Osteogenic differentiation of human amniotic mesenchymal stem cells by phycocyanin and phycoerythrin pigments isolated from Spirulina platensis and Gracilaria gracilis algae.

Bone regeneration is a multistep and regular physiological process that occurs normally in fracture repair and bone defects. However, some factors such as aging, particular diseases and some drugs prevent or slowdown bone natural healing. Cell therapy using stem cells and differentiation activating factors is an effective treatment method for bone regeneration triggering in unusual conditions. Therefore, in the present study the effect of phycocyanin and phycoerythrin pigments which isolated from Spirulina platensis and Gracilaria gracilis algae was investigate on osteogenic differentiation potency of human Amniotic Mesenchymal Stem Cells (hAMSCs). For this purpose, hAMSCs were exposed to 300, 500, and 700 µg/ml concentrations of phycocyanin and phycoerythrin pigments and then the cells viability was measured with MTT assay in 48 and 72 h after treatment. The osteo-differentiation level of cells was studied by measuring ALP activity using calorimetric method and Alizarin red staining for calcium deposition in 7 and 21 days after treatment. Also, total RNA of cells was extracted in different time periods and then cDNA synthesized with specific primers, and relative expression of Runx2, ß-catenin and Osteocalcin genes were investigated using SYBR Green RT-qPCR technique. Osteogenic differentiation of hAMSCs that treated with pigments was confirmed by mineral deposits staining and increased level of ALP activity. Furthermore, these pigments elevated significantly the expression of osteogenic marker genes compared to control samples and caused hAMSCs to differentiate into osteoblast cells. According to these results, phycocyanin and phycoerythrin may suggest as suitable osteogenic supplements with low toxicity, low cost and high efficiency, although the molecular mechanism of its efficacy is not available yet.

Mechanisms of action of non-enzymatic antioxidants to control oxidative stress during in vitro follicle growth, oocyte maturation, and embryo development.

In vitro follicle growth and oocyte maturation still has a series of limitations, since not all oocytes matured in vitro have the potential to develop in viable embryos. One of the factors associated with low oocyte quality is the generation of reactive oxygen species (ROS) during in vitro culture. Therefore, this review aims to discuss the role of non-enzymatic antioxidants in the control of oxidative stress during in vitro follicular growth, oocyte maturation and embryonic development. A wide variety of non-enzymatic antioxidants (melatonin, resveratrol, L-ascorbic acid, L-carnitine, N-acetyl-cysteine, cysteamine, quercetin, nobiletin, lycopene, acteoside, mogroside V, phycocyanin and laminarin) have been used to supplement culture media. Some of them, like N-acetyl-cysteine, cysteamine, nobiletin and quercetin act by increasing the levels of glutathione (GSH), while melatonin and resveratrol increase the expression of antioxidant enzymes and minimize oocyte oxidative stress. L-ascorbic acid reduces free radicals and reactive oxygen species. Lycopene positively regulates the expression of many antioxidant genes. Additionally, L-carnitine protects DNA against ROS-induced damage, while acteoside and laminarin reduces the expression of proapoptotic genes. Mogrosides increases mitochondrial function and reduces intracellular ROS levels, phycocyanin reduces lipid peroxidation, and lycopene neutralizes the adverse effects of ROS. Thus, it is very important to know their mechanisms of actions, because the combination of two or more antioxidants with different activities has great potential to improve in vitro culture systems.

Phycocyanin improved alcohol-induced hepatorenal toxicity and behavior impairment in Wistar rats.

The aim of this study was to investigate a potential preventive effect of phycocyanin extract from Spirulina platensis against ethanol- induced hepatorenal toxicity and cognitive behavior impairment in male Wistar rats. The animals were randomly and equally divided into four groups (six animals each): control group received saline solution, ethanol (EtOH) group was injected intraperitoneally with 1 ml/kg of ethanol solution 38% (w/v), phycocyanin groups were treated with 25 (PC1) or 50 (PC2) mg/kg phycocyanin extract followed by ethanol administration. All treatments were conducted for 14 successive days. Results revealed that ethanol induced oxidative stress in brain, liver, and kidney by increasing lipid peroxidation level and SOD and CAT activities. Serum biochemical perturbations were also observed in EtOH group, which was indicated by a significant elevation in ALT, AST, cholesterol, triglycerides, creatinine, and urea levels. Combined exposure to EtOH with phytocyanin contracted these biochemical alterations. Phycocyanin decreased also EtOH-induced anxiety and ameliorated exploratory behavior assessed by the elevated-plus maze and open field tests respectively.

Combination of Phycocyanin, Zinc, and Selenium Improves Survival Rate and Inflammation in the Lipopolysaccharide-Galactosamine Mouse Model.

Sepsis is related to systemic inflammation and oxidative stress, the primary causes of death in intensive care units. Severe functional abnormalities in numerous organs can arise due to sepsis, with acute lung damage being the most common and significant morbidity. Spirulina, blue-green algae with high protein, vitamins, phycocyanin, and antioxidant content, shows anti-inflammatory properties by decreasing the release of cytokines. In addition, zinc (Zn) and selenium (Se) act as an antioxidant by inhibiting the oxidation of macromolecules, as well as the inhibition of the inflammatory response. The current study aimed to examine the combined properties of Zn, Se, and phycocyanin oligopeptides (ZnSePO) against lipopolysaccharide-D-galactosamine (LPS-GalN)-induced septic lung injury through survival rate, inflammatory, and histopathological changes in Balb/c mice. A total of 30 mice were allocated into three groups: normal control, LPS-GalN (100 ng of LPS plus 8 mg of D-galactosamine), LPS-GalN + ZnSePO (ZnPic, 52.5 µg/mL; SeMet, 0.02 µg/mL; and phycocyanin oligopeptide (PO), 2.00 mg/mL; at 1 h before the injection of LPS-GalN). Lung tissue from mice revealed noticeable inflammatory reactions and typical interstitial fibrosis after the LPS-GalN challenge. LPS-GalN-induced increased mortality rate and levels of IL-1, IL-6, IL-10, TGF-ß, TNF-α, and NF-κB in lung tissue. Moreover, treatment of septic mice LPS-GalN + ZnSePO reduced mortality rates and inflammatory responses. ZnSePO considerably influenced tissue cytokine levels, contributing to its capacity to minimize acute lung injury (ALI) and pulmonary inflammation and prevent pulmonary edema formation in LPS-GalN-injected mice. In conclusion, ZnSePO treatment enhanced the survival rate of endotoxemia mice via improving inflammation and oxidative stress, indicating a possible therapeutic effect for patients with septic infections.

Iron Content, Iron Speciation and Phycocyanin in Commercial Samples of Arthrospira spp.

Cyanobacteria are characterized by high iron content. In this research, we collected ten commercial samples of Arthrospira spp. sold as food supplement to determine iron content and assess whether iron speciation showed variability among samples and changed respect to A. platensis grown in controlled conditions. Particular attention was also paid to phycocyanin, as an iron-binding protein. In six of the ten samples, 14 essential and non-essential trace elements were analysed using ICP-MS. Iron content measured in samples using atomic absorption spectrometry (AAS) varied from 353 (sample S5) to 1459 (sample S7) µg g-1 dry weight and was in the range of those reported by other authors in commercial supplements. Iron speciation was studied using size exclusion chromatography followed by the analysis of the collected fraction for the determination of iron by AAS and for protein separation using SDS-PAGE. Overlapping chromatographic profiles were obtained for total proteins, phycocyanin and iron, although quantitative differences were evidenced among the samples analysed. In most samples, iron was mainly bound to ligands with high molecular mass; however, in four samples iron was also bound to ligands with low molecular mass. In fractions containing the most relevant iron burden, the principal protein was phycocyanin, confirming its role as an iron-binding protein in commercial samples.

Food Matrices Affect the Peptides Produced during the Digestion of Arthrospira platensis-Based Functional Aliments.

Arthrospira platensis (Spirulina) has been credited with multiple beneficial effects, many of which are attributed to bioactive peptides produced during the gastrointestinal digestion of this micro-alga. Many Spirulina-based nutraceuticals have been produced, and numerous functional foods enriched with Spirulina are available on the market. These are subjected to checks aimed at verifying the amount of algae actually present, but few studies relating to the bioavailability of the bioactive compounds in these products have been carried out. However, such investigations could be very important to elucidate the possible critical effects exerted by food matrices on protein digestion and bioactive peptide production. Here, in order to assess the suitability of Spirulina-enriched foods as a source of potentially bioactive peptides, a simulated digestion protocol was used in combination with mass spectrometry quantitative analysis to analyze functionalized pasta and sorbets. In the case of the pasta enriched with Spirulina, the production of peptides was quite similar to that of the Spirulina powder. On the other hand, the type of fruit present in the food matrix influenced the digestion of Spirulina inside the sorbets. In particular, the high concentration of protease inhibitors in kiwifruit drastically reduced the production of peptides from Spirulina in kiwi sorbet.

Comparative study on the effect of dietary ß-carotene and phycocyanin extracted from Spirulina platensis on immune-oxidative stress biomarkers, genes expression and intestinal enzymes, serum biochemical in Nile tilapia, Oreochromis niloticus.

The current trial investigated the roles of ß-carotene and phycocyanin extracted from Spirulina platensis on growth, serum biochemical, digestive enzymes, antioxidant defense, immune responses, and immune gene expression in Nile tilapia (Oreochromis niloticus). Fish (1.52 ± 0.10 g) were randomly stocked to three treatments with three replicates (12 fish per replicate) in nine aquaria (60 L glass aquarium for each), and reared for 70-days. Three tested diets were formulated to be isonitrogenous and isolipidic, and were offered for experimental fish until ad-libitum three times daily at 09:00 a.m., 11.00 a.m. and 3:00 p.m. The first diet (control) was without supplementation. About 50 mg ß-carotene and 50 mg phycocyanin kg-1 were supplemented to the other experimental diets, respectively. Results indicated that feed intake was not (P > 0.05) differ among experimental diets. Compared to control diet wight gain and specific growth rate were significantly (P < 0.05) in fish fed diet containing ß-carotene, while, the highest weight gain and the best FCR were detected in phycocyanin diet. Survival fish among treatments was significantly (P < 0.05) differ and the highest survival rate was showed in fish fed diet supplemented with phycocyanin. Either ß-carotene or phycocyanin significantly (P < 0.05) improved the intestinal digestive enzymes compared with control diet, where the highest values of chymotrypsin, trypsin, lipase and amylase were noticed in fish fed phycocyanin. Diets supplemented with ß-carotene and phycocyanin significantly (P < 0.05) improved hematology parameters contents compared with to the control diet, and the best contents were detected in fish fed diet supplemented with phycocyanin. The highest significant (P < 0.05) phagocytic, lysozyme, immunoglobulin M (IgM), superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and total antioxidant capacity (T-AOC) activities were recorded in diet supplemented with phycocyanin. The transcripts of interferon gamma and interleukin 1ß genes were (P < 0.05) up-regulated in the liver of fish fed diet supplemented with ß-carotene and phycocyanin, but expression of HSP70 gene down-regulated in fish fed ß-carotene and phycocyanin containing diet compared control. The highest gene expression of the interferon gamma and interleukin 1ß was observed in fish fed phycocyanin.

Nutraceutical targeting of TLR4 signaling has potential for prevention of cancer cachexia.

The mechanisms underlying cancer cachexia - the proximate cause of at least 20% of cancer-related deaths - have until recently remained rather obscure. New research, however, clarifies that cancers evoking cachexia release microvesicles rich in heat shock proteins 70 and 90, and that these extracellular heat shock proteins induce cachexia by serving as agonists for toll-like receptor 4 (TLR4) in skeletal muscle, macrophages, and adipocytes. Hence, safe nutraceutical measures which can down-regulate TLR4 signaling can be expected to aid prevention and control of cancer cachexia. There is reason to suspect that phycocyanobilin, ferulic acid, glycine, long-chain omega-3s, green tea catechins, ß-hydroxy-ß-methylbutyrate, carnitine, and high-dose biotin may have some utility in this regard.

Complementary chromatic and far-red photoacclimations in Synechococcus ATCC 29403 (PCC 7335). I: The phycobilisomes, a proteomic approach.

Synechococcus ATCC 29403 (PCC 7335) is a unicellular cyanobacterium isolated from Puerto Peñasco, Sonora Mexico. This cyanobacterium performs complementary chromatic acclimation (CCA), far-red light photoacclimation (FaRLiP), and nitrogen fixation. The Synechococcus PCC 7335 genome contains at least 31 genes for proteins of the phycobilisome (PBS). Nine constitutive genes were expressed when cells were grown under white or red lights and the resulting proteins were identified by mass spectrometry in isolated PBS. Five inducible genes were expressed under white light, and phycoerythrin subunits and associated linker proteins were detected. The proteins of five inducible genes expressed under red light were identified, the induced phycocyanin subunits, two rod linkers and the rod-capping linker. The five genes for FaRLiP phycobilisomes were expressed under far-red light together with the apcF gene, and the proteins were identified by mass spectrometry after isoelectric focusing and SDS-PAGE. Based on in silico analysis, Phylogenetic trees, and the observation of a highly conserved amino acid sequence in far-red light absorbing alpha allophycoproteins encoded by FaRLiP gene cluster, we propose a new nomenclature for the genes. Based on a ratio of ApcG2/ApcG3 of six, a model with the arrangement of the allophycocyanin trimers of the core is proposed.

Role of calcium in the mitigation of heat stress in the cyanobacterium Anabaena PCC 7120.

The effects of exogenously added CaCl2 (0.25mM) on photopigments, photosynthetic O2-evolution, antioxidative enzyme activity, membrane damage, expression of two heat shock genes (groEL and groES) and apoptotic features in Anabaena 7120 under heat stress (45°C) for up to 24h were investigated. Heat stress lowered the level of photopigments; however, Ca2+--supplemented cultures showed a low level reduction in Chl a but induced accumulation of carotenoids and phycocyanin under heat stress. Photosynthetic O2-evolving capacity was maintained at a higher level in cells from Ca2+-supplemented medium. Among the antioxidative enzymes, superoxide dismutase activity was unaffected by the presence or absence of Ca2+ in contrast to increases in catalase, ascorbate peroxidase and glutathione reductase activities in cells grown in Ca2+-supplemented medium. Lower levels of lipid peroxidation were recorded in Anabaena cells grown in Ca2+-supplemented medium in comparison to cells from Ca2+--deprived medium. Target cells grown in Ca2+-deprived medium developed apoptotic features in the early stages of heat shock, while Ca2+ application seemed to interfere with apoptosis because only a few cells showed such features after 24 h of heat exposure, indicating a role for Ca2+ in maintaining cell viability under heat stress. There was also continuous up regulation of two important heat shock genes (groEL and groES) in Ca2+-supplemented cultures, exposed to heat shock, again indicating a role for Ca2+ in stress management.

Effect of lambda cyhalothrin on Calothrix sp. (GUEco 1001), an autochthonous cyanobacterium of rice fields of Brahmaputra floodplain.

Pesticide contamination in the rice fields has manifested into a serious global environmental concern. Application of pesticides in the rice fields has deleterious effects on non-target organisms including nitrogen-fixing cyanobacteria which help to maintain the rice field fertility. In the present research endeavor, the effect of lambda cyhalothrin (5% EC), a synthetic pyrethroid insecticide, has been studied on the growth and pigments content of Calothrix sp. (GUEco 1001), an indigenous strain isolated from rice grown areas of Brahmaputra floodplain. To study the toxic effect of lambda cyhalothrin, the test organism was exposed to varying concentrations of the insecticide i.e., 20 ppm, 40 ppm, 80 ppm, and 160 ppm based upon the determination of LC50 for a period of 20 days. The result obtained in the laboratory showed a progressive decrease in the growth and pigments content by the test organism with increasing concentrations of the lambda cyhalothrin against time dose-dependent manner. At high dose (160 ppm), the test organism showed significant decrease in dry weight biomass (54.5%), chlorophyll-a (68%), carotenoids (38%), phycocyanin (80%), and nitrogen contents (55%) over the control. A little but insignificant stimulatory effect on growth and chlorophyll-a contents was recorded in 20 ppm treatment of the insecticide that, however, was reversed in case of carotenoids and phycocyanin contents.

Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942.

The direct conversion of carbon dioxide into biofuels by photosynthetic microorganisms is a promising alternative energy solution. In this study, a model cyanobacterium, Synechococcus elongatus PCC 7942, is engineered to produce free fatty acids (FFA), potential biodiesel precursors, via gene knockout of the FFA-recycling acyl-ACP synthetase and expression of a thioesterase for release of the FFA. Similar to previous efforts, the engineered strains produce and excrete FFA, but the yields are too low for large-scale production. While other efforts have applied additional metabolic engineering strategies in an attempt to boost FFA production, we focus on characterizing the engineered strains to identify the physiological effects that limit cell growth and FFA synthesis. The strains engineered for FFA-production show reduced photosynthetic yields, chlorophyll-a degradation, and changes in the cellular localization of the light-harvesting pigments, phycocyanin and allophycocyanin. Possible causes of these physiological effects are also identified. The addition of exogenous linolenic acid, a polyunsaturated FFA, to cultures of S. elongatus 7942 yielded a physiological response similar to that observed in the FFA-producing strains with only one notable difference. In addition, the lipid constituents of the cell and thylakoid membranes in the FFA-producing strains show changes in both the relative amounts of lipid components and the degree of saturation of the fatty acid side chains. These changes in lipid composition may affect membrane integrity and structure, the binding and diffusion of phycobilisomes, and the activity of membrane-bound enzymes including those involved in photosynthesis. Thus, the toxicity of unsaturated FFA and changes in membrane composition may be responsible for the physiological effects observed in FFA-producing S. elongatus 7942. These issues must be addressed to enable the high yields of FFA synthesis necessary for large-scale biofuel production.

Dynamics of microcystin production and quantification of potentially toxigenic Microcystis sp. using real-time PCR.

Cyanobacterial blooms in eutrophied water body are generally composed of various genotypes with or without microcystin-producing genes (mcy gene cluster). Thus there is a need for quantification of potent toxin producing strains. The present study aimed at identifying microcystin variants and its producer strains in Durgakund pond, Varanasi, India, based on quantification of cpcBA-IGS and mcyA (condensation domain) genes using real-time PCR and LC-MS. Increase in microcystin concentrations was correlated with increase in mcyA copy number and the level of pigments (chlorophyll a, phycocyanin and carotenoids). Also, selected environmental factors (water temperature, light irradiance, rainfall, pH, N and P) and the concentration of microcystin variants (MC-LR, -RR and -YR) were also assessed in samples during May 2010 to April 2011 to establish the possible correlation among these parameters. Nutrients favored cyanobacterial bloom but it could not be correlated with the levels of microcystin variants and seemed to be geographically specific. Microcystis sp. dominant in the pond comprised potentially toxigenic cells. The ratio of potentially toxigenic Microcystis sp. to that of total Microcystis sp. ranged from 0% to 14%. Such studies paved the way to identify and quantify the most potent microcystin producer in the tropical aquatic body.

Effects of gibberellin A(3) on growth and microcystin production in Microcystis aeruginosa (cyanophyta).

Environmental factors that affect the growth and microcystin production of microcystis have received worldwide attention because of the hazards microcystin poses to environmental safety and public health. Nevertheless, the effects of organic anthropogenic pollution on microcystis are rarely discussed. Gibberellin A(3) (GA(3)) is a vegetable hormone widely used in agriculture and horticulture that can contaminate water as an anthropogenic pollutant. Because of its common occurrence, we studied the effects of GA(3) on growth and microcystin production of Microcystis aeruginosa (M. aeruginosa) PCC7806 with different concentrations (0.001-25mg/L) in batch culture. The control was obtained without gibberellin under the same culture conditions. Growth, estimated by dry weight and cell number, increased after the GA(3) treatment. GA(3) increased the amounts of chlorophyll a, phycocyanin and cellular-soluble protein in the cells of M. aeruginosa PCC7806, but decreased the accumulation of water-soluble carbohydrates. In addition, GA(3) was observed to affect nitrogen absorption of the test algae, but to have no effect on the absorption of phosphorus. The amount of microcystin measured by enzyme-linked immunosorbent assay (ELISA) increased in GA(3) treatment groups, but the stimulatory effects were different in different culture phases. It is suggested that GA(3) increases M. aeruginosa growth by stimulating its absorbance of nitrogen and increasing its ability to use carbohydrates, accordingly increasing cellular pigments and thus finally inducing accumulation of protein and microcystin.

Interannual variation in diatom bloom dynamics: roles of hydrology, nutrient limitation, sinking, and whole lake manipulation.

Spring development of diatoms in Ford Lake, Michigan, USA was markedly different in 2004 from 2005 and 2006. In 2004, diatom biovolume surpassed 15 mm(3)l(-1) but in 2005 and 2006 maximum biovolume was less than 5 mm(3)l(-1). Soluble reactive silica (SRSi) in 2004 fell below 5 microM whereas in 2005 and 2006, SRSi remained above 30 microM. Taxonomic composition was similar among years and consisted mainly of Asterionella, Cyclotella, Fragilaria, Aulacoseira, and Synedra. Bioassay experiments in 2005 demonstrated that P rather than Si was the element most limiting biomass development. However, P supply rate did not account for the differences among years. Model simulations of Si uptake, washout rates, and sinking implicated hydrologic differences among years as the cause of differential success by diatom populations in April of each year. Bioassay experiments performed after overturn demonstrated that diatoms could grow well in unamended lake water, but they did not flourish in the lake; model simulations implicated sinking losses as the reason. In summer 2006, we performed a selective withdrawal of hypolimnetic water from the outlet dam and weakened density stratification. An Aulacoseira bloom resulted in early to mid-August, depleting SRSi to less than 30 microM. The lake, which had been acting as a P source, changed to a P sink during the bloom, and cyanobacteria did not develop as they had in all previous years. Stoichiometric calculations indicate that the net SRSi uptake and the net DP uptake during the induced bloom were consistent with diatom production.

[Purification and properties of Se-containing allophycocyanins from selenium rich Spirulina platensis].

Three Se-containing allophycocyanins (Se-APC) with high purity were purified from Se rich Spirulina platensis (Se-sp.) by hydroxyapatite chromatography, DEAE-52 anion-exchange chromatography and native gel preparative electrophoresis. Their biochemicial properties were explored by spectral scanning and electrophoresis analysis of Native-PAGE, SDS-PAGE and IEF on thin slab gel. Protein molecular weight (MW) of APC aggregation was determined by gel filter on Sephadex G-200 column. Se content of native and denatured Se-APC was detected by 2, 3-DAN fluorocence method. According to visible and fluorescence spectral character, three purified fractions of APC were identified to be APCI, APCII and APCIII. Native-PAGE and SDS-PAGE analysis revealed that they all shaped trimer (alphabeta) 3 of alpha and beta subunit with molecular mass of 18.3kDa and 15.7kDa, whereas APCI contains gamma subunit (about 32kDa) visibly and APCIII maybe contain the linker peptide of L(C)(8 - 10 kDa) based on their MW to be determined of 130.9, 98.1 and 106.30 kDa. IEF detection showed that the pl of Se-APCs was 4.76, 4.85 and 5.02 respectively. Se content of three purified Se-APCs were 316, 273 and 408 microg/g, which decreased about 25% after deaggregation treatment by 0.50 mol/L NaSCN and decreased more than 50% after denaturation treatment by 2-mercaptoethanol and reached to a steady content of 132 microg/g on average. These results indicated that Se incorporation into APC had no influence on function of energy transfer as well as biochemical property of APCs, and Se binding with APCs was highly relevant to its aggregation states whereas Se integrated steadily with its subunits.

Protective effect of Spirulina against doxorubicin-induced cardiotoxicity.

The generation of reactive oxygen species and mitochondrial dysfunction has been implicated in doxorubicin (DOX)-induced cardiotoxicity. The aim of the present study was to determine whether Spirulina, a blue-green algae, could serve as a cardioprotective agent during DOX treatment in a mouse model. Mice were treated with DOX (4 mg/kg bw, intraperitoneally), weekly, for 4 weeks. Spirulina was administered orally for 3 days twice daily, then for 7 weeks along with the four equal injections of DOX. Cardiotoxicity was assessed, at 3 weeks after the end of the DOX-treatment period, by mortality, volume of ascites, liver congestion, oxidative stress and ultrastructural changes of heart tissue. The DOX-treated animals showed higher mortality (53%) and more ascites. Myocardial damage, as assessed by ultrastructural changes, showed loss of myofibrils, cytoplasmic vacuolization and mitochondrial swelling. Myocardial superoxide dismutase and glutathione peroxidase activities were decreased and lipid peroxidation was increased. Pretreatment with Spirulina significantly protected the mice from DOX-induced cardiotoxic effects as evidenced from lower mortality (26%), less ascites, lower levels of lipid peroxidation, normalization of antioxidant enzymes and ultrastructural studies showing minimal damage to the heart. In vitro cytotoxic studies using ovarian cancer cells demonstrated that Spirulina did not compromise the anti-tumor activity of doxorubicin. These results suggest that Spirulina has a protective effect against cardiotoxicity induced by DOX and it may, therefore, improve the therapeutic index of DOX.

Large contact surface interactions between proteins detected by time series analysis methods: case study on C-phycocyanins.

A purely sequence-dependent approach to the modeling of protein-protein interaction was applied to the study of C-phycocyanin alphabeta dimers. The interacting pairs (alpha and beta subunits) share an almost complete structural homology, together with a general lack of sequence superposition; thus, they constitute a particularly relevant example for protein-protein interaction prediction. The present analysis is based on a description posited at an intermediate level between sequence and structure, that is, the hydrophobicity patterning along the chains. Based on the description of the sequence hydrophobicity patterns through a battery of nonlinear tools (recurrence quantification analysis and other sequence complexity descriptors), we were able to generate an explicit equation modeling alpha and beta monomers interaction; the model consisted of canonical correlation between the hydrophobicity autocorrelation structures of the interacting pairs. The general implications of this holistic approach to the modeling of protein-protein interactions, which considers the protein primary structures as a whole, are discussed.

Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer.

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.

Recombinant type A and B phytochromes from potato. Transient absorption spectroscopy.

The cDNAs encoding full-length type A and B phytochromes (phyA and phyB, respectively) from potato were expressed in inducible yeast systems (Saccharomyces cerevisiae and Pichia pastoris). In addition, a deletion mutant of phyB (delta 1-74) was expressed. The apoproteins were reconstituted into chromoproteins by incorporation of the native chromophore, phytochromobilin (P phi B), and of phycocyanobilin (PCB). The incorporation of P phi B yielded chromoproteins with difference absorptions lambda max at 660 and 712 nm (Pr and Pfr, respectively) for phyA, and at 665 and 723 nm for phyB. All difference maxima of PCB phytochromes are blue-shifted by several nanometers with respect to those obtained with the P phi B chromophore. The deletion construct with PCB shows difference absorption maxima at 652 and 705 nm with the Pfr absorbance considerably reduced. Time-resolved kinetic analysis of a phyB-type phytochrome by nanosecond flash photolysis was performed for the first time. Recombinant full-length phyB afforded transient absorbance changes similar (but not identical) to those of phyA from Avena, whereas the kinetic behavior of these intermediates was very different. Contrary to phyA from Avena, the I700 intermediate from phyB reconstituted with either PCB or P phi B decayed following single exponential kinetics with a lifetime of 87 or 84 microseconds, respectively, at 10 degrees C. The formation of Pfr of PCB-containing recombinant phyB (phyB-PCB) could be fitted with three lifetimes of 9, 127, and 728 ms. The corresponding lifetimes of phyB-P phi B are 22.5, 343, and 2083 ms. Whereas for phyB-PCB all three millisecond lifetimes are related to the formation of Pfr, the 2 s component of phyB-P phi B is concomitant with a rapid recovery of Pr. For recombinant potato phyA and delta 1-74 phyB, no time-resolved data could be obtained due to the limited quantities available. As described for phytochromes of other dicotelydons, the Pfr forms of full-length phyA and PhyB of potato underwent rapid dark conversion to Pr.