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.     

Efficient purification of transglutaminase from recombinant <Emphasis Type="Italic">Streptomyces platensis</Emphasis> at various scales

An efficient system for the fast and efficient purification of transglutaminase from recombinant Streptomyces platensis and expressed in Streptomyces lividans 25-2 is described. Because the purification procedure of this system is flexible, culture broth from laboratory (20 l) and pilot-plant (130 l) fermentations were used to purify the enzyme to electrophoretic homogeneity with high purity (90–95%) and yield (61–77%) within 1 or 2 days.     

A <Emphasis Type="Italic">Magnetospirillum</Emphasis> strain WM-1 from a freshwater sediment with intracellular magnetosomes

Summary A helical shaped bacterium capable of producing magnetosomes, designated WM-1, was isolated from freshwater sediment through an improved isolated method that combined magnetic separation and the “race track” method. The strain WM-1 was Gram-negative, 0.2–0.4 μm in diameter and 3–4 μm in length. The strain WM-1 was identified as genus Magnetospirillum in the α-Proteobacteria according to the sequence analysis of the 16S rDNA, the morphology and physiological characteristics. The shape of the magnetosomes in WM-1was cuboidal by electron microscopy. Statistical analysis of WM-1 magnetosome crystals showed that the average number of magnetosomes in a WM-1 bacterium was 8 ± 3.4, and the average length was 54 ± 12.3 nm, and the average width was 43 ± 10.9 nm.     

Optimisation of the purification process of a tumour-targeting antibody produced in <Emphasis Type="Italic">N. benthamiana</Emphasis> using vacuum-agroinfiltration

It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 μg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15–20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 μg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels (<1 EU/ml).     

Purification and characterization of a mucin-binding mycelial lectin from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> with potent mitogenic activity

Mucin-specific lectin from mycelium of Aspergillus nidulans was purified using anion exchange and gel filtration chromatographic techniques with an overall recovery of 32% and 21.97-fold purification. The purified lectin migrated as a single band in SDS–PAGE with an apparent molecular mass of 34 kDa. Carbohydrate analysis revealed that it is a glycoprotein with total sugar content of 2.54%. Optimal agglutination was observed when serially diluted lectin was incubated with human type O erythrocyte suspension at pH 7.0–8.0 and temperature 20–30°C. Lectin was found to be completely stable within pH 5.0–8.0 and temperature at or below 40°C. Demetallization by extensive dialysis against EDTA did not alter its haemagglutination activity. Lectin activity was reduced to half after 24 h incubation with urea and thiourea, with no such effect of guanidine HCl. The lectin showed potent mitogenic response towards mouse splenocytes, attaining a maximum at 200 μg/ml as compared to untreated control cells. Mitogenic lectins are invaluable tools to assess the functioning of immune cells. None of the microfungal lectin has yet been investigated for mitogenic activity. This is the first report on mitogenic activity of lectin from Aspergillus sp.     

Pretreatment incubation for culture and cryopreservation of <Emphasis Type="Italic">Sabal</Emphasis> embryos

Zygotic embryos from mature seeds of Sabal jamaicensis, S. minor, S. umbraculifera and S. yapa were cultured in vitro and cryopreserved successfully. Seed pretreatment prior to embryo isolation was shown to be crucial. Soaking seeds in water or sowing on 1% agar followed by incubation at 30°C for 1 day (S. jamaicensis, S. minor and S. umbraculifera) or 3 days (S. yapa only) prior to embryo isolation increased the percentage emergence of cultured embryos from less than 20% to more than 94%. Without this pretreatment, most isolated embryos turned brown and died soon after culture. Through preincubation combined with partial dehydration to 18–28% moisture content, direct freezing and rapid thawing, isolated embryos were successfully cryopreserved and 31–44% emergence was achieved from postthawed embryos for the four Sabal species.     

Exploring the potential of clay in mitigating <Emphasis Type="Italic">Pyrodinium bahamense</Emphasis> var. <Emphasis Type="Italic">compressum</Emphasis> and other harmful algal species in the Philippines

Harmful algal bloom occurrences worldwide have prompted the testing and use of methods to control and mitigate their detrimental effects. This study investigates the potential of Philippine clay minerals to physically remove phytoplankton cells under laboratory conditions. Ball clay had the highest removal efficiency (∼95%) for Pyrodinium bahamense (paralytic shellfish poisoning causative organism) cells. A slight decrease in the efficiency by 10–20% was seen when culture volume was increased from 50 mL to 1 L. Removal efficiency was reduced to ∼95% when water motion was introduced. Removal of other phytoplankton species (Gymnodinium sanguineum, Amphidinium carterae, Pyrophacus horologium, Chatonella marina, and Alexandrium sp.) using ball clay was less efficient (<70%). Cell removal efficiencies differed with phytoplankton species belonging to the same taxonomic group. Possible mechanisms for cell removal are described.     

The effect of cocultivation treatments on transformation efficiency in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The effect of chemical additives (acetosyringone, AS; L-cysteine, CYS; dithiothreitol, DTT; glutathione, GSH; cellulase, CEL; pectinase, PEC) and light regimes (16/8 light/dark photoperiod, 16L/8D; continuous light, 24L; continuous dark, 24D) applied during cocultivation procedure of pea explants with Agrobacterium tumefaciens on transformation efficiency was studied. A hypervirulent strain of A. tumefaciens EHA 105 with two plasmids, namely pGT89 and pBIN19, both carrying reporter gus-int gene, and bar or nptII selectable marker gene, respectively, was used for genetic transformation of cotyledonary node explants of three dry seed pea cultivars Adept, Komet and Menhir. The focus was laid on cocultivation step (48 h) of transformation protocol. After chemical or physical treatments, transient GUS expression was recorded 20 days after cocultivation as a measure of successful transformation, using a four category scale (0 – without GUS expression, 1 – weak, 2 – medium and 3 – strong GUS expression) for calculation of IGE (Intensity of GUS Expression). Of the tested chemical cocultivation additives, 100 μM AS and 50 mg CYS significantly improved GUS expression (IGE value), while DTT, GSH and both macerating enzymes (CEL, PEC used either separately or in combination) either had no positive effect or were even negative. There were no statistically significant differences between the light regimes tested. Nevertheless, cocultivation in 24L, without chemical additives, reproducibly resulted in the highest frequency of explants scored in category 3 of GUS expression (followed by 24D and 16L/8D treatment). However, application of 100 μM AS reverted this trend. Cv. Adept yielded higher transformation frequencies than cvs. Menhir and Komet. Plasmid pGT89 produced a higher IGE value than pBIN19. Based on our results, the improved cocultivation step for pea consists of 48 h cocultivation at 20 ± 2°C, with 50 mg l⁻¹ CYS and 100 μM AS, 16L/8D photoperiod (or without AS in continuous light).     

Bioremediation of nitroexplosive wastewater by an yeast isolate <Emphasis Type="Italic">Pichia sydowiorum</Emphasis> MCM Y-3 in fixed film bioreactor

Nitroexplosives are essential for security and defense of the nation and hence their production continues. Their residues and transformed products, released in the environment are toxic to both terrestrial and aquatic life. This necessitates remediation of wastewaters containing such hazardous chemicals to reduce threat to human health and environment. Bioremediation technologies using microorganisms become the present day choice. High Melting Explosive (HMX) is one of the nitroexplosives produced by nitration of hexamine using ammonium nitrate and acetic anhydride and hence the wastewater bears high concentration of nitrate and acetate. The present investigation describes potential of a soil isolate of yeast Pichia sydowiorum MCM Y-3, for remediation of HMX wastewater in fixed film bioreactor (FFBR). The flask culture studies showed appreciable growth of the organism in HMX wastewater under shake culture condition within 5–6 days of incubation at ambient temperature (28 ± 2°C). The FFBR process operated in both batch and continuous mode, with Hydraulic Retention Time (HRT) of 1 week resulted in 50–55% removal in nitrate, 70–88% in acetate, 50–66% in COD, and 28–50% in HMX content. Continuous operation of the reactor showed better removal of nitrate as compared to that in the batch operation, while removal of acetate and COD was comparable in both the modes of operation of the reactor. Insertion of baffles in the reactor increased efficiency of the reactor. Thus, FFBR developed with baffles and operated in continuous mode will be beneficial for bioremediation of high nitrate and acetate containing wastewater using the culture of P. sydowiorum.     

Improved procedure for separation and purification of <Emphasis Type="Italic">Arthronema africanum</Emphasis> phycobiliproteins

A rapid, inexpensive and reliable procedure for separation and purification of C-phycocyanin (C-PC) and allophycocyanin (APC) from Arthronema africanum based on a previously described rivanol-sulfate method for C-PC purification was developed. Exclusion of NaCl from the extraction buffer resulted in complete separation of APC and C-PC, two-fold reduction of rivanol treatments, and a higher yield and purity of C-PC. Pure C-PC (A₆₂₀/A₂₈₀ of 4.52) and APC (A₆₅₂/A₂₈₀ of 2.41) were obtained. The estimated molecular masses of the α and β subunits were 17 and 19 kDа for С-phycocyanin and 16 and 18 kDа for APC, respectively. The overall C-PC recovery of 55% (w/w) from its content (100 mg) in the crude extract was 10–20% higher than so far reported. The procedure appears promising for scaling up and broader applications.     

Degradation of chloroform by immobilized cells of <Emphasis Type="Italic">Bacillus</Emphasis> sp. in calcium alginate beads

A Bacillus sp., capable of degrading chloroform, was immobilized in calcium alginate. The beads in 20 g alginate l⁻¹ (about 2 × 10⁸ cells/bead) could be re-used nine times for degradation of chloroform at 40 μM. The immobilized cells had a higher range of tolerance (pH 6.5–9 and 20–41°C) than free cells (pH 7–8.5 and 28–32°C). At 5 g alginate l⁻¹, leakage of the cells from the beads was 0.51 mg dry wt ml⁻¹. This species is the first reported Bacillus that can degrade chloroform as the sole carbon source.     

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.     

A new continuous cell line from larval ovaries of silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

A new continuous cell line from ovarian tissue of commercial variety “Kolar Gold” of silkworm, Bombyx mori, was established and designated as DZNU-Bm-12. The tissue was grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat-inactivated B. mori hemolymph at 25 ± 1°C. The migration of partially attached small round refractive cells from the fragments of ovarioles began from the beginning of explantation. The cells multiplied partially attached in the primary culture initially, and some of them become freely suspended after 20 passages. The cells were adapted to MGM-448 and TNM-FH media each with 10% FBS and the population doubling time of cell line was about 36 and 24 hr, respectively. The chromosome number was near diploid at initial passages and slightly increased at 176th passage, but a few tetraploids and hexaploids were also observed. DNA profiles using simple sequence repeat loci established the differences between DZNU-Bm-12 and DZNU-Bm-1 and most widely used Bm-5 and BmN cell lines. The cell line was found susceptible to B. mori nucleopolyhedrovirus (BmNPV) with 85–90% of the cells harboring BmNPV and having an average of 3–17 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV-based baculoviral expression system and also for studying in vitro virus replication.     

Rapid and repetitive plant regeneration of <Emphasis Type="Italic">Aralia elata</Emphasis> Seem. via somatic embryogenesis

In this work, we established a rapid and repetitive plant regeneration system for Aralia elata Seem. via primary and secondary somatic embryogenesis. Primary somatic embryogenesis was induced using leaf disks, petiole, and root segments, individually cultured for 5 weeks on Schenk and Hildebrandt (SH) (1972) medium with 0–5.0 mg/l indolebutyric acid (IBA). Our investigation demonstrated that optimal IBA concentrations of 3.0, 2.0, and 0.3 mg/l resulted in 100% somatic embryogenesis rates and averages of 11.3, 10.0, and 8.6 somatic embryos per explant for leaf disks, petiole, and root segments, respectively. The primary somatic embryos were used to conduct secondary somatic embryogenesis and the following treatments, in a gradient series, were examined: 0.3–4.0 mg/l IBA, 10–70 g/l sucrose and 0.2–3.0 mg/l abscisic acid (ABA). The results indicated that IBA was more effective than sucrose and ABA, and 3.0 mg/l IBA was the most suitable concentration for secondary somatic embryogenesis. Histological preparations indicated a multi-cellular origin of secondary somatic embryos and different morphological developmental stages during secondary somatic embryogenesis. Primary and secondary somatic embryos germinated readily and developed into normal plantlets after 2 weeks in woody plant medium (WPM, Lloyd and McCown 1980) with 20 g/l sucrose. At 4–5 cm in length, plantlets were transferred to soil (1:1 v/v of peat moss and sand) and the survival rate was 89% after 4 weeks under greenhouse conditions. This system provides a viable contribution to A. elata gene transformation, breeding and regeneration.     

Production of glutamine synthetase in <Emphasis Type="Italic">Escherichia coli</Emphasis> using SUMO fusion partner and application to <Emphasis Type="SmallCaps">l</Emphasis>-glutamine synthesis

l-glutamine (Gln) is an important conditionally necessary amino acid in human body and potential demand in food or medicine industry is expected. High efficiency of l-Gln production by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system has been developed. We report here first the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Bacillus subtilis GS. In order to obtain GS with high Gln-forming activity, safety and low cost for food and pharmaceutics industry, 0.1% (w/v) lactose was selected as inducer. The fusion protein was expressed in totally soluble form in E. coli, and expression was verified by SDS–PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel nitrilo-triacetic acid (Ni–NTA) resin chromatography with a yield of 625 mg per liter fermentation culture. After the SUMO/GS fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 121 mg recombinant GS was obtained from 1 l fermentation culture with no less than 96% purity. The recombinant purified GS showed great transferase activity (23 U/mg), with 25 U recombinant GS in a 50 ml reaction system, a biosynthesis yield of 27.5 g/l l-Gln was detected by high pressure liquid chromatography (HPLC) or thin-layer chromatography. Thus, the application of SUMO technology to the expression and purification of GS potentially could be employed for the industrial production of l-Gln.     

Purification of the photosynthetic reaction center from <Emphasis Type="Italic">Heliobacterium modesticaldum</Emphasis>

We have developed a purification protocol for photoactive reaction centers (HbRC) from Heliobacterium modesticaldum. HbRCs were purified from solubilized membranes in two sequential chromatographic steps, resulting in the isolation of a fraction containing a single polypeptide, which was identified as PshA by LC–MS/MS of tryptic peptides. All polypeptides reported earlier as unknown proteins (in Heinnickel et al., Biochemistry 45:6756–6764, 2006; Romberger et al., Photosynth Res 104:293–303, 2010) are now identified by mass spectrometry to be the membrane-bound cytochrome c ₅₅₃ and four different ABC-type transporters. The purified PshA homodimer binds the following pigments: 20 bacteriochlorophyll (BChl) g, two BChl g′, two 8¹-OH-Chl a _F, and one 4,4′-diaponeurosporene. It lacks the PshB polypeptide binding the F_A and F_B [4Fe–4S] clusters. It is active in charge separation and exhibits a trapping time of 23 ps, as judged by time-resolved fluorescence studies. The charge recombination rate of the P₈₀₀ ⁺F_X⁻ state is 10–15 ms, as seen before. The purified HbRC core was able to reduce cyanobacterial flavodoxin in the light, exhibiting a K _M of 10 μM and a k _cat of 9.5 s⁻¹ under near-saturating light. There are ~1.6 menaquinones per HbRC in the purified complex. Illumination of frozen HbRC in the presence of dithionite can cause creation of a radical at g = 2.0046, but this is not a semiquinone. Furthermore, we show that high-purity HbRCs are very stable in anoxic conditions and even remain active in the presence of oxygen under low light.     

Molecular cloning,expression in <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> of Attacin A gene from <Emphasis Type="Italic">Drosophila</Emphasis> and detection of biological activity

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.     

A study of the process of synchronisation and micronucleation in <Emphasis Type="Italic">Beta vulgaris</Emphasis> and the monitoring of an isolation procedure for micro-nuclei and micro-protoplasts by confocal laser scanning microscopy and flow cytometry

The process of synchronization and micro-nuclei induction in a suspension culture of Beta vulgaris, was induced by the sequential treatment with the DNA-synthesis inhibitor aphidicolin (30 μM, 24 h) and the spindle-toxin amiprophos-methyl (32 μM, 24 h). Mitotic arrest of divisions, spreading of G2-metaphase chromosomes, re-grouping of chromosomes, formation of a nuclear cell wall around single and re-grouped chromosomes and restitution of nuclei with a doubled DNA content was observed. The process of micro-nucleation was induced much less efficiently in Beta vulgaris than in Nicotiana plumbaginifolia. Cytological observations and monitoring of the process with flow cytometry and confocal laser scanning microscopy, was essential to follow up the course of events and to monitor the development of an efficient procedure for micro-protoplast isolation. Micro-nucleated protoplasts were fractioned by iso-osmotic Percoll gradient centrifugation to obtain heterogeneous micro-protoplast populations with cytoplasts and micro-protoplasts of different size. An enriched fraction with small sub-diploid micro-protoplasts was obtained with the equivalent DNA content of 1–4 chromosomes, as revealed by confocal laser scanning microscopy and flow cytometry. Sub-diploid micro-protoplasts with DNA amounts equivalent to 1–4 chromosomes were predominantly observed in the size classes of 1.8–10.2 μm at a frequency of 34.2–34.5%. The DNA measurements of micro-nuclei and micro-protoplasts, confirmed the hypothesis that the process of micro-nucleation followed the same course of cellular events as observed in N. plumbaginifolia. The correlation between DNA content and size of micro-nuclei and micro-protoplasts was not linear and affected by the degree of DNA condensation, total amount of DNA, and the presence of cytoplasm.