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Functional complementation of a nitrate reductase defective mutant of a green alga Dunaliella viridis by introducing the nitrate reductase gene.

Nitrate reductase (NR) catalyzes NAD (P) H dependent reduction of nitrate to nitrite. Transformation systems have been established in several species of green algae by nitrate reductase gene functional complementation. In this report, an endogenous NR cDNA (3.4 kb) and a genomic fragment (14.6 kb) containing the NR gene (DvNIA1) were isolated from the D. viridis cDNA and genomic libraries respectively. Southern blot and Northern blot analyses showed that this gene exists as a single copy in D. viridis and is induced by nitrate. To obtain a NR defective mutant as a recipient strain, D. viridis cells were treated with a chemical mutagen and then cultured on a chlorate-containing plate to enrich chlorate tolerant mutants. Southern analysis showed that one isolate, B14, had a deletion in the DvNIA1 gene region. Using electroporation conditions determined in this laboratory, plasmid pDVNR containing the intact DvNIA1 gene has been electroporated into the defective mutant B14. Strains retaining a nitrate assimilation phenotype were obtained from nitrate plates after spreading the electroporated cells. In some individual strains, transcription of the introduced gene was detected. NR activity in these strains was slightly higher than that in the defective B14 cell, but excretion of nitrite into culture media was almost as high as that of the wild-type cell. Possible episomal presence of the introduced DNA in D. viridis is discussed.

Amino Acid Sequence↗

Pheophytin a, a low molecular weight compound found in the marine brown alga Sargassum fulvellum, promotes the differentiation of PC12 cells.

We identified and characterized a neurodifferentiation compound from the marine brown alga Sargassum fulvellum collected from the Japanese coastline. Several instrumental analyses revealed the compound to be pheophytin a. Pheophytin a did not itself promote neurite outgrowth of PC12 cells. However, when PC12 cells were treated with a low concentration of pheophytin a (3.9 microg/ml) in the presence of a low level of nerve growth factor (10 ng/ml), the compound produced neurite outgrowth similar to that produced by a high level of nerve growth factor (50 ng/ml). Pheophytin a also enhanced signal transduction in the mitogen-activated protein kinase signaling pathway, which is also induced by nerve growth factor. The effect of pheophytin a on neurite outgrowth of PC12 cells was completely blocked by U0126, a representative mitogen-activated protein kinase kinase inhibitor. These results suggest that pheophytin a enhances the neurodifferentiation of PC12 cells in the presence of a low level of nerve growth factor and that this effect is mediated by activation of a mitogen-activated protein kinase signaling pathway.

Animals↗

Purification and characterization of phycocyanin from the blue-green alga Aphanizomenon flos-aquae.

Aphanizomenon flos-aquae (AFA) is a blue-green alga and represents a nutrient-dense food source. In this study the presence of phycocyanin (PC), a blue protein belonging to the photosynthetic apparatus, has been demonstrated in AFA. An efficient method for its separation has been set up: PC can be purified by a simple single step chromatographic run using a hydroxyapatite column (ratio A620/A280 of 4.78), allowing its usage for health-enhancing properties while eliminating other aspecific algal components. Proteomic investigation and HPLC analysis of purified AFA phycobilisomes revealed that, contrary to the well-characterized Synechocystis and Spirulina spp., only one type of biliprotein is present in phycobilisomes: phycocyanins with no allo-phycocyanins. Two subunit polypeptides of PC were also separated: the beta subunit containing two bilins as chromophore and the alpha subunit containing only one.

Chromatography, High Pressure Liquid↗

Radiocarbon behaviour in seawater and the brown algae Fucus serratus in the vicinity of the COGEMA La Hague spent fuel reprocessing plant (Goury)--France.

Extensive studies of the radiocarbon (14C) distribution and transfer in the marine environment of the North-Cotentin peninsula and along the English Channel have been carried out. The main aims of these studies have been to estimate the spatial and temporal variation of the 14C concentration in seawater and to calculate 14C concentration factors for some biological species. Such information will be helpful in order to calculate precisely radiation doses to humans. First results obtained in the vicinity of the COGEMA La Hague nuclear plant (Goury) indicate a 14C labelling of the dissolved inorganic carbon (DIC) in seawater (8.0-26.2 Bq.m(-3)) and a tight relationship between the 14C in the liquid releases from the plant and the 14C concentrations in DIC. The particulate organic carbon (POC) is also labelled. The concentration factor calculations for the brown algae (Fucus serratus) sampled from Goury, and also along the English Channel, give 14C values around 3000 Bq.kg(-1) fresh weight / Bq.L(-1).

Atlantic Ocean↗

Batch and column studies on biosorption of acid dyes on fresh water macro alga Azolla filiculoides.

The biosorption of Acid red 88 (AR88), Acid green 3 (AG3) and Acid orange 7 (AO7) by deactivated fresh water macro alga Azolla filiculoides was investigated in batch mode. Langmuir and Freundlich adsorption models were used for the mathematical description of the batch biosorption equilibrium data and model constants were evaluated. The adsorption capacity was pH dependent with a maximum value of 109.0 mg/g at pH 7 for AR88, 133.5 mg/g at pH 3 for AG3 and 109.6 mg/g at pH 3 for AO7, respectively, was obtained. The pseudo first and second order kinetic models were also applied to the experimental kinetic data and high correlation coefficients favor pseudo second order model for the present systems. The ability of A. filiculoides to biosorb AG3 in packed column was also investigated. The column experiments were conducted to study the effect of important design parameters such as initial dye concentration (50-100 mg/L), bed height (15-25 cm) and flow rate (5-15 mL/min) to the well-adsorbed dye. At optimum bed height (25 cm), flow rate (5 mL/min) and initial dye concentration (100 mg/L), A. filiculoides exhibited 28.1mg/g for AG3. The Bed Depth Service Time model and the Thomas model were used to analyze the experimental data and the model parameters were evaluated.

Adsorption↗

Removal of copper from aqueous solution using Ulva fasciata sp.--a marine green algae.

Batch adsorption experiments were carried out for the removal of copper from its aqueous solution using Ulva fasciata sp. a marine green algae as adsorbent. The adsorption of Cu(II) by Ulva fasciata sp. was investigated as a function of pH, contact time, initial Cu(II) and adsorbent concentrations and adsorbent size. About 0.1 g of Ulva fasciata sp. was found to be enough to remove 95% of 20 mg/L copper from 30 mL aqueous solution in 20 min. The optimum pH value was found to be 5. The dynamic data fitted to the pseudo-second order kinetic model. The Langmuir and Freundlich adsorption models were applied to describe the equilibrium isotherms and the isotherm constants were determined. The experimental adsorption data were fitted to the Langmuir adsorption model. The maximum adsorption capacity was 26.88 mg/g. The applicability of Lagergren kinetic model was also investigated.

Adsorption↗

Kinetics and equilibrium modelling of lead uptake by algae Gelidium and algal waste from agar extraction industry.

Pb(II) biosorption onto algae Gelidium, algal waste from agar extraction industry and a composite material was studied. Discrete and continuous site distribution models were used to describe the biosorption equilibrium at different pH (5.3, 4 and 3), considering competition among Pb(II) ions and protons. The affinity distribution function of Pb(II) on the active sites was calculated by the Sips distribution. The Langmuir equilibrium constant was compared with the apparent affinity calculated by the discrete model, showing higher affinity for lead ions at higher pH values. Kinetic experiments were conducted at initial Pb(II) concentrations of 29-104 mgl(-1) and data fitted to pseudo-first Lagergren and second-order models. The adsorptive behaviour of biosorbent particles was modelled using a batch mass transfer kinetic model, which successfully predicts Pb(II) concentration profiles at different initial lead concentration and pH, and provides significant insights on the biosorbents performance. Average values of homogeneous diffusivity, D(h), are 3.6 x 10(-8); 6.1 x 10(-8) and 2.4 x 10(-8)cm(2)s(-1), respectively, for Gelidium, algal waste and composite material. The concentration of lead inside biosorbent particles follows a parabolic profile that becomes linear near equilibrium.

Adsorption↗

The structural bases of the processive degradation of iota-carrageenan, a main cell wall polysaccharide of red algae.

iota-Carrageenans are sulfated 1,3-alpha-1,4-beta-galactans from the cell walls of red algae, which auto-associate into crystalline fibers made of aggregates of double-stranded helices. iota-Carrageenases, which constitute family 82 of glycoside hydrolases, fold into a right-handed beta-helix. Here, the structure of Alteromonas fortis iota-carrageenase bound to iota-carrageenan fragments was solved at 2.0A resolution (PDB 1KTW). The enzyme holds a iota-carrageenan tetrasaccharide (subsites +1 to +4) and a disaccharide (subsites -3, -4), thus providing the first direct determination of a 3D structure of iota-carrageenan. Electrostatic interactions between basic protein residues and the sulfate substituents of the polysaccharide chain dominate iota-carrageenan recognition. Glu245 and Asp247 are the proton donor and the base catalyst, respectively. C-terminal domain A, which was highly flexible in the native enzyme structure, adopts a alpha/beta-fold, also found in DNA/RNA-binding domains. In the substrate-enzyme complex, this polyanion-binding module shifts toward the beta-helix groove, forming a tunnel. Thus, from an open conformation which allows for the initial endo-attack of iota-carrageenan chains, the enzyme switches to a closed-tunnel form, consistent with its highly processive character, as seen from the electron-microscopy analysis of the degradation of iota-carrageenan fibers.

Alteromonas↗

Analysis of cell-seeded 3-dimensional bone constructs manufactured in vitro with hydroxyapatite granules obtained from red algae.

PURPOSE: Bone tissue engineering is a promising approach for the treatment of defective or lost bone in the maxillofacial region. Biocompatible and biodegradable scaffolds seeded with living cells are used to create functional tissue for load-bearing bone reconstruction. The aim of this study was to manufacture cell-seeded 3-dimensional bone constructs based on hydroxyapatite ceramic granule calcified from red algae and mesenchymal cambial-layer precursor cells. The ability of these cells to grow on hydroxyapatite ceramic was quantitatively investigated to evaluate 3-dimensional bone constructs for their potential use in bone tissue engineering. MATERIALS AND METHODS: Mesenchymal cambial-layer precursor cells were isolated from mandibular periosteum biopsy samples of 3 patients. To manufacture 72 bone constructs, these cells and hydroxyapatite ceramic granules (C GRAFT/Algipore; Clinician's Preference LLC, Golden, CO) were cultivated under osteogenic differentiation conditions in a rotating wall vessel system. After 6 and 21 days, histologic examination and scanning electron microscopy were performed. The absolute DNA content, protein synthesis, and alkaline phosphatase activity were also quantified. The osteoblastic phenotype of the constructs was confirmed by the expression of bone-specific genes (osteocalcin, osteonectin, osteopontin, and core binding factor alpha1) using semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis. RESULTS: Cells within the constructs showed good viability, which was evidenced by an increase in DNA content over the culture period. The decrease in alkaline phosphatase-specific activity could be an indicator of the maturation of cells and the induction of mineralization. The osteoblastic phenotype of the constructs was demonstrated on protein and at the RNA level over the entire culture period. CONCLUSION: We observed a positive effect of hydroxyapatite ceramic granules on mesenchymal cambial-layer precursor cell behavior in cell-seeded 3-dimensional bone constructs, indicating the potential applicability of C GRAFT/Algipore composites in bone tissue engineering.

Blotting, Western↗

Maxilla sinus grafting with marine algae derived bone forming material: a clinical report of long-term results.

PURPOSE: Autogenous bone grafting continues to be considered the gold standard for sinus grafting. For the past 15 years the author has used an alternative graft material and followed-up the input/output statistic of implants to evaluate if this material results similar to the autogenous bone graft. Histomorphometric evaluations of graft materials show how much new bone is formed and if the graft material is resorbed. MATERIALS AND METHODS: In our study we used a marine derived carbonated red algae that is chemically converted into hydroxyapatite (HA). This material is distributed worldwide as the Communauté Européenne approved material AlgiPore (Dentsply Friadent, Mannheim, Germany), as the US Food and Drug Administration approved material C GRAFT (The Clinician Preference LLC, Golden, CO), and the Russian approved material AlgOss (Unexim Co, Moscow, Russia). A total of 209 sinus grafts were performed on 118 patients who presented with a severely resorbed maxillary alveolar process with 1 to 5 mm (mean, 3.6 mm) of remaining bone. The available bone was comparable to Class D bone as described by Simion et al. After 6 months implants were placed and 6 months later the implants were loaded. RESULTS: From September 5, 1990, to September 1, 2004, the author performed 209 sinus grafts on 118 patients. The longest observation period of loaded implants in this study is 156 months (13 years). Implant loss was 27 out of 614 loaded implants (4.4%), showing a survival rate of 95.6%. Smokers and women over 50 are included. Although AlgiPore/C GRAFT/AlgOss (ACA) undergoes a resorption process, we found only 14% volume loss after 6.4 months compared with 49.5% after 6 months when autogenous bone was used. CONCLUSION: This retrospective study of over 14 years shows once again that the sinus lift procedure with grafting of the sinus floor and subsequent implant placement is a proven method. This 14-year longitudinal study shows that the marine derived HA material ACA in a mixture with approximately 10% autogenous collector bone and blood or platelet rich plasma is able to enhance enough new bone in 6 months to allow implant osseointegration after 6 more months with a high implant survival rate.

Absorbable Implants↗

Effects of solar ultraviolet radiation on photosynthesis of the marine red tide alga Heterosigma akashiwo (Raphidophyceae).

In order to assess the short- and long-term impacts of UV radiation (UVR, 280-400nm) on the red tide alga, Heterosigma akashiwo, we exposed the cells to three different solar radiation treatments (PAB: 280-700nm, PA: 320-700nm, P: 400-700nm) under both solar and artificial radiation. A significant decrease in the effective quantum yield (Y) during high irradiance periods (i.e., local noon) was observed, but the cells partially recovered during the evening hours. Exposure to high irradiances for 15, 30, and 60min under a solar simulator followed by the recovery (8h) under dark, 9 and 100micromolphotonsm(-2)s(-1) of PAR, highlighted the importance of the irradiance level during the recovery period. Regardless the radiation treatments, the highest recovery (both in rate and total Y) was found at a PAR irradiance of 9micromolphotonsm(-2)s(-1), while the lowest was observed at 100micromolphotonsm(-2)s(-1). In all experiments, PAR was responsible for most of the observed inhibition; nevertheless, the cells exposed only to PAR had the highest recovery in any condition, as compared to the other radiation treatments. In long-term experiments (10 days) using semi-continuous cultures, there was a significant increase of UV-absorbing compounds (UV(abc)) per cell from 1.2 to >4x10(-6)microgUV(abc)cell(-1) during the first 3-5 days of exposure to solar radiation. The highest concentration of UV(abc) was found in samples exposed in the PAB as compared to PA and P treatments. Growth rates (mu) mimic the behavior of UV-absorbing compounds, and during the first 5 days mu increased from <0.2 to ca. 0.8, and stayed relatively constant at this value during the rest of the experiment. The inhibition of the Y decreased with increasing acclimation of cells. All our data indicates that H. akashiwo is a sensitive species, but was able acclimate relatively fast (3-5 days) synthesizing UV-absorbing compounds and thus reducing any impact either on photosystem II or on growth.

Chlorophyll↗

In vitro assay and light regulation of nitrate reductase in red alga Gracilaria chilensis.

Nitrate reductase (NR) is the first enzyme in the nitrogen assimilation pathway. The in vitro NR activity of Gracilaria chilensis was assayed under different conditions to reveal its stability and biochemical characteristics, and an optimized in vitro assay is described. Maximal NR activities were observed at pH 8.0 and 15 degrees C. The apparent Km value for NADH was 8 microM and for nitrate 680 microM. Crude extracts of G. chilensis stored at 4 degrees C showed a 50% decrease of NR activity after 24 h. The highest NR activity value (253.20+/-2.60 x 10(-3) U g(-1)) was obtained when 100% von Stosch medium (500 microM NO3-) was added before extraction of apical parts. Algae under light:dark cycles of 12:12h exhibited circadian fluctuation of NR activity and photosynthesis with more than 2 times higher levels in the light phase. No evidence of endogenous diel rhythm controlling NR activity or photosynthesis was observed. Light pulses lasting 10 or 60 min during the darkness increased the NR activity by 30% and 45%, respectively. The results indicate that NR and photosynthesis are regulated mainly by light and not by a biological clock.

Circadian Rhythm↗

Antioxidant properties of a novel phycocyanin extract from the blue-green alga Aphanizomenon flos-aquae.

Aphanizomenon flos-aquae (AFA) is a fresh water unicellular blue-green alga (cyanophyta) rich in phycocyanin (PC), a photosynthetic pigment with antioxidant and anti-inflammatory properties. The purpose of this study was to evaluate the ability of a novel natural extract from AFA enriched with PC to protect normal human erythrocytes and plasma samples against oxidative damage in vitro. In red blood cells, oxidative hemolysis and lipid peroxidation induced by the aqueous peroxyl radical generator [2,2'-Azobis (2-amidinopropane) dihydrochloride, AAPH] were significantly lowered by the AFA extract in a time- and dose-dependent manner; at the same time, the depletion of cytosolic glutathione was delayed. In plasma samples, the natural extract inhibited the extent of lipid oxidation induced by the pro-oxidant agent cupric chloride (CuCl2); a concomitant increase of plasma resistance to oxidation was observed as evaluated by conjugated diene formation. The involvement of PC in the antioxidant protection of the AFA extract against the oxidative damage was demonstrated by investigating the spectral changes of PC induced by AAPH or CuCl2. The incubation of the extract with the oxidizing agents led to a significant decrease in the absorption of PC at 620 nm accompanied with disappearance of its blue color, thus indicating a rapid oxidation of the protein. In the light of these in vitro results, the potential clinical applications of this natural compound are under investigation.

Amidines↗

Improving glycogenesis in Streptozocin (STZ) diabetic mice after administration of green algae Chlorella.

Chlorella, a type of unicellular fresh water algae, has been a popular foodstuff in Japan and Taiwan. Studies have shown the hypoglycemic effects of Chlorella in alloxan-induced and Streptozocin (STZ)-induced diabetic animals. However, the mechanisms by which Chlorella treatment affects blood glucose homeostasis have not been studied. Diabetes in ICR mice was induced by injection of STZ. Lipogenesis in vivo was measured by incorporating 3H-H2O into lipids in brown and white adipose tissues. Glucose uptake in the liver and soleus muscles was measured by assaying 2-deoxy-D-[1,2-3H] glucose levels. The effects of Chlorella on serum non-esterified fatty acids (NEFA) were measured with commercial assay kits. Insulin-stimulated lipogenic rates in brown and white adipose tissues were unaffected by Chlorella. However, Chlorella increased 2-deoxyglucose uptake in the livers and soleus muscles in normal and STZ mice compared to that in their respective controls (p < 0.01). In addition, fasting NEFA levels were lower in Chlorella-treated STZ mice compared to H2O-treated STZ mice (p < 0.005). The current results suggest that the hypoglycemic effects of Chlorella are due to an enhancement of glucose uptake in the liver and in soleus muscles. The improved insulin sensitivity after Chlorella treatment could be also due to lower NEFA levels, since insulin sensitivity is usually blunted by elevated NEFA in diabetes.

Adipose Tissue↗

Synergistic effects of diuron and sedimentation on photosynthesis and survival of crustose coralline algae.

Effects of short-term exposure to sedimentation and diuron, separately and in combination, on the photophysiology and survival of crustose coralline algae (CCA) were examined in controlled time-course experiments, using pulse-amplitude modulation (PAM) chlorophyll fluorometry. These experiments indicated that the effects of sediments and diuron, when applied in isolation, were often reversible, with recovery time dependent upon sediment type and diuron concentration. Exposure to fine (<63 microm grain size), nutrient-rich estuarine sediments reduced effective quantum yields (Delta F/F(m')) of photosystem II in CCA species more than exposure to the same amount of fine (<63 microm grain size) calcareous sediments. Significant inhibition of photosynthesis (Delta F/F(m')) was also observed at diuron concentrations > or =2.9 microg L(-1). Fine estuarine sediments in combination with 0.79 microg L(-1) dissolved diuron, caused yields (Delta F/F(m')) to drop by 60% compared with controls after 24 h. The combined exposure to sediments and diuron also retarded recovery, thus Delta F/F(m') values were still only 60% of the controls after 9 days recovery in clean seawater. Mortality of CCA was observed in some fragments treated with combinations of sediment and diuron. Our results suggest that sediment deposition and exposure to diuron can negatively affect the photosynthetic activity of CCA, with sedimentation stress being significantly enhanced by the presence of trace concentrations of diuron.

Animals↗

Comparative effects of the blue green algae Nodularia spumigena and a lysed extract on detoxification and antioxidant enzymes in the green lipped mussel (Perna viridis).

Nodularia spumigena periodically proliferates to cause toxic algal blooms with some aquatic animals enduring and consuming high densities of the blue green algae or toxic lysis. N. spumigena contains toxic compounds such as nodularin and lipopolysaccharides. This current work investigates physiological effects of exposure from bloom conditions of N. spumigena cells and a post-bloom lysis. Biochemical and antioxidative biomarkers were comparatively studied over an acute 3-day exposure. In general, a post-bloom N. spumigena lysis caused opposite physiological responses to bloom densities of N. spumigena. Specifically, increases in glutathione (GSH) and glutathione peroxidase (GPx) and decreases in glutathione S-transferase (GST) were observed from the N. spumigena lysis. In contrast, N. spumigena cell densities decreased GSH and increased GST and lipid peroxidation (LPO) in mussels. Findings also suggest that at different stages of a toxic bloom, exposure may result in toxic stress to specific organs in the mussel.

Analysis of Variance↗

Therapeutic potentials of unicellular green alga Chlorella in advanced glycation end product (AGE)-related disorders.

Reducing sugars can react non-enzymatically with amino groups of protein to form Amadori products. These early glycation products undergo further complex reaction such as rearrangement, dehydration, and condensation to become irreversibly cross-linked, heterogeneous fluorescent derivatives, termed advanced glycation end products (AGEs). The formation and accumulation of AGEs in various tissues has been known to progress at an accelerated rate under hyperglycemic conditions in diabetes. Recent understanding of this process has revealed that AGEs have been implicated in the development of many of the pathological sequelae of diabetes and aging, such as atherosclerosis and diabetic microangiopathy. Furthermore, recently, AGE-their receptor (RAGE) interaction was also involved in neurodegenerative diseases, melanoma growth, expansion and metastasis. These observations suggest that blockade of AGE formation may be a novel promising target for therapeutic intervention in these devastating AGE-related disorders. We have recently found that unicellular green alga Chlorella inhibited the formation of AGEs in vitro. Since several lines of evidence have shown anti-atherogenic effects of Chlorella on animal models, we hypothesize here that the beneficial aspects of Chlorella on atherosclerosis could be ascribed, at least in part, to its AGE inhibitory property and that Chlorella may have therapeutic potentials in treatment of patients with other AGE-related disorders such as diabetic microangiopathy and Alzheimer's disease. In this paper, we would like to propose the possible ways of testing our hypotheses. Does daily intake of Chlorella reduce the risk of the incidence and progression of diabetic vascular complications including atherosclerosis? Does Chlorella treatment prevent the development of Alzheimer's disease and/or improve the cognitive impairment of patients with this disorder? If the answers are yes, are plasma or tissue levels of AGE in these patients actually suppressed by Chlorella treatment? And, does the extent of the AGE reduction by Chlorella predict the beneficial effects of Chlorella on these disorders? These prospective studies will provide further valuable information whether blockade by Chlorella of the AGE formation could be clinically relevant.

Administration, Oral↗

A CARD-FISH protocol for the identification and enumeration of epiphytic bacteria on marine algae.

A CARD-FISH protocol was developed and applied to analyse surface-associated bacteria on the marine algae Ulva lactuca, Delisea pulchra, Corallina officinalis, Amphiroa anceps, Porphyra sp. and Sargassum linearifolium. The combination of Alexa(546)-labelled tyramide as the reporter molecule with SYBR Green II counterstain allowed for superior detection of the hybridised probe fluorescence against plant tissue from which pigment autofluorescence has been reduced.

Bacteria↗