Single-step chromatography for simultaneous purification of C-phycocyanin and allophycocyanin with high purity and recovery from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

The cyanobacterium Spirulina (Arthrospira) platensis is a good source of phycobiliprotein purification. C-phycocyanin (C-PC) is the major phycobiliprotein, while allophycocyanin (APC) is less abundant in S. platensis. Previously reported methods for C-PC purification are only able to offer either high purity or high efficiency. This paper describes one-step anion exchange chromatography method with continuous pH gradient elution for simultaneous purification of C-PC and APC with high purity and high recovery. Crude C-PC and APC were extracted and concentrated by ammonium sulfate fractionation at saturation of 25% and 60%, then purified on a DEAE-Sepharose Fast Flow chromatography column with continuous pH gradient elution from pH 5.0 to 3.6. After this single-step chromatography, C-PC and APC with high purity and recovery were simultaneously obtained. The purity ratios of C-PC and APC reached 5.59 (A₆₂₀/A₂₈₀) and 5.19 (A₆₅₀/A₂₈₀), respectively. Their purity was further demonstrated by electrophoresis and fluorescence emission spectroscopy. Moreover, the total recovery yield of pure C-PC and APC were 67.04% and 80.0%, representing 111.83 and 29.28 mg·g⁻¹ lyophilized weight, respectively. The obtained C-PC and APC remained stable over a pH range of 4–9. This purification method for high purity and recovery of C-PC and APC proved to be fairly efficient compared with previously reported methods.     

Efficient separation and purification of allophycocyanin from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

ALLOPHYCOCYANIN FROM A LOCAL ISOLATE GEITLERINEMA SP. A28DM (CYANOBACTERIA): A SIMPLE AND EFFICIENT PURIFICATION PROCESS1

Allophycocyanin (APC) is the least‐studied cyanobacterial bile‐pigment invariably present within the phycobilisome core of cyanobacteria. In the present study, we describe a simple, cost‐effective, and reproducible method for the purification of APC from a local isolate, Geitlerinema sp. A28DM. The pigment was extracted from the algal biomass and precipitated with 0.25% aqueous solution of the highly aromatic cationic dye “ethodin.” The precipitated APC was then subjected to a single size‐exclusion chromatographic step using Sephadex G‐100. Pure cyanobacterial APC (C‐APC) (A₆₅₂/A₂₈₀ of 3.2) was obtained and characterized by its absorption spectrum with maximum at 652 nm and a shoulder at 620 nm, and by SDS‐PAGE, showing two bands with molecular masses of 15 and 17.5 kDa, corresponding to α and β subunits of the biliprotein. The final yield of C‐APC was 66% from its content in the crude extract. The procedure appears to be promising for wider applications and larger production of APC.     

Efficient isolation of high quality nucleic acids from different tissues of <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively.     

An improved method for isolating high-quality DNA from<Emphasis Type="Italic">Vitis vinifera</Emphasis> nuclei

We have developed a simple and highly efficient protocol for isolating large quantities (150–400 μg/g leaf tissue) of high-quality DNA from fresh and frozenVitis vinifera leaves. Isolated DNA is essentially free of polysaccharides, polyphenols, and other major contaminants as judged by viscosity, clear color, A_260/280 ratio, digestibility by restriction enzymes for Southern blot analysis, and PCR suitability.     

Studies on the <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> CCT-4518 laccase purified by hydrophobic interaction chromatography

A laccase from Pycnoporus sanguineus was purified by two steps using phenyl-Sepharose columm. A typical procedure provided 54.1-fold purification, with a yield of 8.37%, using syringaldazine as substrate. The molecular weight of the purified laccase was 69 and 68 kDa as estimated by 12% (w/v) SDS-PAGE gel and by gel filtration, respectively. The K _m values for the substrates ABTS, syringaldazine, and guaiacol were 58, 8.3, and 370 μM, respectively. The enzyme’s pH optimum for syringaldazine was 4.2 and optimal activity was 50°C. The enzyme showed to be thermostable because when kept at 50°C for 24 and 48 h it retained 93 and 76% activity. This laccase was inhibited by l-cysteine, β-mercaptoethanol, NaN₃, NaF, and HgCl₂.     

Rapid and Effective Method of RNA Isolation from Green Microalga <Emphasis Type="Italic">Ankistrodesmus convolutus</Emphasis>

The rapid and effective method for the isolation of RNA from green microalga Ankistrodesmus convolutus based on homogenization in a simple CTAB buffer and selective precipitation of RNA with lithium chloride is developed. This procedure avoids the use of toxic chaotropic agents and phenol while high concentration of dithiothreitol is used to inhibit RNase activity and prevent oxidative cross-linking of nucleic acids by phenolics. The extraction procedure was able to produce high quality and intact RNA from A. convolutus. The yield of total RNA was 0.69–0.73 mg/g of fresh weight, with A₂₆₀/A₂₈₀ ratio of 1.79–1.86. The obtained RNA was of sufficient quality and suitable for downstream application such as RT-PCR and cDNA library construction. The procedure may also have wider applicability for total RNA isolation from other green microalgae species.     

Isolation of C-phycocyanin from <Emphasis Type="Italic">Synechococcus</Emphasis> sp., (<Emphasis Type="Italic">Anacystis nidulans</Emphasis> BD1)

We report a procedure for obtaining fairly pure phycocyanin from a local isolate of the cyanobacterium Synechococcus sp (Anacystis nidulans BD1). Cells were incubated with 1 mg∙mL⁻¹ of lysozyme at 37°C for 16 h with shaking. The cell-free extract was treated with activated charcoal and chitosan. The purity (A _620/280) of phycocyanin obtained after lysozyme treatment was up to 2.18, which could be improved to 4.72 after incubation with activated charcoal and chitosan. The yield of phycocyanin was 80–100 mg∙g⁻¹ dry weight of cells. The method reported here is a single-step and efficient procedure and has the potential to be adopted for large-scale production of phycocyanin.     

A new procedure for fast isolation and purification of plastocyanin from the cyanobacterium Anabaena variabilis

Methods are described for growing the cyanobacterium A. variabilis and for the isolation and purification of plastocyanin from the grown culture. Cell paste which had been stored at –35°C was suspended in 1 mM MES buffer, pH 6.5 and centrifuged. The supernatant was diluted to a conductivity of 0.12 mS, [Fe(CN)₆]^3- added to a concentration of 0.5 mM and the solution loaded on a S Sepharose Fast Flow column. After elution and ultrafiltration, the plastocyanin containing fractions were reloaded on a S Sepharose Fast Flow column for final purification. A typical yield in three days from cells harvested from 3×20 l of medium was 32 mg plastocyanin with a minimum absorbance ratio A₂₇₈/A₅₉₇=1.14. This procedure is faster and the yield higher than for previous procedures.Abbreviations MES 2(N-morpholino)ethanesulfonic acid - PC plastocyanin     

Extraction and purification of an unusual phycoerythrin in a terrestrial desiccation tolerant cyanobacterium <Emphasis Type="BoldItalic">Lyngbya arboricola</Emphasis>

Presence and stability of an unusual phycoerythrin (PE) characteristically similar to R-PE are described in a terrestrial, desiccation-tolerant cyanobacterium, Lyngbya arboricola. Extraction and purification of the PE by using acetone precipitation, gel filtration and ion-exchange chromatography resulted in achieving a purity index (A₅₆₀/A₂₈₀) of up to 5.2. SDS-PAGE of the PE showed presence of 18 kDa, 20 kDa and 32 kDa bands corresponding to α, β and γ subunits of R-PE without any other contaminating phycobiliproteins (PBPs). The absorption spectrum of the PE was distinguished by two major peaks at 499 and 559 nm. The maximum fluorescence emission at room temperature was 578 nm. Spectroscopic and electrophoresis characteristics of PE in the dry mats on storage at 25 ± 1°C over silica gel for 2 years remained almost unaffected. Quantitatively, storage stability of the PE was in the order of dry mats > lyophilized > liquid state and the impact of temperature on loss of PE was in the order of 25°C > −20°C > 4°C. The relevance of L. arboricola for production of stable unusual PE is discussed.     

Genomic DNA extraction from<Emphasis Type="Italic">Victoria amazonica</Emphasis>

DNA extraction is difficult in many plants because of metabolites that interfere with DNA isolation procedures and subsequent applications such as DNA restriction, amplification, and cloning. We developed the first reliable and efficient method for isolatingVictoria amazonica genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 1.5 M NaCl, 2% polyvinylpyrrolidone (PVP) (Mr 1000), 5% mercaptoethanol, 0.12% sodium sulfite, and an incubation at 65°C for 4 h. The purity of isolated genomic DNA was confirmed by means of high-performance liquid chromatography (HPLC) profile and spectrophotometric analyses (A_260/230 ratio of 1.836, A_260/280 of 1.842). DNA was obtained in the amount of 387 μg per gram of leaf material, and it proved amenable to restriction digestion.     

Chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence induction kinetics in leaves predicted from a model describing each discrete step of excitation energy and electron transfer associated with Photosystem II

Chlorophyll a fluorescence induction (FI) is widely used as a probe for studying photosynthesis. On illumination, fluorescence emission rises from an initial level O to a maximum P through transient steps, termed J and I. FI kinetics reflect the overall performance of photosystem II (PSII). Although FI kinetics are commonly and easily measured, there is a lack of consensus as to what controls the characteristic series of transients, partially because most of the current models of FI focus on subsets of reactions of PSII, but not the whole. Here we present a model of fluorescence induction, which includes all discrete energy and electron transfer steps in and around PSII, avoiding any assumptions about what is critical to obtaining O J I P kinetics. This model successfully simulates the observed kinetics of fluorescence induction including O J I P transients. The fluorescence emission in this model was calculated directly from the amount of excited singlet-state chlorophyll in the core and peripheral antennae of PSII. Electron and energy transfer were simulated by a series of linked differential equations. A variable step numerical integration procedure (ode15s) from MATLAB provided a computationally efficient method of solving these linked equations. This in silico representation of the complete molecular system provides an experimental workbench for testing hypotheses as to the underlying mechanism controlling the O J I P kinetics and fluorescence emission at these points. Simulations based on this model showed that J corresponds to the peak concentrations of Q _A ⁻ Q_B (Q_A and Q_B are the first and second quinone electron acceptor of PSII respectively) and Q _A ⁻ Q _B ⁻ and I to the first shoulder in the increase in concentration of Q _A ⁻ Q _B ²⁻ . The P peak coincides with maximum concentrations of both Q _A ⁻ Q _B ²⁻ and PQH₂. In addition, simulations using this model suggest that different ratios of the peripheral antenna and core antenna lead to differences in fluorescence emission at O without affecting fluorescence emission at J, I and P. An increase in the concentration of Q_B-nonreducing PSII centers leads to higher fluorescence emission at O and correspondingly decreases the variable to maximum fluorescence ratio (F _v/F _m).     

<Emphasis Type="Italic">Drosophila simulans Lethal hybrid rescue</Emphasis> mutation (<Emphasis Type="Italic">Lhr</Emphasis>) rescues inviable hybrids by restoring X chromosomal dosage compensation and causes fluctuating asymmetry of development

The Drosophila simulans Lhr rescues lethal hybrids from the cross of D. melanogaster and D. simulans. We describe here, the phenotypes of Lhr dependent rescue hybrids and demonstrate the effects of Lhr on functional morphology of the salivary chromosomes in the hybrids. Our results reveal that the phenotypes of the ‘Lhr dependent rescued’ hybrids were largely dependent on the genetic background and the dominance in species and hybrids, and not on Lhr. Cytological examination reveal that while the salivary chromosome of ‘larval lethal’ male carrying melanogaster X chromosome was unusually thin and contracted, in ‘rescued’ hybrid males (C _mel X _mel Y _sim ; A _mel A _sim ) the X chromosome showed typical pale staining, enlarged diameter and incorporated higher rate of ³H-uridine in presence of one dose Lhr in the genome. In hybrid males carrying simulans X chromosome (C _mel X _sim Y _mel ; A _mel A _sim ), enlarged width of the polytene X chromosome was noted in most of the nuclei, in Lhr background, and transcribed at higher rate than that of the single X chromosome of male. In hybrid females (both viable, e.g., C _mel X _mel X _sim ; A _mel A _sim and rescued, e.g., C _mel X _mel X _mel ; A _mel A _sim ), the functional morphology of the X chromosomes were comparable to that of diploid autosomes in presence of one dose of Lhr. In hybrid metafemales, (C _mel X _mel X _mel X _sim ; A _mel A _sim ), two dose of melanogaster X chromosomes and one dose of simulans X chromosome were transcribed almost at ‘female’ rate in hybrid genetic background in presence of one dose of Lhr. In rescued hybrid males, the melanogaster-derived X chromosome appeared to complete its replication faster than autosomes. These results together have been interpreted to have suggested that Lhr suppresses the lethality of hybrids by regulating functional activities of the X chromosome(s) for dosage compensation.     

Two-stage method for purification of ceruloplasmin based on its interaction with neomycin

A two-stage chromatography that yields highly purified ceruloplasmin (CP) from human plasma and from rat and rabbit serum is described. The isolation procedure is based on the interaction of CP with neomycin, and it provides a high yield of CP. Constants of inhibition by gentamycin, kanamycin, and neomycin of oxidase activity of CP in its reaction with p-phenylenediamine were assayed. The lowest K _i for neomycin (11 μM) corresponded to the highest specific adsorption of CP on neomycin-agarose (10 mg CP/ml of resin). Isolation of CP from 1.4 liters of human plasma using ion-exchange chromatography on UNO-Sphere Q and affinity chromatography on neomycin-agarose yields 348 mg of CP with 412-fold purification degree. Human CP preparation obtained with A ₆₁₀/A ₂₈₀ ∼ 0.052 contained neither immunoreactive prothrombin nor active thrombin. Upon storage at 37°C under sterile conditions, the preparation remained stable for two months. Efficient preparation of highly purified CP from rat and rabbit sera treated according to a similar protocol suggests the suitability of our method for isolation of CP from plasma and serum of other animals. The yield of CP in three separate purifications was no less than 78%.     

A simple extraction method suitable for PCR-based analysis of plant,fungal, and bacterial DNA

A simple and easy protocol for extracting high-quality DNA from microorganisms and plants is presented. The method involves inactivating proteins by using SDS/proteinase K and precipitating polysaccharides in the presence of high salt. Further purification is based on differential solubility of DNA and high-molecular-weight polysaccharides in aqueous media. The procedure does not use the toxic and potentially hazardous phenol and chloroform, and as many as 100 samples can be processed per day. Absorbency ratios (A₂₆₀/A₂₈₀) of 1.6–2.0 indicated a minimal presence of contaminating metabolites. The DNA was completely digested with 5 restriction enzymes:EcoR I,RsaI,TaqI,EcoR V, andHind III. PCR analysis using enterobacterial repetitive intergenic consensus (ERIC) sequence, sequence-characterized amplified region (SCAR), and random amplified microsatellite (RAMS) primers showed the DNA's compatibility with downstream applications. This procedure is applicable to a range of pathogens and plants and thus may find wide application in quarantine services and marker-assisted selection (MAS) breeding.     

Application of TMA-PEG to promote C-phycocyanin extraction from S. platensis in the PEG ATPS

A novel aqueous two phase system (ATPS) using trimethylamine-polyethylene glycols (TMA-PEG) to promote the extraction of C-phycocyanin (C-PC) from S.platensis was introduced. The purity of C-PC (EP) obtained in the ATPS of PEG1000/Na₃PO₄ was increased 2.1 times by the addition of TMA-PEG1000. The purification factor was enhanced from 2.9 to 10.1 when 65% TMA-PEG1000 was added in the system. The ATPS operation must be carried out in the pH range of 6.0-7.0 and at temperatures less than 35 °C for maintaining the stability of C-PC. The partition coefficient and recovery ratio of C-PC increased with the increasing concentration of TMA-PEG. The system parameters like TMA-PEG1000 content, tie line length (TLL), pH, temperature and phase volume ratio (V_r) were screened and optimized using the fractional factorial design and Box-Behnken experiment design. The optimized system is composed of 11.8% PEG1000/TMA-PEG1000 (w/w), 64.42% TMA-PEG1000 (w/w PEG1000) and 9.5% Na₃PO₄ (w/w) with 38.19% TLL (w/w) and 0.89 V_r at pH 6.5 and 25 °C. The obtained value of EP was 5.21 in one-stage ATPS and 6.7 in two-stage ATPS. The recovery ratio of C-PC in the new ATPS extraction system was more than 97%.     

Photosensitized formation of singlet oxygen by phycobiliproteins in neutral aqueous solutions

Phycobiliproteins (PBPs) are a type of promising sensitizers for photodynamic therapy (PDT). Upon irradiation (λ>500nm) of an oxygen-saturated aqueous solution of phycobiliproteins, particularly, C-phycocyanin (C-PC), allophycocyanin (APC) or R-phycoerythrin (R-PE), the formation of singlet oxygen (¹O₂) was detected by using imidazole in the presence of p-nitrosodimethylaniline (RNO). The bleaching of RNO caused by the presence of imidazole in our system showed typical concentration dependence with a maximum at about 8mM imidazole, which is in agreement with the formation of ¹O₂. In addition, the generation of ¹O₂ was verified further in the presence of D₂O and specific singlet oxygen quencher — 1,4-diazabicyclo [2,2,2] octane (DABCO) and sodium azide (NaN₃). Our experimental results indicated that APC possesses high ability to generate reactive oxygen species and the relative quantum yields of photogeneration of ¹O₂ by PBPs are as follows: APC > C-PC > R-PE.     

Water potential and gas exchange did not reflect performance of Pinus radiata D. Don in an agroforestry system under conditions of soil-water deficit in a temperate environment

In order to understand how radiata pines respond to declining supply of soil-water in agroforestry systems, we monitored water potential in xylem (ψ _x ), osmotic potential (ψ) and relative water content (q) for fascicles at pre-dawn and at mid-day for 3-year-old trees that were raised from either seedlings (Seedling) or from tissue culture (TC3 and TC4), and grown either alone (Control) or over lucerne (Medicago sativa) pasture (Lucerne). Water relations at dawn were mostly similar for all the pines, except late in the season when ψ was lower, bulk turgor pressure (P), deduced as the difference between ψ _x and ψ, was higher, for TC3 than for the other two pines. At mid-day, Seedling often had higher ψ _x and ψ, but because of its poor osmotic adjustment (OA) had lower P, than either TC3 or TC4. The cell walls were more elastic in Seedling with modulus of elasticity (e) of 6.5 MPa compared with 8.1 MPa for both TC3 and TC4, while loss of turgor was estimated to occur at ψ _x of −1.45 MPa for Seedling, −1.38 MPa for TC3 and −1.35 MPa for TC4. All trees irrespective of their origin had higher ψ _x , P, CO₂ assimilation (A), and stomatal conductance (g _s ), but lower ψ, in Control than in Lucerne in which the soil profile was consistently drier. The trends in ψ _x , ψ, q and A did not reflect the known differences in dry weight of trees, P was in the order TC3 > TC4 > Seedling, consistent with previously reported tree weights. Both TC3 and TC4 had higher P, due to their larger OA, than Seedling, although the latter had higher A. Thus ψ _x and A that are routinely measured may not always adequately explain differences in growth amongst pines; it is advisable that ψ be determined to allow deductions of P be made when using water relations to analyse plant growth.