Effective transformation of the cyanobacterium Spirulina platensis using electroporation

Although Spirulina (Arthrospira) is expected to be a suitableorganism for producing recombinant proteins, a gene transfer system hasnot yet been established, due to a lack of suitable vectors and because Spirulina appears refractory to common genetic manipulations. As theinitial stages of the development of recombinant DNA methodology, weexamined the effects on transformation efficiency of electroporationconditions such as electric-field strength (2, 4, 6, 8, 10, 12kV cm^-1) and time constant (2.5, 5 ms). At a time constant of2.5 ms, few transformants were observed regardless of the field strength.The longer time constant of 5.0 ms reproducibly yielded transformants atthe middle field strength of 4 - 8 kV cm^-1, but gave high killingand no transformation at the higher field strength of 10 - 12kV cm^-1. Chloramphenicol acetyltransferase (CAT) activities wereincreased only in the transformants from 2–6 kV cm^-1 and 5.0 ms.The density of the transformants was significantly correlated with therelative value of CAT activity (r = 0.89, n = 11, p < 0.01),suggesting that the chloramphenicol resistance was due to CAT activity. Weconcluded that transformation of S. platensis was most effective at apulse duration 5.0 ms with an electric field of 4 kV cm^-1, and thatforeign genes can be expressed in this organism.     

Monolayer Film of Phycobilisome-Thylakoid Membrane Complexes from Spirulina platensis

Monolayer films of phycobilisome-thylakoid membrane complexes isolated from Spirulina platensis were prepared at air/aqueous solution interface by using the Langmuir-Blodgett technique. The film preparation was optimized with 0.5 M phosphate buffer (pH 7.0) as sub-phase at 20 °C. The monolayer was transferred into grids and into mica surface for observing the surface image of the complexes by transmission electron microscopy and atomic force microscope, respectively. The shape of complexes was disk-like with the diameter of about 50 nm and the thickness of about 35 nm. The absorption and fluorescence spectra of the complexes in the monolayer were consistent with those in buffer solution, which suggests that the complexes in the monolayer preserve the basic functional groups of photosynthetic apparatus and can be used as a model to investigate the structural connection and functional association of the light-harvesting antenna with the reaction centres.     

Clarification and concentration with membrane technology of a phycocyanin solution extracted from Spirulina platensis

Membrane technologies were investigated with the aim to improve stability of C-Phycocyanin extracts resulting from ultrasonic breakage of Spirulina platensis. Five membranes, ranging from microfiltration to reverse osmosis, were utilized both for clarification and concentration steps. Nanofiltration with tubular organic membranes exhibited good performances: pigment recovery was 100%, mean permeation flux was 85 l h^–1 m^–2 for achieving a concentration factor of 7 with 30×10⁵ Pa pressure and 1.5 m s^–1 tangential velocity (turbulent flow).     

李氏块菌提取物抗氧化活性的研究

对李氏块菌Tuber liyuanum子实体的不同提取物,包括甲醇提取物(ME)、乙醇提取物(EE)、丙酮提取物(AE)、正丁醇提取物(BAE)和乙酸乙酯提取物(EAE),进行清除DPPH自由基、羟基自由基、超氧阴离子自由基、铁离子螯合能力、还原力以及总酚含量的测定和研究,发现ME对DPPH自由基、超氧阴离子自由基清除及还原力活性最高,EC50值分别为23.37mg/m L、11.65mg/m L和24.47mg/m L;EE对羟基自由基清除和铁离子螯合能力活性最高,EC50值分别为7.24mg/m L和小于0.5mg/m L;ME的总酚含量最高(3.08mg GAE/g提取物),其次是EE(1.34mg GAE/g提取物),提取物总酚含量与抗氧化活性呈现出一定的正相关性。     

Antioxidant activities of phlorotannins isolated from Japanese Laminariaceae

The antioxidant activities of brown algal phlorotannins were evaluated using the inhibition of phospholipid peroxidation in the liposome system, and by determining radical scavenging activities against the superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH). Oligomers of phloroglucinol (1,3,5-trihydroxybenzene), eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol and 8,8′-bieckol (hexamers), isolated from the Laminarian brown algae Eisenia bicyclis, Ecklonia cava and Ecklonia kurome, showed potent inhibition of phospholipid peroxidation at 1 μM in the liposome system. The phlorotannins had significant radical scavenging activities against the superoxide anion (50% effective concentration values: 6.5–8.4 μM) and DPPH (50% effective concentration values: 12–26 μM), and were more effective compared to ascorbic acid and α-tocopherol. For the purpose of using phlorotannins as functional food ingredients, the antioxidant activity of a complex of crude phlorotannins and soybean protein was examined. The complex had a pronounced DPPH-radical scavenging activity. These results suggest that phlorotannins are potent anti-inflammatory substances, and that the Laminariaceous brown algae, which are abundant in phlorotannins, may be useful as a new functional foodstuff or supplement with anti-inflammatory activity.     

Antioxidant and hepatoprotective activities of low molecular weight sulfated polysaccharide from Laminaria japonica

A low molecular weight sulfated polysaccharide (LMWF) was prepared from Laminaria japonica by mild acid hydrolysis. The antioxidant activity of LMWF in vitro was studied using three kinds of oxygen free radical systems. LMWF had effective scavenging abilities on superoxide radical, hydroxyl radical and hypochlorous acid directly in vitro. The hepatoprotective effect of LMWF was studied using two acute liver injury mice models induced by carbon tetrachloride (CCl₄) and D-galactosamine (D-GalN). Addition of LMWF significantly lowered the content of serum malonaldehyde and markedly increased the activities of superoxide dismutase and glutathione peroxidase, compared with the model groups in both kinds of liver injury mice. Moreover, administration of LMWF significantly inhibited the elevation of glutamate pyruvate transaminase induced by CCl₄ and D-GalN in mice. The results suggest that the antioxidant activity of LMWF plays an important role in its hepatoprotective effect in the liver injury mice induced by CCl₄ and D-GalN.     

Effects of carotenoids from Deinococcus radiodurans on protein oxidation

Aims:  To evaluate the antioxidant effect of carotenoids from Deinococcus radiodurans on protein.
Methods and Results:  Deinococcus radiodurans strain R1 (ATCC 13939) and its mutant strain R1ΔcrtB were used for this study. The total carotenoids (R1ex) from D. radiodurans were obtained by extraction with acetone/methanol (7 : 2, by vol), and their antioxidant activity was measured using the DPPH˙ (2,2-diphenyl-1-picrylhydrazyl) system. The protein oxidation level, in vitro and in the cell, was measured using the DNPH (2,4-dinitrophenyl hydrazine) method. The carotenoid extract R1ex scavenged 40·2% DPPH˙ radicals compared to β-carotene (31·7%) at a concentration of 0·5 mg ml⁻¹. The intracellular level of protein oxidation in mutant R1ΔcrtB, which does not contain carotenoid, was 0·0212 mmol mg⁻¹ protein which is significantly greater than that in the wild type (0·0169 mmol mg⁻¹ protein) following the treatment with H₂O₂. The purified major carotenoid product (deinoxanthin) from the wild type showed a greater inhibition of oxidative damage in bovine serum albumin than lycopene or lutein.
Conclusions:  Carotenoids prevent protein oxidation and contribute to the resistance to cell damage in D. radiodurans .
Significance and Impact of the Study:  Our results provide the evidence that carotenoids can protect proteins in D. radiodurans against oxidative stress.     

Generation of active oxygen species during enzymic isolation of protoplasts from oat leaves

Summary Protoplasts were isolated from oat (Avena sativa L.) leaves by the combination of highly purified preparations of pectin lyase, xylanase, and cellulase C₁. During the enzymic isolation, superoxide radical (O ₋ ² ) was generated from the tissues. Both the protoplasts themselves and the cell walls, exposed to enzyme treatment, produced O ₋ ² . Hydrogen peroxide (H₂O₂) apparently accumulated in the reaction mixture due to the spontaneous dismutation reaction of O ₋ ² , while a part of H₂O₂ may have been produced directly from cell walls by the action of enzymes. Singlet molecular oxygen (¹O₂) generated in the reaction mixture was detected by cholesterol oxidation in small unilamellar liposomes. It seems likely that¹O₂ may be generated by the peroxidase-H₂O₂-halide system during enzymic treatment of the leaves. The work was partially supported by the Research Project “Research and development of the improvement of bacterial and plant cells by cell fusion” of the Food and Agriculture Research and Development Association (Japan).     

Free radical and reactive oxygen species scavenging activities of the extracts from seahorse, Hippocampus kuda Bleeler

Seahorse, Hippocampus kuda (SH) a marine teleost fish, is well known not only for its special medicinal composition and used as one of the most famous and expensive materials of traditional Chinese medicine. It was extracted with water (SHW), methanol (SHM), and ethanol (SHE), respectively and evaluated by various antioxidant assays. The including reducing power, total antioxidant, DPPH radical scavenging, hydroxyl radical scavenging, superoxide anion radical scavenging, alkyl radical scavenging, and protective effect on DNA damage caused by hydroxyl radicals generated. Further, the ROS level was detected using a fluorescence probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), which could be converted to highly fluorescent dichlorofluorescein (DCF) with the presence of intracellular ROS on mouse macrophages, RAW264.7 cell and inhibited myeloperoxidase (MPO) activity in human myeloid, HL60 cells, respectively. Those various antioxidant activities were compared to standard antioxidants such as α-tocopherol. Among SHM exhibited the highest antioxidant activity in linoleic acid system, effective reducing power, DPPH radical scavenging, hydroxyl radical scavenging, superoxide radical scavenging, alkyl radical scavenging, inhibitory intracellular ROS, and inhibited MPO activity. Furthermore, MTT assay showed no cytotoxicity on mouse macrophages cell (RAW264.7) and human cell lines (MRC-5, HL60, U937). This antioxidant property depends on concentration and increasing with increased amount of extracts. The results obtained in the present study indicated that the see horse (Hippocampus kuda Bleeker) is a potential source of natural antioxidant.