Purification and partial characterization of an extracellular alginate lyase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> isolated from brown seaweed

The extracellular enzyme alginate lyase produced from marine fungus Aspergillus oryzae isolated from brown alga Dictyota dichotoma was purified, partially characterized, and evaluated for its sodium alginate depolymerization abilities. The enzyme characterization studies have revealed that alginate lyase consisted of two polypeptides with about 45 and 50 kDa each on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and showed 140-fold higher activity than crude enzyme under optimized pH (6.5) and temperature (35°C) conditions. Zn²⁺, Mn²⁺, Cu²⁺, Mg²⁺, Co²⁺ and NaCl were found to enhance the enzyme activity while (Ca²⁺, Cd²⁺, Fe²⁺, Hg²⁺, Sr²⁺, Ni²⁺), glutathione, and metal chelators (ethylenediaminetetraacetic acid and ethylene glycol tetraacetic acid) suppressed the activity. Fourier transform infrared and thin-layer chromatography analysis of depolymerized sodium alginate indicated the enzyme specificity for cleaving at the β-1,4 glycosidic bond between polyM and polyG blocks of sodium alginate and therefore resulted in estimation of relatively higher polyM content than polyG. Comparison of chemical shifts in ¹³C nuclear magnetic resonance spectra of both polyM and polyG from that of sodium alginate also showed further evidence for enzymatic depolymerization of sodium alginate.     

Mesograzers prefer mostly native seaweeds over the invasive brown seaweed <Emphasis Type="Italic">Sargassum muticum</Emphasis>

The destruction of wetlands due to afforestation areas is a common activity in temperate and subtropical regions in Southern America. The expansion of pine (Pinus elliottii Engelm.) in the Coastal Plain of Southern Brazil is critical and its impacts on aquatic biodiversity are little known. We tested the hypotheses that: (1) pine occurrence diminishes the macrophyte richness and changes the macrophyte composition in ponds; (2) beta-diversity between natural and pine ponds is determined mainly by species nestedness (species loss). Sampling was carried out from 2007 to 2009 in five ponds in pine invasion matrix and five ponds in native grassland matrix. In natural ponds, the total richness was 87 species, followed by 51 species in pine ponds. From the total richness, 42 species were shared between natural and pine ponds. The natural ponds were richer than the pine ponds along the entire study, and the composition of macrophyte species was different between natural and pine ponds. Comparing natural ponds with each other and pine ponds with each other, the species turnover (species replacement) was determinant for beta-diversity, however, when we compared natural and pine ponds, we found that nestedness and species turnover were equivalent for beta-diversity. The increase in the nestedness mechanism indicates that the pine occurrence implies in species loss in Southern Brazil ponds. The change of hydroperiod may be one of the causes for the macrophyte species loss. The removal of pine from areas destined to conservation in Southern Brazil is urgent as well as a proper management of pine plantations in order to minimise its expansions and impacts in the aquatic biodiversity, since 90% of its wetlands have been already lost.     

Peroxynitrite-scavenging constituents from the brown alga<Emphasis Type="Italic">Sargassum thunbergii</Emphasis>

Peroxynitrite formationin vivo is implicated in numerous human diseases and there is considerable interest in the use of antioxidants and natural products for their treatment. The three components (1–3) isolated fromSargassum thunbergii as well as the organic solventsoluble fractions and the aqueous layer ofS. thunbergii were evaluated for their potential to scavenge authentic ONOO and ONOO derived from 3-morpholinosydnonimine (SIN-1). The antioxidant activity of the individual fractions was in the order of 85% aquaous (aq.) MeOH>n-BuOH>n-hexane> H₂O. The three known compounds sargahydroquinoic acid (1), sargaquinoic acid (2) and sargachromenol (3) showed peroxynitrite-scavenging activities comparable to those of L-ascorbic acid and penicillamine. These results showed a possible antioxidant activity in major constituents ofS. thunbergii.     

Development and characterization of <Emphasis Type="Italic">japonica</Emphasis> rice lines carrying the brown planthopper-resistance genes <Emphasis Type="Italic">BPH12</Emphasis> and <Emphasis Type="Italic">BPH6</Emphasis>

The brown planthopper (Nilaparvata lugens Stål; BPH) has become a severe constraint on rice production. Identification and pyramiding BPH-resistance genes is an economical and effective solution to increase the resistance level of rice varieties. All the BPH-resistance genes identified to date have been from indica rice or wild species. The BPH12 gene in the indica rice accession B14 is derived from the wild species Oryza latifolia. Using an F₂ population from a cross between the indica cultivar 93-11 and B14, we mapped the BPH12 gene to a 1.9-cM region on chromosome 4, flanked by the markers RM16459 and RM1305. In this population, BPH12 appeared to be partially dominant and explained 73.8% of the phenotypic variance in BPH resistance. A near-isogenic line (NIL) containing the BPH12 locus in the background of the susceptible japonica variety Nipponbare was developed and crossed with a NIL carrying BPH6 to generate a pyramid line (PYL) with both genes. BPH insects showed significant differences in non-preference in comparisons between the lines harboring resistance genes (NILs and PYL) and Nipponbare. BPH growth and development were inhibited and survival rates were lower on the NIL-BPH12 and NIL-BPH6 plants compared to the recurrent parent Nipponbare. PYL-BPH6 + BPH12 exhibited 46.4, 26.8 and 72.1% reductions in population growth rates (PGR) compared to NIL-BPH12, NIL-BPH6 and Nipponbare, respectively. Furthermore, insect survival rates were the lowest on the PYL-BPH6 + BPH12 plants. These results demonstrated that pyramiding different BPH-resistance genes resulted in stronger antixenotic and antibiotic effects on the BPH insects. This gene pyramiding strategy should be of great benefit for the breeding of BPH-resistant japonica rice varieties.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Seasonal variation in nutritional composition and anti-proliferative activity of brown seaweed, <Emphasis Type="Italic">Sargassum oligocystum</Emphasis>

This study investigated the nutritional components and anti-proliferative activity of Sargassum oligocystum against an adriamycin-resistant human small cell lung carcinoma cell line (GLC4/Adr). The seaweed samples were collected from Nang Rong Beach, Chonburi Province, Thailand in February (the hot/dry season), May (the early monsoon season), August (the monsoon season) and December (the cool/dry season) of 2012. In general, the nutritional composition of S. oligocystum was high during the hot dry and early monsoon season. Additionally, four different types of extract were obtained: ethanolic extract (E1), lipophilic extract (E2), acidic extract (E3) and alkali extract (E4). The highest anti-proliferative activity was found in samples collected during the monsoon season. It is noteworthy that the E2 extract collected during the monsoon season was the most effective, with an IC₅₀ (half maximal inhibitory concentration) value of 2.52 ± 0.54 μg mL^?1. The anti-proliferative activity showed a positive correlation with vitamin E (α-tocopherol) in the extracts. However, there were no clear correlations between the total phenolic or fucoxanthin contents and anti-proliferative activity. These results indicate that S. oligocystum has potential as an alternative source for functional food or cancer treatments in the future.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

Antiviral polysaccharides isolated from Hong Kong brown seaweed <Emphasis Type="Italic">Hydroclathrus clathratus</Emphasis>

Two relatively pure polysaccharides H3-a1 and H3-b1 had been isolated from the brown seaweed Hydroclathrus clathratus. They were characterized by HPLC, ultraviolet scanning, gas chromatography, infrared spectroscopy and elemental analysis, and shown to be two different sulfated polysaccharides with different monosaccharide content, but both with high relative molecular mass. They contained some proteins and uronic acid respectively. The sulfate content and bioactivity of these polysaccharides varied during purification. The fractions derived from the hot water extract also exhibited low anticoagulant effect. This is the first time that the antiherpetic and anticoagulant activities were evaluated for the polysaccharides from the Hong Kong brown seaweed Hydroclathrus clathratus.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Polysaccharide and lipid composition of the brown seaweed <Emphasis Type="Italic">Laminaria gurjanovae</Emphasis>

Polysaccharide and lipid composition of the Pacific brown seaweed Laminaria gurjanovae is determined. Alginic acid is shown to be the main polysaccharide of its biomass (about 28%); it consists of mannuronic and guluronic acid residues at a ratio of 3: 1. The yield of water-soluble polymannuronic acid is low and does not exceed 1.1% of dry biomass. High laminaran content (about 22%) is found, whereas the yield of fucoidan is no more than 3.6%. Laminaran consists of two fractions, soluble and insoluble in cold water, their ratio is 2.5: 1. Insoluble laminaran is a practically linear 1,3-β-D-glucan, and the soluble fraction was shown to be 1,3;1,6-β-D-glucan. The oligosaccharide products of desulfation or partial acidic hydrolysis of fucoidan were studied by MALDI TOF MS; they were found to be fuco- and galactooligosaccharides. The fucoidan is suggested to be a highly sulfated partially acetylated galactofucan (Fuc/Gal is ～1: 1). The main lipid components of the dried L. gurjanovae are neutral lipids and glyceroglycolipids, whereas phospholipids are found in minor amounts. The main fatty acid components of lipids are 14:0, 16:0, 16:1 ω-7, 18:1 ω-7 and 18:2 ω-6 acids.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

Purification and characterization of cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript> from <Emphasis Type="Italic">Acaryochloris marina</Emphasis>

Cytochrome c ₆ , (cyt c ₆) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E _m) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron transfer processes is discussed.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.