Dark induction of nitrate reductase in the halophilic alga Dunaliella salina

The effect of nitrogen starvation on the NO₃-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH ₄ ⁺ as the only nitrogen source (NH ₄ ⁺ -cells) were transferred into NO ₃ ^? medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO ₃ ^? , NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m^?2 s^?1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m^?2 s^?1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO ₃ ^? -induced NR synthesis when the cells, previously grown in NH ₄ ⁺ medium, were transferred into NO ₃ ^? medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH ₄ ⁺ to NO ₃ ^? medium (at time 0 h), NO ₃ ^? induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO ₃ ^? to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO ₃ ^? medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO ₃ ^? uptake and NR induction due to the expression of NR and NO ₃ ^? -transporter mRNAs.     

Role of external carbonic anhydrase in light-dependent alkalization byFucus serratus L. andLaminaria saccharina (L.) Lamour. (Phaeophyta)

It has been proposed that many marine macroalgae are able to utilize HCO ₃ ^– for photosynthesis and growth, and that energy-dependent ion pumping is involved in this process. We have therefore studied the light-dependent alkalization of the surrounding medium by two species of marine macroscopic brown algae,Fucus serratus L. andLaminaria saccharina (L.) Lamour. with the aim of investigating the role of extracellular carbonic anhydrase (EC 4.2.1.1.) in the assimilation of inorganic carbon from the seawater medium. In particular, the influence of membrane-impermeable or slowly permeable carbonic-anhydrase inhibitors on the rate of alkalization of the seawater has been investigated. Inhibition of the alkalization rate occurred in both species at an alkaline pH (pH 8.0) but no inhibition was observed at an acidic pH (pH 6.0). The alkalization was found to be light-dependent and inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and, thus, correlated with photosynthesis. Alkalization by macroalgae has previously been shown to be proportional to inorganiccarbon uptake. We suggest that alkalization of the medium at alkaline pH in both of the species examined is mainly the consequence of an extracellular reaction. The reaction is catalyzed by extracellular carbonic anhydrase which converts HCO ₃ ^– to OH^– and CO₂; CO₂ is then taken up through the plasmalemma. However, we do not exclude the involvement of other mechanisms of inorganic-carbon uptake.Abbreviations AZ acetazolamide - CA carbonic anhydrase - CAext extracellular carbonic anhydrase - C_i inorganic carbon - DBS dextran-bound sulfonamide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PPFD photosynthetic photon flux density This study was carried out with financial support by SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Trygger's Fund for Scientific Research (Sweden), SJFR (Swedish Council for Forestry and Agricultural Research) and CICYT (Spain). Z. Ramazanov is an invited professor of Ministerio de Educación y Ciencia, Spain.     

Involvement of photorespiration and glycolate pathway in carbonic anhydrase induction and inorganic carbon concentration in Chlamydomonas reinhardtii

Interaction between induction of carbonic anhydrase (CA) activity, induction of inorganic carbon (C_i) concentrating mechanisms and the photorespiratory glycolate pathway has been studied in wild type 6145c and photorespiratory mutant 18–7F (low in phosphoglycolate phosphatase activity) cells of C. reinhardtii . Cell transfer from high CO₂ (5%, v/v) to low CO₂ (0.03%) provoked an increase of extracellular and total (extracellular plus intracellular) CA in both wild type and mutant cells. During adaptation to low CO₂ conditions, both strains excreted ammonium to the medium at a similar rate in the presence of l -methionine- d-l -sulfoximine (MSX), an inhibitor of glutamine synthetase (GS). MSX also provoked ammonium excretion by air adapted wild type and mutant cells, even though both strains had high levels of CA activity and of C_i concentrating activities.
GS increased in both strains after transfer from high to low CO₂ conditions. However, this increase was abolished by aminooxyacetate, an inhibitor of the glyoxylate-serine aminotransferase, and by glycolaldehyde, an inhibitor of triose phosphate to ribulose 1,5-bisphosphate conversion. CA synthesis did not occur in the presence of either aminooxyacetate or glycolaldehyde. Algae grown in high CO₂ in the presence of aminooxyacetate did not induce C_i concentrating mechanisms. Integration of these three processes, i.e., CA synthesis, C_i-concentration, and photorespiratory glycolate pathway is proposed in the framework of carbon metabolism of the alga.     

Intracellular Localization of Three l-Glutamate Dehydrogenase Isozymes from Chlamydomonas reinhardtii

The intracellular localization of the activity and synthesis of three isozymes of NAD(P)⁺-glutamate dehydrogenase from the unicellular green alga Chlamydomonas reinhardtii cw-92 has been established. Isozyme activities have been located within mitochondria by using differential centrifugation techniques and discontinuous Percoll gradient separations. Experiments with protein synthesis inhibitors cycloheximide, rifampicin, chloramphenicol, and actinomycin D, under dark and carbon starvation conditions, revealed that synthesis of the three isozymes was likely to occur in cytosol as precursor proteins that are then transported and processed inside the mitochondria.     

Phenolic substances of brown algae and their antioxidant activity

The composition and content of phenolic substances were studied in 14 species of marine brown algae of the Canary Islands littoral (Spain). The highest content of phenolic substances was found inCystoseira compressia andSargassum furcatum. A high antioxidant activity was found in florotannin isolated fromCystoseira sp.     

Isolation and characterization of a starchless mutant of Chlorella pyrenoidosa STL-PI with a high growth rate, and high protein and polyunsaturated fatty acid content

The growth characteristics, biochemical composition and ultrastructure of a novel starchless mutant of Chlorella pyrenoidosa, designated as STL‐PI, are compared to the same characteristics of its parental strain, C. pyrenoidosa 82T. The STL‐PI mutant had a 22 ± 5% higher growth rate, and 24.5 ± 4.2% more protein than the parental strain, 82T. Furthermore, the STL‐PI mutant accumulated 20.4% more polyunsaturated fatty acids and 18% less saturated fatty acids than the parental 82T. When the parental 82T was cultured in a nitrogen‐free media, their starch content increased from 6.8 ± 2.8% to 22.5 ± 3.1%. In contrast, the STL‐PI mutant produced no starch, regardless of the growth conditions. Instead, the mutant cells responded to nitrogen limitation by further increasing their lipid content from 25.2 ± 1.2% to 38.0 ± 2.3% per dry weight. Transmission electron micrography revealed that nitrogen limitation typically stimulates the formation of starch granules in the chloroplast of 82T cells. Yet no starch granules were observed in the STL‐PI cells. Instead, only the formation of large lipid globules was observed in the mutant cells. These results demonstrate that the starchless mutant STL‐P1 possesses novel physiological and phytochemical properties distinct from the 82T cells: their cells were deficient in starch synthesis and showed higher growth rates and productivity than 82T cells.     

pH-sensitive starch hydrogels via free radical graft copolymerization,synthesis and properties

Natural polymers are considered high value polymeric materials because of their potential as biocompatible materials with medical applications. The chemical modification of natural polymers by grafting has received considerable attention in recent years because of the wide variety of monomers available. As the first part of a continued research on conversion of carboxymethyl starch (CMS) to useful biopolymer-based materials, large numbers of carboxylic functional groups were introduced onto CMS by grafting with polymethacrylic acid (PMAA). Free radical graft copolymerization was carried out at 70 °C, bis-acrylamide as a crosslinking agent and persulfate as an initiator. Equilibrium swelling studies were carried out in enzyme-free simulated gastric and intestinal fluids (SGF and SIF, respectively). Also, the sodium dicofenac as a model drug was entrapped in these nano-gels and the in vitro release profiles were established separately in both enzyme-free SGF and SIF. The drug release was found to be faster in SIF. The drug-release profiles indicate that amount drug release depends on their degree of swelling, and crosslinking. This hydrogel converted to nano by freeze-drying method and characterized by scanning electron microscopy, differential scanning calorimetry and FT-IR spectrometry.     

Synthesis and in vitro evaluation of carboxymethyl starch-chitosan nanoparticles as drug delivery system to the colon

The purpose of this study was to examine chitosan (CS)-carboxymethyl starch (CMS) nanoparticles as drug delivery system to the colon. The 5-aminosalicylic acid (5-ASA) was chosen as model drug molecule. CS-CMS nanoparticles were formulated by a complex coacervation process under mild conditions. The influence of process variables, including the two ionic polymers, on particle size, and nanoparticles entrapment of 5-ASA was studied. In vitro release of 5-ASA was also evaluated, and the integrity of 5-ASA in the release fraction was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The release of 5-ASA from nanoparticle was based on the ion-exchange mechanism. The CS-CMS nanoparticles developed based on the modulation of ratio show promise as a system for controlled delivery of drug to the colon.     

Synthesis and in vitro studies of biodegradable thiolated chitosan hydrogels for breast cancer therapy

Local drug delivery strategies have gained momentum recently as a promising modality in cancer therapy. In order to deliver Letrozole (LTZ) at the tumor site in therapeutically relevant concentrations, acetyl-polyamidoamine (Ac-PAMAM)-thiolated chitosan (TCS) films were fabricated. LTZ could be loaded at 31% wt/wt in films, which were translucent and flexible. Physicochemical characterization of LTZ via thermal technique revealed information on solid-state properties of LTZ as well as thiolated chitosan in films. While thiolated chitosan was in amorphous form, LTZ seemed to be present in both amorphous and crystalline forms in film. The lack of formulation-induced local inflammatory responses of LTZ-acetyl-polyamidoamine (Ac-PAMAM)-thiolated chitosan (TCS) films a new paradigm for localized chemotherapy based on breast delivery systems.     

Ulva rigida (Ulvales,Chlorophyta) tank culture as biofilters for dissolved inorganic nitrogen from fishpond effluents

Ulva rigida was cultivated in 7501 tanks at different densities with direct and continuous inflow (at 2, 4, 8 and 12 volumes d^–1) of the effluents from a commercial marine fishpond (40 metric tonnes, Tm, of Sparus aurata, water exchange rate of 16 m³ Tm^–1) in order to assess the maximum and optimum dissolved inorganic nitrogen (DIN) uptake rate and the annual stability of the Ulva tank biofiltering system. Maximum yields (40 g DW m^–2 d^–1) were obtained at a density of 2.5 g FW 1^–1 and at a DIN inflow rate of 1.7 g DIN m^–2 d^–1. Maximum DIN uptake rates were obtained during summer (2.2 g DIN M^–2 d^–1), and minimum in winter (1.1 g DIN m^–2 d^–1) with a yearly average DIN uptake rate of 1.77 g DIN m^–2 d^–1 At yearly average DIN removal efficiency (2.0 g DIN m^–2 d^–1, if winter period is excluded), 153 m² of Ulva tank surface would be needed to recover 100% of the DIN produced by 1 Tm of fish.Abbreviations DIN= dissolved inorganic nitrogen (NH _inf4 ^sup+ + NO _inf3 ^sup– + NO _inf2 ^sup– ); - FW= fresh weight; - DW= dry weight; - PFD= photon flux density; - V= DIN uptake rate