed birds to estimate time–activity budgets and derive time–energy budgets enabling the modelling of daily energy expenditure (DEE). Annual pattern in ‘foraging’ and ‘resting’ matched perfectly the annual pattern of the natural regime flow and there was a subtle relationship between water stage and time spent ‘diving’ the later increasing with rising discharge up to a point where it fell back. Thus, time–activity budgets meet the main prediction of the NRFP. For males and females Dippers, estimates of feeding rates (ratio E_obs/E_req = observed rate of energy gain / required foraging rate) and energy stress (M = DEE / Basal Metabolic Rate) also partly matched the NFRP. Maximum value for the ratio E_obs/E_req was registered in May whilst M peaked in spring. These ratios indicated that Pyrenean Dippers could face high energy stress during winter but paradoxically none during high snowmelt spates when food is expected to be difficult to obtain in the channel and when individual birds were observed spending ca 75% of the day ‘resting’. Annual pattern in DEE did not match the NFRP ; two phases were clearly identified, the first between January to June (with oscillating values 240–280 kJ d^− 1 ind^− 1) and the second between July and December (200–220 kJ d^− 1 ind^− 1). As total energy expenditure was higher during the most constraining season or life cycle, we suggest that energy management by Dippers in Pyrenean mountain streams may fit the ‘peak total demand’ hypothesis. At this step of the study, it is not possible to tell whether Dippers use an ‘energy-minimisation’ or an ‘energy-maximisation' strategy.     

Long-Term Temperature Acclimation of Photosynthesis in Steady-State Cultures of the Polar Diatom <Emphasis Type="Italic">Fragilariopsis cylindrus</Emphasis>

Cultures of the obligate psychrophilic diatom Fragilariopsis cylindrus (Grunow) were grown for 4 months under steady-state conditions at −1 °C and +7 °C (50 μmol photons m⁻² s⁻¹) prior to measurements in order to investigate long-term acclimation of photosynthesis to both temperatures. No differences in maximum intrinsic quantum yield of PS II (F_V/F_M) and relative electron transport rates could be detected at either temperature after 4 months of acclimation. Measurements of photosynthesis (relative electron transport rates) vs. irradiance (P vs. E curves) revealed similar values for relative light utilization efficiency (α = 0.57 at −1 °C, α = 0.60 at +7 °C) but higher values for irradiance levels at which photosynthesis saturates (E_K) at −1 °C and, therefore, higher maximum photosynthesis (P_MAX = 54 (relative units) at −1 °C, P_MAX = 49 at +7 °C). Nonphotochemical quenching (NPQ) measurements at 385 μmol photons m⁻² s⁻¹ indicated higher (37%) NPQ for diatoms grown at −1 °C compared to +7 °C, which was possibly related to a 2-fold increase in the concentration of the pigment diatoxanthin and a 9-fold up-regulation of a gene encoding a fucoxanthin chlorophyll a,c-binding protein. Expression of the D1 protein encoding gene psbA was ca. 1.5-fold up-regulated at −1 °C, whereas expression levels of other genes from Photosystem II (psbC, psbU, psbO), as well as rbcL, the gene encoding the Rubisco large subunit were similar at both temperatures. However, a 2-fold up-regulation of a plastid glyceraldehyde-P dehydrogenase at −1 °C indicated enhanced Calvin cycle activity. This study revealed for the first time that a polar diatom could efficiently acclimate photosynthesis over a wide range of polar temperatures given enough time. Acclimation of photosynthesis at −1 °C was probably regulated similarly to high light acclimation.