Photobiological Effects on Biochemical Composition in Porphyridium cruentum (Rhodophyta) with a Biotechnological Application.

This study describes the relation of photosynthetic capacity, growth and biochemical compounds in the microalgae Porphyridium cruentum under saturated irradiance (200 µmol m-2  s-1 ) by white light (WL) and low-pressure sodium vapor lamps (SOX lamps-control) and supplemented by fluorescent lamps (FLs) with different light qualities (blue: λmax = 440 nm; green: λmax = 560 nm; and red: λmax = 660 nm). The maximum photosynthetic efficiency (Fv / Fm ) showed a positive correlation with the light quality by saturating light SOX in mixture with stimulating blue light than the white light (WL) at the harvest day (10 days). The production, that is maximal electron transport rate (ETRmax ), and energy dissipation, that is maximal nonphotochemical quenching (NPQmax ), had the same pattern throughout the time (3-6 days) being the values higher under white light (WL) compared with SOX and SOX plus supplemented different light qualities. Total protein levels increased significantly in the presence of SOX light, while phycoerythrin (B-PE) showed significant differences under SOX+ blue light. Arachidonic acid (ARA) was higher under SOX and SOX plus supplemented different light qualities than that under WL, whereas eicosapentaenoic acid (EPA) was the reverse. The high photomorphogenic potential by SOX light shows promising application for microalgal biotechnology.

Using a B-Phycoerythrin Extract as a Natural Colorant: Application in Milk-Based Products.

Nowadays, there is a growing interest in finding new coloring molecules of natural origin that can increase and diversify the offer of natural food dyes already present in the market. In the present work, a B-phycoerythrin extract from the microalgae Porphyridium cruentum was tested as a food colorant in milk-based products. Using spectroscopy and colorimetry, the extract was characterized and gave evidence of good properties and good stability in the pH range between 4.0 and 9.0. Coloring studies were conducted to demonstrate that samples carrying the pink extract could be used for simulating the pink color of marketed milk-based products. The staining factors, representing the amount of pink protein to be added to reproduce the color of strawberry commercial products, ranged between 1.6 mg/L and 49.5 mg/L, being sufficiently low in all samples. Additionally, color stability during a short period of cold storage was studied: it demonstrated that the three tested types of dairy products remained stable throughout the 11-day analysis period with no significant changes. These results prove the potential of the B-phycoerythrin extract as a natural colorant and alternative ingredient to synthetic coloring molecules.

Protein Cohabitation: Improving the Photochemical Stability of R-Phycoerythrin in the Solid State.

The poor photochemical stability of R-phycoerythrin (R-PE) has been a bottleneck for its broad-spectrum applications. Inspired by nature, we studied a sustainable strategy of protein cohabitation to enhance R-PE stability by embedding it in a solid matrix of gelatin. Both pure R-PE and fresh phycobiliprotein (PBP) extracts recovered from Gracilaria gracilis were studied. The incorporation of R-PE in the gelatin-based films (gelatin-RPE and gelatin-PBPs) has improved its photochemical stability for at least 8 months, the longest time period reported so far. These results were evidenced by not only absorption but also emission quantum yield measurements (Φ). Moreover, the photostability of gelatin-RPE films upon continuous excitation with an AM1.5G solar simulator was tested and found to remain stable for 23 h after initial decreasing up to 250 min. In the end, another approach was established to allow 100% photostability for a 3 h exposure to an AM1.5G solar simulator by doping the gelatin-based film including R-Phycoerythrin with n-propyl gallate stabilized with Tween 80, allowing their use as naturally based optically active centers in photovoltaic applications.

Biogenic iron-silver nanoparticles inhibit bacterial biofilm formation due to Ag+ release as determined by a novel phycoerythrin-based assay.

Silver nanoparticles (Ag-NPs) can be considered as a cost-effective alternative to antibiotics. In the presence of Fe(III)-citrate and Ag+, Klebsiella oxytoca DSM 29614 produces biogenic Ag-NPs embedded in its peculiar exopolysaccharide (EPS). K. oxytoca DSM 29614 was cultivated in a defined growth medium-containing citrate (as sole carbon source) and supplemented with Ag+ and either low or high Fe(III) concentration. As inferred from elemental analysis, transmission and scanning electron microscopy, Fourier transform infrared spectrometry and dynamic light scattering, Ag-EPS NPs were produced in both conditions and contained also Fe. The production yield of high-Fe/Ag-EPS NPs was 12 times higher than the production yield of low-Fe/Ag-EPS NPs, confirming the stimulatory effect of iron. However, relative Ag content and Ag+ ion release were higher in low-Fe/Ag-EPS NPs than in high-Fe/Ag-EPS NPs, as revealed by emission-excitation spectra by luminescent spectrometry using a novel ad hoc established phycoerythrin fluorescence-based assay. Interestingly, high and low-Fe/Ag-EPS NPs showed different and growth medium-dependent minimal inhibitory concentrations against Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 15442. In addition, low-Fe/Ag-EPS NPs exert inhibition of staphylococcal and pseudomonal biofilm formation, while high-Fe/Ag-EPS NPs inhibits staphylococcal biofilm formation only. Altogether, these results, highlighting the different capability of Ag+ release, support the idea that Fe/Ag-EPS NPs produced by K. oxytoca DSM 29614 can be considered as promising candidates in the development of specific antibacterial and anti-biofilm agents.Key points • Klebsiella oxytoca DSM 29614 produces bimetal nanoparticles containing Fe and Ag.• Fe concentration in growth medium affects nanoparticle yield and composition.• Phycoerythrin fluorescence-based assay was developed to determine Ag+release.• Antimicrobial efficacy of bimetal nanoparticle parallels Ag+ions release.

Purification of R-phycoerythrin from Gracilaria lemaneiformis by centrifugal precipitation chromatography.

Centrifugal precipitation chromatography (CpC) is a powerful chromatographic technique invented in the year 2000 but so far very little applied. The method combines dialysis, counter-current and salting out processes. The separation rotor consists of two identical spiral channels separated by a dialysis membrane (6-8 K MW cut-off) in which the upper channel is eluted with an ammonium sulfate gradient and the lower channel with water, and the mixtures are separated according to their solubility in ammonium sulfate as a chromatographic technique. In the present study, the method was successfully applied for separation and purification of R-phycoerythrin (R-PE), a protein widely used as a fluorescent probe, from the red alga Gracilaria lemaneiformis. The separation was performed with the elution of ammonium sulfate from 50% to 0% in 21.5 h at a flow rate of 0.5 ml/min, while the lower channel was eluted with water at a flow rate of 0.05 ml/min after sample charge, and the column was rotated at 200 rpm. After a single run, the absorbance ratio A565/A280 (a criterion for the purity of R-PE) was increased from 0.5 of the crude to 6.5. The purified R-PE exhibited a typical "three peaks" spectrum with absorbance maximum at 497, 538 and 565 nm. The Native-PAGE showed one single protein band and 20 kDa (subunits α and ß) and 30 kDa (subunit γ) can be observed in SDS-PAGE analysis which were consistent with the (αß)6γ subunit composition of R-PE. The results indicated that CpC is an efficient method to obtain protein with the high purity from a complex source.

Phycoerythrin-Derived Tryptic Peptide of a Red Alga Pyropia yezoensis Attenuates Glutamate-Induced ER Stress and Neuronal Senescence in Primary Rat Hippocampal Neurons.

SCOPE: Glutamate excitotoxicity has been observed in association with neurodegenerative disorders. This study aimed to investigate whether a phycoerythrin-derived tryptic peptide of Pyropia yezoensis (PYP) reduces glutamate-induced excitotoxicity and neuronal senescence in primary rat hippocampal neurons. METHODS AND RESULTS: Glutamate exposure (100 µm) decreased cell viability and increased expression of endoplasmic reticulum (ER) stress response protein glucose-regulated protein 78 (GRP78) starting at 60 min following glutamate exposure, which was prevented by pretreating the neurons with PYP (1 µg mL-1 ). The glutamate-induced increase in GRP78 expression was downregulated by blocking N-methyl-d-aspartate (NMDA) receptor with MK801 (10 µm) and inhibiting c-Jun N-terminal kinase (JNK) phosphorylation with SP600125 (10 µm). Moreover, phosphorylation of JNK was decreased by blockade of NMDA receptor. The PYP pretreatment downregulated glutamate-induced increase in GRP78 expression and JNK phosphorylation, and this effect was abolished by inhibiting tropomyosin-related kinase B (TrkB) receptor, phosphatidylinositiol 3-kinase, and extracellular signal-regulated kinase (ERK)1/2 using cyclotraxin B (200 nm), LY294002 (20 µm), and SL327 (10 µm), respectively. In addition, PYP downregulated increase in GRP78 expression, senescence-associated ß-galactosidase activity, and neurite degeneration in aging hippocampal neurons. CONCLUSION: These findings indicate that activation of TrkB receptor-mediated ERK1/2 by PYP attenuates glutamate-induced ER stress, which may improve the survival of hippocampal neurons with age.

Combination of palladium nanoparticles and tubastatin-A potentiates apoptosis in human breast cancer cells: a novel therapeutic approach for cancer.

BACKGROUND: Breast cancer is the most common malignant disease that occurs in women. Histone deacetylase (HDAC) inhibition has recently emerged as an effective and attractive target for the treatment of cancer. The aim of this study was to investigate the efficacy of a combined treatment of tubastatin A (TUB-A) and palladium nanoparticles (PdNPs) against MDA-MB-231 human breast cancer cells using two different cytotoxic agents that work by two different mechanisms, thereby decreasing the probability of chemoresistance in cancer cells and increasing the efficacy of toxicity, to provide efficient therapy for advanced stage of cancer without any undesired side effects. METHODS: PdNPs were synthesized using a novel biomolecule called R-phycoerythrin and characterized using various analytical techniques. The combinatorial effect of TUB-A and PdNPs was assessed by various cellular and biochemical assays and also by gene expression analysis. RESULTS: The biologically synthesized PdNPs had an average size of 25 nm and were spherical in shape. Treatment of MDA-MB-231 human breast cancer cells with TUB-A or PdNPs showed a dose-dependent effect on cell viability. The combination of 4 µM TUB-A and 4 µM PdNPs had a significant inhibitory effect on cell viability compared with either TUB-A or PdNPs alone. The combinatorial treatment also had a more pronounced effect on the inhibition of HDAC activity and enhanced apoptosis by regulating various cellular and biochemical changes. CONCLUSION: Our results suggest that there was a strong synergistic interaction between TUB-A and PdNPs in increasing apoptosis in human breast cancer cells. These data provide an important preclinical basis for future clinical trials on this drug combination. This combinatorial treatment increased therapeutic potentials, thereby demonstrating a relevant targeted therapy for breast cancer. Furthermore, we have provided the first evidence for the combinatorial effect and mechanism of toxicity of TUB-A and PdNPs in human breast cancer cells. The novelties of the study were identification of a combination therapy that consists of suitable therapeutic molecules that kill cancer cells and also exploration of two different possible mechanisms involved to reduce chemoresistance in cancer cells.

One-step purification of R-phycoerythrin from the red edible seaweed Grateloupia turuturu.

A one-step chromatographic method for the purification of R-phycoerythrin (R-PE) of Grateloupia turuturu Yamada is described. Native R-PE was obtained with a purity index of 2.89 and a recovery yield of 27% using DEAE-Sepharose Fast Flow chromatography with a three-step increase in ionic strength. The analysis by SDS electrophoresis showed a broad band between 18 and 21kDa in size corresponding to subunits α and ß and a low intensity band of 29kDa corresponding to the γ subunit. Two forms of R-PE were identified by gel filtration chromatography: a native form with a molecular weight of 260±5kDa and a dissociated form with a molecular weight of 60±2kDa. The native form presented the characteristic absorption spectrum of R-PE with three absorbance maxima at 498, 540 and 565nm, whereas the dissociated form presented only the 498 and 540nm peaks. Moreover, the two forms displayed two different fluorescence maxima.

Isolation and characterization of macromolecular protein R-Phycoerythrin from Portieria hornemannii.

R-Phycoerythrin (R-PE) is one of the three phycobiliproteins which are extensively used as fluorescent probes, and it is prepared from red macro-algae. This macromolecular protein has gained importance in many biotechnological applications in food science, immunodiagnostic, therapy, cosmetics, protein and cell labeling, and analytical processes. In the present investigation, R-PE was isolated and purified from a red alga Portieria hornemannii. R-PE extracted and purified through ammonium sulfate precipitation (55%) followed by Q-Sepharose column chromatography had yielded a maximum purity of 5.2%. R-PE exhibited a typical "three-peak" with absorption maxima at 499, 545 and 565 nm. CD spectrum of R-PE yielded the following secondary structure data: alpha helix (14.30%), beta helix (28.10%), turn helix (19.20%) and random coil helix (38.40%). The molecular mass of R-PE was 240 kDa under Native-PAGE. Three different subunits such as α, ß and γ of 16 kDa, 21 kDa and 39 kDa were segregated under SDS-PAGE. On two dimensional gel electrophoresis, one basic and four acidic subunits were detected. Five different tryptic peptides were assigned under MALDI-TOF. The sequences of N-terminus of R-PE of 10 different amino acids are Met Lys Gln Met Trp Asp Arg Met Val Val. The preparative procedures of the R-PE extraction and purification established based on the experiments exhibit advantages and can offer a reference for R-PE preparation from other marine red macro-alga P. hornemannii.

Enhanced production of biomass, pigments and antioxidant capacity of a nutritionally important cyanobacterium Nostochopsis lobatus.

A diazotrophic cyanobacterium Nostochopsis lobatus was evaluated for enhanced production of biomass, pigments and antioxidant capacity. N. lobatus showed potentially high antioxidant capacity (46.12 microM AEAC) with significant improvement under immobilized cell cultures (87.05 microM AEAC). When a mixture of P and Fe was supplemented, biomass, pigments, nutritive value and antioxidant capacity increased substantially at pH 7.8. When considered separately, P appeared to be a better supplement than Fe for the production of biomass, chlorophyll and carotenoids. However, for phycocyanin, phycoerythrin, nutritive value and antioxidant capacity, Fe appeared more effective than P. Our study indicates N. lobatus to be a promising bioresource for enhanced production of nutritionally rich biomass, pigments and antioxidants. The study also suggests that P and Fe are potentially effective supplements for scale-up production for commercial application.

Research on double-probe, double- and triple-tip effects during atomic force microscopy scanning.

Information obtained by atomic force microscopy (AFM) depends strongly on the kind of probe or tip used; therefore, probe and tip effects have to be taken into account when verifying or interpreting the data acquired. In many papers, double-tip effects have been mentioned while other research was done; however, there are only a few special reports on double- or triple-tip effects, especially double-probe effects. In our paper, metaphase chromosomes of Chinese hamster ovary (CHO) cells, aggregates of pectin molecules, membrane surface of mouse embryonic stem cells, and R-phycoerythrin-conjugated immunoglobulin G complexes were imaged by AFM with high-quality probes, double-probe cantilever, and double-tip and triple-tip probes, respectively, in order to determine double-probe, double-tip, and triple-tip effects during AFM scanning. We found that the double-probe, double-tip, and triple-tip effects share the same principle, and that these effects correlate with distance and height differences between probes of double-probe cantilever or tips of double-tip or multiple-tip probes. Since many other factors influence double-probe or double-tip effects, more in-depth studies must be undertaken. However, this initial research will make all users of AFM techniques aware of double-probe and double-tip or triple-tip effects during AFM scanning and aid in verifying or interpreting the data acquired.

The use of ozone as an oxidizing agent to evaluate antioxidant activities of natural substrates.

Ozone, the main component of photochemical smog and air pollution, can damage the skin by oxidizing stratum corneum enzymes, lipids and structural proteins. We have developed a rapid screening assay to determine free radical scavenging capacity of various active ingredients that are frequently used in personal care products. Several known antioxidants including vitamin C, vitamin E analog Trolox, walnut seed extract, lipoic acid and ergothioneine inner salt were assayed for their ability to neutralize ozone-induced oxidation of beta-phycoerythrin, a fluorescent reporter protein derived from algae. The free radical scavenging capacities of these antioxidants were quantified and compared. The results demonstrate that this assay is a valuable primary screening tool for identifying antioxidant activity of natural or synthetic substrates that can be used in personal care products to protect the uppermost layer of our skin from oxidizing damage induced by O3.

Peroxyl radical scavenging activity of Ginkgo biloba extract EGb 761.

Antioxidant mechanisms have been proposed to underlie the beneficial pharmacological effects of EGb 761, an extract from Ginkgo biloba leaves used for treating peripheral vascular diseases and cerebrovascular insufficiency in the elderly. In vitro evidence has been reported that EGb 761 scavenges various reactive oxygen species, i.e. nitric oxide, and the superoxide, hydroxyl, and oxoferryl radicals. However, the ability of EGb 761 to scavenge peroxyl radicals (reactive species mainly involved in the propagation step of lipid peroxidation) has not been investigated. To characterize further the antioxidant action of EGb 761, we measured the protective effects of EGb 761 during: (1) the oxidation of B-phycoerythrin by peroxyl radicals generated in aqueous solution by 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH); and (2) the reaction of luminol or cis-parinaric acid with peroxyl radicals generated from 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) in liposomes or in human low density lipoprotein (LDL), respectively. To evaluate the peroxyl radical scavenging activity of EGb 761 in a more physiologically relevant model of damage to lipid-containing systems, we also analyzed the effect of the extract on the oxidation of human LDL exposed to the azo-initiators in terms of: (1) accumulation of cholesterol linoleate ester hydroperoxides, (2) depletion of alpha-tocopherol and beta-carotene, and (3) changes in intrinsic tryptophan fluorescence. EGb 761 afforded protection against oxidative damage in all the systems we analyzed; thus, it is an efficient scavenger of peroxyl radicals. This result extends the oxygen radical scavenging properties of the extract and supports the hypothesis of an antioxidant therapeutic action of EGb 761.