Establishment of Agrobacterium tumefaciens-mediated genetic transformation in Dunaliella bardawil

Dunaliella bardawil, a unicellular microalga, grows in relatively high concentrations of salt and has so far been refractory to Agrobacterium-mediated transformation. An inverse relationship between salt concentration and hygromycin resistance was observed. Co-cultivation at 0.2?M NaCl allowed growth of both D. bardawil and A. tumefaciens. Lowering salt concentrations also enabled the use of lower concentrations of hygromycin, the selection agent. Cells resistant to 100?mg?l^?1 hygromycin were selected and growth of Agrobacterium was completely eliminated in these cells using cefotaxime/potassium clavulanate. The concentration of sodium chloride was gradually increased to 1.0?M with simultaneous reduction of hygromycin concentration for better growth of D. bardawil. Agrobacterium was unable to survive in the growth medium used for Dunaliella. Expression of β-glucuronidase (uidA), green fluorescent protein (GFP) and hygromycin phosphotransferase (hpt) in the hygromycin-resistant culture was detected using X-gluc as substrate and Western blotting using GFP antibodies and RT-PCR respectively. Cells growing in 1.0?M NaCl (in the absence of hygromycin) retained their ability to grow in hygromycin even after 18 months of cultivation. These cells expressed GFP and PCR for hpt gene was positive. The stability of the integrated transgene and resistance to hygromycin in three different transformation events were ascertained periodically. Southern blotting of DNA extracted from hygromycin resistant cells (HRC) that were 15–18 months old established the presence of the integrated transgene in the DNA of D. bardawil. Results of the present study substantiate A. tumefaciens-mediated transformation of the unicellular marine alga D. bardawil. Agrobacterium tumefaciens-mediated transgene integration along with the massive outdoor cultivation methods used for D. bardawil may allow the commercial synthesis of secondary metabolites and heterologous proteins.     

Antioxidant-mediated protective effect of potato peel extract in erythrocytes against oxidative damage

Potato peels are waste by-product of the potato processing industry. They are reportedly rich in polyphenols. Our earlier studies have shown that extracts derived from potato peel (PPE) possess strong antioxidant activity in chemical and biological model systems in vitro, attributable to its polyphenolic content. The main objective of this study was to investigate the ability of PPE to protect erythrocytes against oxidative damage, in vitro. The protection rendered by PPE in erythrocytes was studied in terms of resistance to oxidative damage, morphological alterations as well as membrane structural alterations. The total polyphenolic content in PPE was found to be 3.93 mg/g powder. The major phenolic acids present in PPE were predominantly: gallic acid, caffeic acid, chlorogenic acid and protocatechuic acid. We chose the experimental prooxidant system: FeSO₄ and ascorbic acid to induce lipid peroxidation in rat RBCs and human RBC membranes. PPE was found to inhibit lipid peroxidation with similar effectiveness in both the systems (about 80–85% inhibition by PPE at 2.5 mg/ml). While PPE per se did not cause any morphological alteration in the erythrocytes, under the experimental conditions, PPE significantly inhibited the H₂O₂-induced morphological alterations in rat RBCs as revealed by scanning electron microscopy. Further, PPE was found to offer significant protection to human erythrocyte membrane proteins from oxidative damage induced by ferrous–ascorbate. In conclusion, our results indicate that PPE is capable of protecting erythrocytes against oxidative damage probably by acting as a strong antioxidant.     

Photoperiod influences endogenous indoleamines in cultured green alga Dunaliella bardawil

Effect of light intensity and photoperiod on growth, indoleamines and carotenoid production was studied in unicellular green algae D. bardawil. Maximum biomass and carotenoid contents were found when cultures were grown in light (intensity of 2.0 Klux) at a photoperiod of 16/8h light and dark cycle. There was a profound influence of tested photoperiod conditions of light:dark viz. 8:16, 10:14, and 12:12 hr, continuous light on indoleamines (SER and MEL) production as estimated by HPLC and confirmed by mass spectral data obtained from LC-MS-ESI studies. Serotonin level increased from 908 to 1765 pg/g fresh wt with increase in light duration and melatonin level increased from 267 to 584 pg/g fresh wt during increase in dark phase. Carotenoids production was high in continuous light than other tested conditions.     

Carotenoids protectPhaffia rhodozyma against singlet oxygen damage

Summary The only known habitat of the astaxanthin-containingPhaffia rhodozyma is in slime fluxes of deciduous trees at high altitudes. In this habitat, the function of carotenoids inP. rhodozyma is probably to provide protection against photogenerated antifungal substances in the tree flux such as singlet oxygen (¹O₂). To investigate the role of carotenoids inP. rhodozyma, genetic selections were employed to determine if carotenogenic yeast strains ofP. rhodozyma have enhanced ability to quench¹O₂. Singlet oxygen was generated in liquid culture by the interaction of visible light (-550 nm) with the photosensitizer rose bengal or by the activation of -terthienyl with ultraviolet light (=366 nm). In each case the treatments selected for growth of pigmented strains ofP. rhodozyma. Albino (carotenoid-less) or yellow (-carotene producing) strains grew less well in media containing¹O₂. Addition of the¹O₂ quencher sodium azide to the medium with -terthienyl allowed growth of non-pigmented strains. Since the ecological niche ofP. rhodozyma is highly specific, we investigated whether extracts of birch trees (Betula), the original source ofP. rhodozyma, contained a compound that would select for pigmented populations of the yeast. WhenP. rhodozyma strains were exposed to ethyl acetate extracts ofBetula papyrifera excited with 366 nm ultraviolet light, only pigmented cells were able to grow. These results suggest that carotenogenesis developed inP. rhodozyma in response to the presence of photoactivatable antifungal compounds produced by the host tree.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Stereoisomers of beta-Carotene and Phytoene in the Alga Dunaliella bardawil

Dunaliella bardawil, a halotolerant green alga, was previously shown to accumulate high concentrations of β-carotene when grown outdoors under defined conditions. The β-carotene of algae cultivated under high light intensity in media containing a high salt concentration is composed of approximately 50% all-trans β-carotene and 40% 9-cis β-carotene. We show here that the 9-cis to all-trans ratio is proportional to the integral light intensity to which the algae are exposed during a division cycle. In cells grown under a continuous white light of 2000 microeinsteins per square meter per second, the ratio reached a value of around 1.5, while in cells grown under a light intensity of 50 microeinsteins per square meter per second, the ratio was around 0.2. As previously shown, algae treated with the herbicide norflurazon accumulate phytoene in place of β-carotene. Electron micrographs showed that the phytoene is accumulated in many distinct globules located in the interthylakoid spaces of the chloroplast. Here too, two isomers are present, apparently all-trans and 9-cis phytoene, and their ratio is dependent upon the integral light intensity to which the algae are exposed during a division cycle. In the presence of norflurazon, Dunaliella bardawil grown under a light intensity of 2000 microeinsteins per square meter per second contained about 8% phytoene with a 9-cis to all-trans ratio of about 1.0. This ratio decreased to about 0.1 when the light intensity was reduced to 50 microeinsteins per square meter per second. These data suggest that the isomerization reaction which leads to the production of the 9-cis isomer occurs early in the path of carotene biosynthesis, at or before the formation of all-trans phytoene. The presence of the 9-cis isomer of β-carotene and the dependence of its preponderance on light intensity seem to be a common feature of many plant parts. Thus carrots which are exposed to minimal light contain no 9-cis isomer while sun-exposed leaves, fruits, and flowers contain 20 to 50% of the 9-cis isomer.     

Production of phytoene by herbicide-treated microalgae Dunaliella bardawil in two-phase systems

In the present study we have optimized the concentration of the bleaching herbicide norflurazon to obtain Dunaliella bardawil cells able to accumulate phytoene without losing viability. The highest concentration of phytoene 10.4 g/gChl was obtained for a concentration of norflurazon of 10 microg/mL. Norflurazon-treated Dunaliella bardawil cells are able to accumulate high concentrations of phytoene if the carotenogenic pathway is stimulated, but the lack of colored carotenoids make these cells particularly sensitive to high light intensities and to UVB radiation, so other stimuli, such as nitrogen starvation, have to be used to force the accumulation of phytoene. Detailed time-course evolution of the carotenoids lutein, violaxanthin, zeaxanthin, phytene and beta-carotene and the photosynthetic pigment chlorophyll was followed upon transfer of Dunaliella bardawil cells to nitrogen starvation in presence and absence of norflurazon. The combined use of the carotenogenic pathway inhibitor norflurazon and biphasic aqueous/organic systems to force the excretion of phytoene into the culture medium has been investigated. Cells cultured in the biphasic system were viable and able to produce phytoene during 3 days. Futhermore the productivity increased from 0.14 g/gChl . h in the aqueous culture to 0.18 g/gChl . h in the biphasic system. About 15% of the total phytoene produced by Dunaliella bardawil was excreted and immediately partionated into the organic phase. The concentration of phytoene in the decane phase was 2.05 g/gChl after 72 h, this means that about 47 g of phytoene per litre of culture were in the organic phase.     

abeo-abietanes from Teucrium polium roots as protective factors against oxidative stress

Six new 17(15→16)-abeo-abietanes, along with seven known compounds, were isolated and characterized from ethyl acetate root extract of Teucrium polium L., a medicinal plant belonging to the Labiatae family reported to have hypolipidemic, hypoglycaemic, anti-nociceptive and anti-inflammatory effects. Their structures were elucidated by 1D (¹H, ¹³C and DEPT) and 2D (COSY, TOCSY, HSQC, HMBC, and NOESY) NMR and mass spectral data. The antioxidative properties of pure metabolites were analyzed on the basis of their DPPH radical scavenging capability. The antioxidant capacity in cell-free systems of the isolated metabolites was carried out by measuring their capabilities to inhibit the synthesis of thiobarbituric acid reactive species in assay media using as oxidable substrates a vegetable fat and the pentose sugar 2-deoxyribose and to prevent oxidative damage of the bovine serum albumin (BSA) hydrosoluble protein. All of the compounds showed a significant and dose–response efficacy although weaker than that exercised by the standard Trolox®.     

Availability and antiperoxidative effects of beta-carotene from Dunaliella bardawil in alcohol-drinking rats

The present study demonstrated the high bioavailability and antiperoxidative capacity of the natural beta-carotene isomer mixture of Dunaliella bardawil compared with synthetic beta-carotene under alcohol-induced oxidative stress. Weanling rats were adapted to ethanol by increasing ethanol levels in their drinking water to 30% at 5% intervals per week; other rats received water with no added ethanol. One water-drinking group and one alcohol-drinking group with no dietary carotene were used as controls. Two water-drinking groups were supplemented with 1 g/kg diet beta-carotene either from Dunaliella or a synthetic source, and due to reduced food intake, two ethanol-fed groups received 2 g beta-carotene per kilogram of diet from each source. Following 3 months of ethanol consumption, both carotene sources were found to prevent ethanol-induced lipid peroxidation as expressed by the hepatic conjugated oxidized dienes level. However, in the algal-fed rats, hepatic carotene and vitamin A levels were higher. In addition to a lower performance of the group fed ethanol and synthetic beta-carotene, there were three deaths in this group.     

Mode of Action of the Massively Accumulated beta-Carotene of Dunaliella bardawil in Protecting the Alga against Damage by Excess Irradiation

When grown under defined conditions Dunaliella bardawil accumulates a high concentration of β-carotene, which is composed primarily of two isomers, all-trans and 9-cis β-carotene. The high β-carotene alga is substantially resistant to photoinhibition of photosynthetic oxygen evolution when compared with low β-carotene D. bardawil or with Dunaliella salina which is incapable of accumulating β-carotene. Protection against photoinhibition in the high β-carotene D. bardawil is very strong when blue light is used as the photoinhibitory agent, intermediate with white light, and nonexistent with red light. These observations suggest that the massively accumulated β-carotene in D. bardawil protects the alga against damage by high irradiation by screening through absorption of the blue region of the spectrum. Irradiation of D. bardawil by high intensity blue light results in the following temporal sequence of events: photoinhibition of oxygen evolution, photodestruction of 9-cis β-carotene, photodestruction of all-trans β-carotene, photodestruction of chlorophyll and cell death.     

In vitro protective effect of Rhodiola rosea extract against hypochlorous acid-induced oxidative damage in human erythrocytes

Rhodiola rosea L. (Crassulaceae) is a plant living at high altitudes in Europe and Asia. Its roots have long been used in the traditional medical system of these geographical areas to increase the organism resistance to physical stress; today, it has become an important component of many dietary supplements. In this study we investigate the antioxidant capacity of the R. rosea aqueous extract evaluating its ability to counteract some of the main damages induced by hypochlorous acid (HOCl), a powerful oxidant generated by activated phagocytes, to human erythrocytes. Ascorbic acid was used as a reference substance because of its physiological HOCl-scavenging ability. Our study demonstrates that R. rosea is able to significantly protect, in a dose-dependent manner, human RBC from glutathione (GSH) depletion, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inactivation and hemolysis induced by the oxidant. Furthermore, we demonstrate that R. rosea aqueous extract acts from the inside of the erythrocyte suggesting a probable involving of cell components. The protection on GSH afforded by the R. rosea extract with respect to ascorbic acid, occurred also if added 2 or 5 min. later than the oxidant, suggesting a more rapid or powerful effect.     

Hydroxy-urea protects erythrocytes against oxidative damage

Hydroxy-urea (OH-U) is used to treat sickle cell anemia by increasing hemoglobin fetal-fraction. It has been suggested that the sickle cell mutations lead to the formation of unstable HbS and release of iron, which can result in lipid peroxidation (LPO), and eventual cell damage. Since oxidative processes might be involved in pathogenesis of sickle cell disease, we investigated the antioxidant property of OH-U in a red blood cell (RBC) model. Intact RBCs or RBC membranes were exposed to t-butyl hydroperoxide (t-BHP, 0.75 mM) or iron (ferrous sulfate; 100 microM) at 37 degrees C for 60 min in the presence or absence of OH-U (1.25 mM). The extent of oxidative damage was measured by LPO (as thiobarbituric acid reactive substances, TBARS), hemoglobin oxidation (as percent of methemoglobin, metHb %), and decrease in the activities of membrane-bound Na+/K+-ATPase and Ca2+-ATPases. Our results show that OH-U inhibited t-BHP-induced LPO in fresh RBC membranes significantly (P <0.01). OH-U significantly inhibited t-BHP-mediated LPO (P <0.01) and metHb formation (P <0.01) in intact RBC. Also, OH-U inhibited iron-induced LPO and metHb formation in intact RBC (P <0.01). In addition, OH-U blocked t-BHP-mediated changes in membrane ATPase activities. Furthermore, OH-U blocked iron-mediated hydroxyl radical generation in a dose-dependent fashion. In conclusion, the observed antioxidant properties of OH-U might contribute to its therapeutic action in sickle cell disease.     

UV-A mediated induction of carotenoid accumulation in Dunaliella bardawil with retention of cell viability

The effect of adding UV-A radiation (320–400 nm) to photosynthetically active radiation (PAR, 400–700 nm) during growth of the photosynthetic marine microalga Dunaliella bardawil was investigated in this work in terms of cell growth and carotenoid production. Although signs of slow cell growth (slight reduction of chlorophyll and protein content) were observed after 24 h of cell exposure to UV-A (40 mol photons m^–2 s^–1 and 70 mol photons m^–2 s^–1) plus 140 mol photons m^–2 s^–1 PAR , 84 h exposure to these UV-A conditions slightly stimulated cell growth and increased the photosynthetic efficiency of the exposed cultures. The enhanced cell growth was coupled with an increase in total carotenoid content. Besides -carotene as the major pigment, increases in the well-known antioxidants lutein and zeaxanthin of about 3-fold and 5-fold, respectively, were determined in cultures exposed to UV-A radiation of 70 mol photons m^–2 s^–1for 84 h. As a consequence, far from being negative to cell growth, low and medium UV-A radiation are stress factors that could be successfully applied to long-term processes for large scale carotenoid production using D. bardawil cultures with retention of cell viability. UV-A exposure has the advantage of being a factor either easily applied or removed as required, in contrast to other nutrient stresses, which require medium replacement for their application.     

Polyhydroxylated 4-thiaflavans as multipotent antioxidants: protective effect on oxidative DNA damage in vitro

Hydroxylated 4-thiaflavans, possessing the antioxidant features of catechol containing flavonoids and/or tocopherols, were evaluated as protective agents against oxidation damage induced in herring sperm DNA by cumene hydroperoxide (CumOOH) or by the glutathione/ferric ion (GSH/Fe(3+)) system. Our data indicate that the effective protection exerted by some of the tested compounds is overall higher than those provided by catechin and alpha-tocopherol, which might be attributed both to the scavenging properties and chelation of Fe(2+) ions.     

Far-red light inhibits growth but promotes carotenoid accumulation in the green microalga Dunaliella bardawil

It has been demonstrated that far-red light reduces growth of marine phytoplankton and that light quality controls growth and photosynthetic metabolism in algae. The green halotolerant microalga, Dunaliella bardawil, accumulates high amounts of β-carotene (up to 10% of its dry weight) under conditions of high light or nutrient limitation. The influence of increasing irradiance and of far-red light in D. bardawil was studied. Continuous irradiance was provided by white fluorescent lamps alone (WL) or supplemented with far-red Linestra lamps (WL+FR). For both types of light, cultures were acclimatized at increasing irradiances (50-300 µmol m^?2 s^?1), and cell density, photosynthetic activity and pigment content were determined. Cell density increased with the photon irradiance, and was higher in WL than in WL+FR under the same irradiance, but the reverse occurred in respect of cell volume. Growth rate was higher under WL+FR. Far-red light induced faster growth but reduced the maximal cell density of the cultures. Chlorophyll a concentration was higher in white light, but total carotenoid content increased dramatically in both far-red light treatments (about 50% on a per cell basis) and with the increase of irradiance. Our results show that far-red light has a significant influence on growth and photosynthesis of D. bardawil, inducing a decrease in cell density, photosynthetic activity and chlorophyll concentration, and an increase in growth rate, cell volume and carotenoid content.     

On the Factors Which Determine Massive beta-Carotene Accumulation in the Halotolerant Alga Dunaliella bardawil

Dunaliella bardawil, a β-carotene-accumulating halotolerant alga, has been analyzed for the effect of various growth conditions on its pigment content, and compared with Dunaliella salina, a β-carotene nonaccumulating species. In D. bardawil, increasing light intensity and light period or inhibiting growth by various stress conditions such as nutrient deficiency or high salt concentration caused a decrease in the content of chlorophyll per cell and an increase in the amount of β-carotene per cell. As a result, the β-carotene-to-chlorophyll ratio increased from about 0.4 to 13 grams per gram and the alga changed its visual appearance from green to deep orange. D. salina grown similarly decreased in content of both chlorophyll and β-carotene per cell and the culture turned from green to yellowish. Low chlorophyll-containing cells of D. bardawil or D. salina exhibit very high photosynthetic rates when expressed on a chlorophyll basis (~600 micromoles O₂ evolved per milligram chlorophyll per hour).

Variation of pigment content in D. bardawil by a large variety of environmental agents has been correlated with the integral irradiance received by the algal culture during a division cycle. The higher the integral irradiance per division cycle, the lower the chlorophyll content per cell; the higher the β-carotene content per cell, and therefore the higher the β-carotene-to-chlorophyll ratio. The results are interpreted as indicating a protecting effect of β-carotene against injury by high irradiance under conditions of impairment in chlorophyll content per cell.

     

Lipid peroxidation as a source of oxidative damage in Helicobacter pylori: protective roles of peroxiredoxins

Oxidative stress conditions lead to enzymatic and non-enzymatic unsaturated fatty acid-initiated lipid peroxidation reactions. One exacerbating product is lipid hydroperoxide (LOOH) which itself promotes formation of several additional peroxyl radicals. Helicobacter pylori mutant strains with disruptions in genes encoding the peroxiredoxins, alkyl hydroperoxide reductase (ahpC) and the bacterioferritin comigratory protein (bcp), were more sensitive than the parent strain to oxidizing agents. These mutant strains were particularly sensitive, compared to the wild type, to killing by the unsaturated fatty acid linolenic acid but were not sensitive to the saturated fatty acid palmitic acid. A double mutant strain (ahpC bcp) accumulated more than 3-fold more lipid peroxides than the parent strain, indicating these peroxiredoxins together play a role in detoxifying lipid peroxides. The level of free iron accumulation, a signature of oxidative stress damage, was correlated specifically to organic peroxide-mediated stress by both in vivo and in vitro approaches. Free iron accumulation and concomitant destruction of [Fe-S] cluster-containing proteins (hydrogenase and aconitase) was correlated to damage mediated by exogenous t-butyl peroxide, or separately to intracellular accumulation of lipid peroxides in mutant strains. A major macromolecular target of accumulating lipid peroxides in H. pylori is DNA, as mutant analysis approaches combined with quantitative DNA fragmentation studies and specific DNA damage assessment (i.e. 8-oxoguanine formation) were used to demonstrate that such damage was especially associated with ahpC and ahpC bcp strains.     

Cold-acclimation protects photosystem II against freezing damage in the halotolerant alga Dunaliella salina

Cold-acclimation (CA) of the halotolerant alga Dunaliella was inhibited by light and by high salt. CA was associated with enhanced resistance to freezing in saline growth solutions, as manifested by protection of photosynthetic oxygen evolution and by reduced permeabilisation of the plasma membrane. Oxygen evolution activity in isolated chloroplasts was not affected by freezing, but was inhibited by high salt and the inhibition could be reversed or protected by glycerol. The activity of chloroplasts from cold-acclimated cells was more resistant to salt than of non-acclimated cells. Electron transport measurements in chloroplasts indicated that high salt inhibited PS-II, but not PS-I electron transport. High salt also inhibited PS-II thermoluminescence (TL) activity in chloroplasts. Similar inhibition of PS-II TL was observed by freezing intact cells in saline solutions. Chloroplasts from cold-acclimated cells had enhanced resistance to inhibition of PS-II electron transport and of PS-II TL by high salt. These results suggest that inhibition of oxygen evolution upon freezing Dunaliella cells may result from inactivation of PS-II due to massive influx of salt and loss of glycerol. The enhanced freeze-resistance of cold-acclimated cells to inhibition of oxygen evolution can be accounted for partly by protection of PS-II against high salt.