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PubMed2005

Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels.

Neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin-2 (ChR2). Here, we quantitatively investigated the rapidness of the light-gated current of ChR2 expressed in PC12 cells using blue light-emitting diode (LED) light. The light-gated current consists of two components, inactivating and non-inactivating. The magnitude of inactivating component was almost linearly related to the light intensity. The non-inactivating component showed a tendency to saturate at high illumination. Both the activation and inactivation rates of the light-gated current were linearly dependent on the light intensity. However, the activation rate (turning-on rate) is about 10-fold faster than the inactivation rate. Although the turning-off time constant was little dependent on the light intensity, that at the end of 1s light pulse was about two-fold larger than that at 20 ms. Neurons are also made photosensitive by the expression of ChR2 in the living animal. Since both the turning-on and turning-off time constants of light-gated current was smaller than the membrane time constant of neurons, the LED light illumination of the photosensitive neurons was enough to evoke action potentials in a pulse-to-pulse manner in an acute slice of hippocampus.

Algorithms↗
PubMed2004

Prospects for molecular farming in the green alga Chlamydomonas.

Protein-based therapeutics have enjoyed great success over the past decade. Unfortunately, this clinical success has come with a heavy price tag that is due to the inherently high costs of the capitalization and production of these complex molecules using current mammalian-based fermentation systems. Recent progress has been made in the production of recombinant proteins, including antibodies, in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. C. reinhardtii offers an attractive alternative to traditional mammalian-based expression systems for several reasons, including its ability to provide stable plastid and nuclear transformants rapidly and its inherently low costs for capitalization and production.

Algal Proteins↗
PubMed2006

Method for large-scale isolation and purification of R-phycoerythrin from red alga Polysiphonia urceolata Grev.

R-phycoerythrin was isolated and purified from a red alga, Polysiphonia urceolata Grev, using Streamline column combined with ion-exchange chromatography or hydroxyapatite chromatography. The purity of R-phycoerythrin isolated by Streamline column was up to 1.66 and the yield of R-phycoerythrin could be as high as 0.68 mg/g frozen P. urceolata. All the eluates from Streamline column were divided into two equivalent parts, respectively. One part was pumped into the ion-exchange column loaded with Q-Sepharose and the other was applied to the adsorption column loaded with hydroxyapatite. The purities of R-phycoerythrin purified using these two methods were both up to 3.26, more than 3.2 the commonly accepted criterion. The yield of purified R-phycoerythrin from the ion-exchange chromatography was 0.40 mg/g frozen P. urceolata and that from the hydroxyapatite chromatography could reach 0.34 mg/g frozen P. urceolata. The purified protein had three absorption peaks at 498, 535, and 565 nm and displayed a fluorescence maximum at 580 nm, which was consistent with the typical spectrum of R-phycoerythrin. The purified R-PE was also identified with electrophoresis. Only one single protein band appeared on native-PAGE with silver staining. SDS-PAGE demonstrated the presence of one 20 kDa major subunit, and one low intensity band corresponding to 33 kDa subunit. The results indicate that using the expanded bed adsorption combined with ion-exchange chromatography or hydroxyapatite chromatography, R-phycoerythrin can be purified from frozen P. urceolata on large scale.

Ammonium Sulfate↗
PubMed2006

Antiviral property and mechanisms of a sulphated polysaccharide from the brown alga Sargassum patens against Herpes simplex virus type 1.

A sulphated polysaccharide (SP-2a) from the brown alga Sargassum patens (Kütz.) Agardh (Sargassaceae) was found to significantly inhibit the in vitro replication of both the acyclovir (ACV)-sensitive and -resistant strains of Herpes simplex virus type 1 (HSV-1), in dose-dependent manners, with 50% inhibitions occurring with 1.5-5.3 microg/ml of the polysaccharide. SP-2a exhibited extracellular virucidal activity only against the ACV-sensitive strains, but not the resistant strain, at the concentration of 100 microg/ml. The strongest antiviral activities against the different strains of HSV-1 were observed when this polysaccharide was present during and after adsorption of the virus to host cells. The inhibitory effect of SP-2a on virus adsorption occurred dose-dependently in all the HSV-1 strains tested, and the adsorption of the ACV-resistant DM2.1 strain was reduced by 81.9% (relative to control) with 4 microg/ml of the polysaccharide. This study clearly demonstrated that the antiviral mode of action of SP-2a is mediated mainly by inhibiting virus attachment to host cells, and this sulphated polysaccharide might have different modes of action against the ACV-sensitive and -resistant strains of HSV-1.

Acyclovir↗
PubMed2004

Trihydroxylated linear diterpenes from the brown alga Bifurcaria bifurcata.

Two novel polar diterpenes were isolated from the brown alga Bifurcaria bifurcata collected off the Atlantic coast of Morocco, and their structures established by spectral methods. Both compounds are trihydroxylated acyclic diterpenes derived from 12-hydroxygeranylgeraniol. They were tested in vitro for their cytotoxicity and proved to be active against the NSCLC-N6 cell line. Their absolute configuration at the C-12 position has been determined with a modified Mosher's method [J. Am. Chem. Soc. 113 (1991) 4092] and that of the 12-hydroxygeranylgeraniol (bifurcadiol) has been revised.

Cytotoxicity Tests, Immunologic↗
PubMed2004

Halogenated diterpenoids from the red alga Laurencia nipponica.

Chemical compositions of three collections of the red alga Laurencia nipponica from the western part of the Sea of Japan were studied. One of them contained a series of the previously known sesquiterpenoids. Another one gave C15 bromoallene ethers, predominantly. Finally, two new halogenated diterpenes, 15-bromoparguer-9(11)-ene-16-ol and 15-bromoparguer-7-ene-16-ol, were isolated from the third collection of the same species. Structures of these diterpenoids were established by 1D and 2D NMR (1H-1H COSY, DEPT, HMQC, HMBC and NOESY) along with molecular calculations for conformations having lowest energies and mass spectroscopy. Diversity and variability of halogenated secondary metabolites in L. nipponica were discussed.

Diterpenes↗
PubMed2005

Plocoralides A-C, polyhalogenated monoterpenes from the marine alga Plocamium corallorhiza.

Three new polyhalogenated monoterpenes, plocoralides A-C (1-3) along with three known compounds (4-6) have been isolated from the organic extract of the red alga P. corallorhiza. Structures of the new compounds were characterized as 4,8-dibromo-1,1-dichloro-3,7-dimethyl-2E,6E-octadiene (1), 4,6-dibromo-1,1-dichloro-3,7-dimethyl-2E,7-octadiene (2) and 4,8-dibromo-1,1,7-trichloro-3,7-dimethyl-2E,5Z-octadiene (3) on the basis of one- and two-dimensional NMR spectroscopic data and MS analyses. Compounds 2-6 show moderate cytotoxicity toward esophageal cancer cells.

Antineoplastic Agents↗
PubMed2006

Aldingenin derivatives from the red alga Laurencia aldingensis.

Three brominated bisabolene-type sesquiterpene derivatives, aldingenin B, C and D, together with cholesterol and palmitic acid, have been isolated from the red alga Laurencia aldingensis (Ceramiales, Rodophyta) and their structures elucidated by spectroscopic methods including NMR analysis.

Laurencia↗
PubMed2005

Trends in biomass and metal sequestration associated with reeds and algae at Wheal Jane Biorem pilot passive treatment plant.

An assessment of the rate of biomass production both of the reeds in the aerobic cells and the algae in the rock filters, which form the final stage in the series of treatment cells, has been undertaken. The biomass production for the reeds was found to be highest for the lime-dosed anoxic limestone drain system, but even this was very low in comparison to values reported for natural and constructed wetlands. The algal coverage of each lagoon was relatively homogeneous, with no significant difference between the three systems studied. However, too many unknown factors suggest that further study is required. The metal uptake was higher in the roots than the stems, although no variation between cells of systems was detectable, and the difference was not as marked as reported by other workers. The metal concentrations in the debris samples were markedly higher than the roots of the reeds. The values for Fe, Al and As were several orders of magnitude larger than the influent minewater. Further study is required here, but this appears to be a key component in the function of the reeds. The lime-dosed system rock filter showed the highest Fe removal rate but the lowest Mn removal rate. Some possible mechanisms are discussed in the paper, but further investigation would be required to test these hypotheses.

Aerobiosis↗
PubMed2005

Trends in biomass and metal sequestration associated with reeds and algae at Wheal Jane Biorem pilot passive treatment plant.

An assessment of the rate of biomass production both of the reeds in the aerobic cells and the algae in the rock filters, which form the final stage in the series of treatment cells, has been undertaken. The biomass production for the reeds was found to be highest for the lime-dosed anoxic limestone drain system, but even this was very low in comparison to values reported for natural and constructed wetlands. The algal coverage of each lagoon was relatively homogeneous, with no significant difference between the three systems studied. However, too many unknown factors suggest that further study is required. The metal uptake was higher in the roots than the stems, although no variation between cells of systems was detectable, and the difference was not as marked as reported by other workers. The metal concentrations in the debris samples were markedly higher than the roots of the reeds. The values for Fe, Al and As were several orders of magnitude larger than the influent minewater. Further study is required here, but this appears to be a key component in the function of the reeds. The lime-dosed system rock filter showed the highest Fe removal rate but the lowest Mn removal rate. Some possible mechanisms are discussed in the paper, but further investigation would be required to test these hypotheses.

Aerobiosis↗
PubMed2005

Expression of human antibodies in eukaryotic micro-algae.

Protein based therapeutics have enjoyed great success over the past decade. Unfortunately, with this clinical success comes a heavy price tag, owing to the inherently high costs of capitalization and production using mammalian cell fermentation. To address this problem, we have begun developing a system for the expression of recombinant proteins in the unicellular eukaryotic green algae, Chlamydomonas reinhardtii, leading to the production of human IgA single chain antibodies. The expression of human monoclonal antibodies in C. reinhardtii offers an attractive alternative to traditional mammalian based expression systems for several reasons, including an ability to rapidly obtain stable plastid and nuclear transformants, coupled with inherently low costs of capitalization and production.

Antibodies↗
PubMed2004

Genome sequence and characterization of a virus (HaRNAV) related to picorna-like viruses that infects the marine toxic bloom-forming alga Heterosigma akashiwo.

Heterosigma akashiwo (Rhaphidophyceae) is a unicellular, flagellated, bloom-forming, toxic alga of ecological and economic importance. Here, we report the results of sequencing and analyzing the genome of an 8.6-kb single-stranded RNA virus (HaRNAV-SOG263) that infects H. akashiwo. Our results show that HaRNAV is related to picorna-like viruses, but does not belong within any currently defined virus family. This is based on the genome organization and sequence comparisons of putative RNA-dependent RNA polymerase (RdRp), helicase, and capsid protein sequences. The genome sequence predicts a single open reading frame (orf) encoding a polyprotein that contains conserved picorna-like protein domains, with putative nonstructural protein domains present in the N-terminus and the structural proteins in the C-terminus of the polyprotein. We have analyzed and compared the virus structural proteins from infectious and noninfectious particles. In this way, we identified structural protein cleavage sites as well as protein processing events that are presumably important for maturation of virus particles. The combination of genome structure and sequence relationships to other viruses suggests that HaRNAV is the first member of a proposed new virus family (Marnaviridae), related to picorna-like viruses.

Amino Acid Sequence↗
PubMed2004

Nitrogen mass balance across pilot-scale algae and duckweed-based wastewater stabilisation ponds.

Nitrogen removal processes and nitrogen mass balances in algae-based ponds (ABPs) and duckweed (Lemna gibba)-based ponds (DBPs) were assessed during periods of 4 months, each under different operational conditions. During periods 1 and 2, the effect of cold and warm temperature was studied. During periods 2 and 3, the effect of low- and high-system organic loading (OL) was studied in warm seasons operation. The pilot-scale systems consisted of four similar ponds in series fed with domestic sewage with hydraulic retention time of 7 days in each pond. Overall nitrogen removal was higher during warm temperature in both ABPs and DBPs, but similar during periods 2 and 3. Nitrogen removal in DBPs was lower than in ABPs by 20%, 12% and 8% during cold temperature, warm temperature and high-OL periods, respectively. Depending on temperature and OL rate, ABPs showed higher nitrogen removal via sedimentation (46-245% higher) compared to DBPs. Also, ABPs also showed higher nitrogen removal via denitrification (7-37% higher) compared to DBPs. Ammonia volatilisation in both systems did not exceed 1.1% of influent total nitrogen during the entire experimental period. N uptake by duckweed corresponds to 30% of the influent nitrogen during warm/low OL period and decreased to 10% and 19% during the cold and warm/high OL period, respectively. Predictive models for nitrogen removal presented a good reflection of nitrogen fluxes on overall nitrogen balance under the prevailing experimental conditions.

Araceae↗
PubMed2005

Equilibrium and kinetic modelling of Cd(II) biosorption by algae Gelidium and agar extraction algal waste.

In this study an industrial algal waste from agar extraction has been used as an inexpensive and effective biosorbent for cadmium (II) removal from aqueous solutions. This biosorbent was compared with the algae Gelidium itself, which is the raw material for agar extraction. Equilibrium data follow both Langmuir and Redlich-Peterson models. The parameters of Langmuir equilibrium model are q(max)=18.0 mgg(-1), b=0.19 mgl(-1) and q(max)=9.7 mgg(-1), b=0.16 mgl(-1), respectively for Gelidium and the algal waste. Kinetic experiments were conducted at initial Cd(II) concentrations in the range 6-91 mgl(-1). Data were fitted to pseudo-first- and second-order Lagergren models. For an initial Cd(II) concentration of 91 mgl(-1) the parameters of the pseudo-first-order Lagergren model are k(1,ads)=0.17 and 0.87 min(-1); q(eq)=16.3 and 8.7 mgg(-1), respectively, for Gelidium and algal waste. Kinetic constants vary with the initial metal concentration. The adsorptive behaviour of biosorbent particles was modelled using a batch reactor mass transfer kinetic model. The model successfully predicts Cd(II) concentration profiles and provides significant insights on the biosorbents performance. The homogeneous diffusivity, D(h), is in the range 0.5-2.2 x10(-8) and 2.1-10.4 x10(-8)cm(2)s(-1), respectively, for Gelidium and algal waste.

Adsorption↗
PubMed2006

Rapid ecotoxicological bioassay using delayed fluorescence in the green alga Pseudokirchneriella subcapitata.

A rapid and simple ecotoxicological bioassay allows quick estimation of the effects of Simazine (CAT) or 3,5-dichlorophenol (3,5-DCP) on the growth of the green alga Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum). The effects of a 15-min exposure to CAT or 3,5-DCP on delayed fluorescence (DF) in P. subcapitata were compared to the results of standard growth inhibition tests involving 72h of exposure to these chemicals at the same concentrations. Integrated DF intensity in the time period from 0.6 to 50s was found to correlate with algal growth inhibition as measured by the standard tests. In addition, the behaviour of DF in this time period exhibited characteristic kinetics indicative of the chemical being tested.

Biological Assay↗
PubMed2026

Investigating overflow metabolism in heterotrophic cultures of the green alga Chromochloris zofingiensis.

Chromochloris zofingiensis is of interest for its ability to perform a reversible trophic switch in the presence of glucose that is characterized by a shutdown of photosynthesis and an accumulation of energy storage metabolites. Previous work has shown that this trophic switch is accompanied by overflow metabolism and the production of lactate in aerobic conditions. This trophic switch is not observed in nutrient replete media. We utilized isotopically assisted metabolic flux analysis to characterize intracellular flux distributions that are associated with different metabolic phenotypes observed in this organism in different media formulations in light and dark conditions. The results of this analysis showed that low iron cultures have no flux through carbon fixation reactions, and that the carbon flux entering the TCA cycle in these cultures is approximately 40 % lower than that in iron replete cultures grown heterotrophically. This analysis was complemented with transcriptomics data collected for C. zofingiensis grown in iron limited conditions to provide further evidence towards the negative impact of iron limitation on both photosynthetic and respiratory activity. Overflow metabolism allows this alga to compensate for the lower energy production that results from iron limitation. This work highlights how nutrient availability can lead to changes in the metabolism of C. zofingiensis.

Iron↗
PubMed2002

Polyenoic fatty acid isomerase from the marine alga Ptilota filicina: protein characterization and functional expression of the cloned cDNA.

The recently described enzyme, polyenoic fatty acid isomerase (PFI), from the marine alga Ptilota filicina J. Argardh has been analyzed with respect to its protein structure and an associated cofactor. The enzyme was purified to homogeneity (as judged by SDS-PAGE and silver staining). By sedimentation equilibrium ultracentrifugation the mass of the native enzyme was estimated to be 125 kDa. The N-terminal peptide sequence derived from this protein was used to isolate two very similar cDNA clones encoding novel 500-amino acid proteins, both with calculated molecular masses of 55.9 kDa and pIs of 4.87. The data predict translation of a preprotein containing a signal peptide of 21 amino acids that is removed during maturation. Deglycosylation assays demonstrate that native PFI from P. filicina is a glycoprotein. The purified protein is chromophoric with a flavin-like UV spectrum and sequence analysis reveals the presence of a flavin-binding motif near the mature N-terminus. Heterologous expression of active PFI in Arabidopsis, using one of the cDNA clones, was successful as evidenced by conversion of arachidonic acid to a conjugated triene in an in vitro assay of the transgenic plant tissues.

Amino Acid Sequence↗
PubMed2003

Isolation of two novel mannan- and L-fucose-binding lectins from the green alga Enteromorpha prolifera: biochemical characterization of EPL-2.

EPL-1 and EPL-2 represent lectins isolated from the green alga Enteromorpha prolifera. Both lectins are 20- to 22-kDa single-chain, nonglycosylated proteins. N-terminal sequence analysis of peptides representing over 70% of their primary structures shows that EPL-1 and EPL-2 represent novel proteins. Sedimentation-diffusion equilibrium experiments showed that EPL-1 and EPL-2 had average apparent molecular masses of 60000+/-6000 Da (EPL-1) and 59500+/-3000 Da (EPL-2), indicating that EPL-1 and EPL-2 have a tendency to self-associate into higher order aggregates, possibly homodimers and homotetramers, in equilibrium. The carbohydrate-binding specificity of EPL-2 was studied by enzyme-linked lectin assay and intrinsic fluorescence measurements. The results show that the combining site of EPL-2 is capable of accommodating both D-mannose and L-fucose, which share the conformation of the hydroxyl groups at positions 2 (axial) and 4 (equatorial), and includes subsites for the substituents at O1 and for branched mannose residues.

Amino Acid Sequence↗