Clonal production of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Doty) Doty in vitro

Micropropagation has proven to be a reliable method to mass produce certain crops. This method also has been applied in macroalgae to produce clones for seaweed farming. Protocols for callus production and shoot regeneration from protoplasts have been established for some seaweed species like Kappaphycus alvarezii. Cells and larger tissues, whether in solid or suspension medium, have been used to propagate clones which were later tested for suitability for farming. Although clonal production was successful, the long duration of culture in vitro limits the production process making the growing of Kappaphycus in vitro an expensive technique to produce clones. In this study, K. alvarezii was grown in vitro to develop a more efficient protocol for the production of clones. Small sections of Kappaphycus were grown in suspension for 1 month under the same temperature, light, and salinity. The type of media, source of explants, length of explants, and stocking density that resulted in the highest growth rate and survival rate were determined. Growth rate of K. alvarezii is significantly higher in media with inorganic nitrogen added than in Grund medium or Ascophyllum nodosum medium only. The appearance of shoot primordia as early as 5 days was observed in media with higher nitrogen concentration. Growth rates of explants approximately 3 and 5 mm are significantly higher than 10 mm sections. Shoots develop significantly faster in explants from tips than sections from older branches. Growth rate of K. alvarezii grown at 0.5, 0.75, 1, 1.25 s 10 mL^?1 of medium is not significantly different. This protocol could significantly reduce the (1) time of culture and (2) cost of plantlets production by not using plant growth regulators and formulated media in vitro. Nursery reared plantlets/propagules for farming would be affordable to the stakeholders for sustainability of seaweed production.     

Effects of extraction parameters on gel properties of carrageenan from <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Rhodophyta)

Carrageenan was isolated under different extraction conditions from Kappaphycus alvarezii collected in North Sulawesi, Indonesia. Its gel properties include very strong elasticity even at low concentrations. Molecular weight and rheological properties were obtained by gel permeation chromatography and dynamic viscoelasticity measurements in order to clarify the average molecular weight at various extraction temperatures (50, 70, 90°C) and times (1, 3, 5 h), as well as gel formation ability. The results showed that both the weight-average and the number-average molecular weight decreased with increasing extraction temperature. However, the gelation rate of the carrageenan was found to be constant at around 40°C, whereas the storage modulus, G′, and loss modulus, G″, of the gels differed from each other.     

Plantlet regeneration of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> var. adik-adik by tissue culture

Three color morphotypes of Kappaphycus alvarezii var. adik-adik (brown, green and red) collected from a farming area in Tictauan Is., Zamboanga City, Philippines were used as explants in the study in order to micropropagate ‘new’ plants. Individual sections of sterile Kappaphycus alvarezii var. adik-adik, initially cultured in a 48-well culture plate containing ESS/2 + E3 + PGR, released callus cells after 4–5 days of incubation at 23–25°C, 13:11H LD cycle and 10–15 μmol photons m⁻² s⁻¹ light intensity. True calli were formed after 29–35 days following dense formation of filaments or undifferentiated round cells at the medullary and inner cortical layers of the section. Plantlets (2–3 mm long) of Kappaphycus alvarezii var. adik-adik were able to regenerate after 98, 150 and 177 days in-vitro among the reds, greens, and browns, respectively. This study established successful methods for the production and regeneration of tissue explants of Kappaphycus alvarezii var. adik-adik which can possibly be used to mass produce ‘new’ cultivars for land- and sea-based nurseries as sources for commercial farming. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

<Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Rhodophyta,Areschougiaceae) cultivated in subtropical waters in Southern Brazil

Four strains of Kappaphycus alvarezii were cultivated in the subtropical waters of Florianópolis, Santa Catarina State, Brazil (27°29′19′′ S/48°32′28′′ W), from February 2009 to February 2010. Seaweeds were cultivated on floating raft near of mussel farms. Salinity ranged from 29 to 36 psu and temperature from 17.1 to 28.5°C. Higher growth rates (5.12–4.29% day⁻¹) were measured in summer and autumn, showing a positive correlation between growth rate and water temperature. Lower growth rates (0.54–0.32% day⁻¹) occurred in winter, resulted mainly by biomass loss. Significant differences were observed among the strains in spring and the brown tetrasporophytic strain was the only one which failed to recover, being excluded of the experiments. The effect of cultivation periods (36, 42, and 97 days) on carrageenan yield, gel strength, and viscosity were analyzed. Carrageenan yields were higher for plants kept 42 days in the sea (28%), against 25% for 36 and 97 days. There were no significant differences in carrageenan yield among the strains analyzed. Viscosity increased with the increase of cultivation period, while gel strength seemed to vary at random. Tetrasporangia and cystocarps were not observed, and lost fragments did not attach outside the raft. In general, dissolved inorganic nitrogen concentration decreased around the cultivation area as compared to the mussel farm. Results show that cultivation of K. alvarezii is technically feasible in subtropical waters and can be associated with local mussel farms, mitigating the eutrophication and, eventually, increasing the economic return of the farmers.     

Preparation,structural characterization and <Emphasis Type="Italic">in vitro</Emphasis> antitumor activity of <Emphasis Type="Italic">kappa</Emphasis>-carrageenan oligosaccharide fraction from <Emphasis Type="Italic">Kappaphycus striatum</Emphasis>

Oligosaccharides were prepared through mild hydrochloric acid hydrolysis of kappa-carrageenan from Kappaphycus striatum to compare the antitumor activity with carrageenan polysaccharides. Oligosaccharide fractions were isolated by gel permeation chromatography and the structure of fraction 1 (F1) was studied by using negative-ion electrospray ionization-mass spectrometry (ESI-MS), and ¹H and ¹³C-NMR spectrometry. The in vitro antitumor effects in three human neoplastic cell lines (KB, BGC, and Hela) of polysaccharides and F1 were investigated. The bioassay results showed that F1 exhibited relatively higher antitumor activity against the three cancer cells than polysaccharides.     

Lectin genes in the <Emphasis Type="Italic">Frankia alni</Emphasis> genome

Frankia alni strain ACN14a’s genome was scanned for the presence of determinants involved in interactions with its host plant, Alnus spp. One such determinant type is lectin, proteins that bind specifically to sugar motifs. The genome of F. alni was found to contain 7 such lectin-coding genes, five of which were of the ricinB-type. The proteins coded by these genes contain either only the lectin domain, or also a heat shock protein or a serine-threonine kinase domain upstream. These lectins were found to have several homologs in Streptomyces spp., and a few in other bacterial genomes among which none in Frankia EAN1pec and CcI3 and two in strain EUN1f. One of these F. alni genes, FRAAL0616, was cloned in E. coli, fused with a reporter gene yielding a fusion protein that was found to bind to both root hairs and to bacterial hyphae. This protein was also found to modify the dynamics of nodule formation in A. glutinosa, resulting in a higher number of nodules per root. Its role could thus be to permit binding of microbial cells to root hairs and help symbiosis to occur under conditions of low Frankia cell counts such as in pioneer situations.     

Cloning,Expression and Purification of <Emphasis Type="Italic">Microcystis viridis</Emphasis> Lectin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Microcystis viridis lectin (MVL), a sugar-binding protein originally isolated from freshwater blue-green algae Microcystis viridis, has been reported to have potent anti-HIV activity. In this paper, we described the expression and purification of recombinant-MVL (R-MVL) gene in E. coli. The results demonstrated that the R-MVL in shake flask cultures was primarily expressed either in the form of inclusion bodies at 37°C or in the soluble fraction at 23 °C. Secondly, a one-step purification based on nickel-affinity chromatography was employed and 15 mg of highly purified (>95%) R-MVL from 1 l of cell cultures was yielded. The purified R-MVL was then subjected to MALDI-TOF–MS analysis for protein identification. In conclusion, for the first time, the R-MVL was successfully cloned and expressed in E. coli, which is useful for further study and large-scale cost-effective production of MVL protein.     

Isolation of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Specific Phages with Broad Activity Spectra

The aim of the study was to screen various kinds of samples for Pseudomonas aeruginosa specific phages and to isolate and partially characterize those with broad activity spectra. The Pseudomonas specific phages were isolated using an enrichment procedure with single strains or the cocktail of P. aeruginosa strains as hosts. Using the described procedure, phages were successfully isolated only from water samples, while in soil and feces no Pseudomonas specific phages were detected. The lytic spectra of isolated phages were determined by spot method on lawns of 33 P. aeruginosa strains and five species belonging to family Enterobacteriaceae. The results showed that among isolated phages, 001A, δ, and I possessed the broad activity spectra, as were able to plaque on more than 50% of tested P. aeruginosa strains, while none of the phages were able to lyse the other tested species. Significant differences in phage activity spectra were not observed when P. aeruginosa cocktail was applied for sample enrichment. The most of the phages examined by electron microscopy belonged to family Siphoviridae, while the broad activity spectra isolates, except for 001A, possessed morphological characteristics of family Podoviridae. Digested DNA of the phages δ and I showed similar patterns, indicating the prevalence and success of this phage type in the environment.     

Isolation of protoplasts from tissue fragments of Philippine cultivars of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Solieriaceae,Rhodophyta)

Protoplasts were isolated from tissue fragments (<1 mm²) of three Philippine cultivars of Kappaphycus alvarezii: the giant cultivar, cultivar L and Bohol wild type, by enzymatic dissolution of cell walls. Yields of viable protoplasts from young and old thalli (apical, middle, basal segments) were compared at various temperatures, duration of treatment and pH using eight combinations of commercial enzymes (abalone acetone powder and cellulase), and prepared extracts from fresh viscera of abalone (Haliotis asinina) and a terrestrial garden snail. Isolated protoplasts were grown in various culture media, temperatures, photoperiods and irradiance values to determine the conditions that favor germination and growth.Protoplast yields in tissues treated with commercial enzymes and the garden snail extract were lower than those obtained in tissues treated with fresh abalone extracts. Generally, the number of viable protoplasts increased with duration of enzyme treatment at 25 °C with a maximum yield of 8.2 × 10³ g⁻¹ tissue at 48 h. Yields were consistently higher in all cultivars at pH 6.1. The yields were also high from the middle segments of the giant cultivar (3.7 × 10³ g⁻¹ tissue) and Bohol wild type (4.5 × 10³ g⁻¹ tissue) treated with fresh abalone extract, and from basal segments of cultivar L and tissues treated with garden snail extract. The germination rate of protoplasts was highest (39.8%) at 25 °C and 20 μmol photon m⁻² s⁻¹, using a 12:12 light dark photoperiod. The filament was 3.7 mm long by Day 5. These findings are relevant to developing cultures from protoplasts for genetic or strain improvement of K. alvarezii cultivars.     

Isolation and Biochemical Characterization of a Galactoside Binding Lectin from <Emphasis Type="Italic">Bauhinia variegata</Emphasis> Candida (BvcL) Seeds

A new lectin (BvcL) from seeds of a primitive Brazilian Caesalpinoideae, the Bauhinia variegata candida was purified and biochemical characterized. BvcL was isolated by gel filtration chromatography on Sephadex G75 and affinity chromatography on immobilized d-lactose column. SDS-PAGE showed that BvcL under non-reducing condition presents two bands of 68 and 32 kDa and a single band of 32 kDa in reducing condition. However, only one band was seen in native PAGE. The hemagglutination activity of BvcL was not specific for any human blood group trypsin-treated erythrocytes. Carbohydrate inhibition analysis indicated that BvcL is inhibited by lactose, galactose, galactosamine and other galactoside derivates. Amino acid analysis revealed a large content of Ser, Gly, Thr, Asp and Glu and low concentrations of Met, Cys and His. Intrinsic fluorescence of BvcL was not significantly affected by sugar binding galactose; and aromatic-region CD is unusually high for plant lectins. The N-terminal amino acid sequence of 17 residues showed 90% sequential homology to galactose-specific legume lectins of the subfamily Caesalpinoideae.     

Effects of UVB radiation on the carragenophyte <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Rhodophyta,Gigartinales): changes in ultrastructure,growth, and photosynthetic pigments

Damage to the ozone layer has led to increased levels of ultraviolet radiation at the earth’s surface. Increased ultraviolet radiation can affect macroalgae in many important ways, including reduced growth rate, changes in cell biology and ultrastructure. Kappaphycus alvarezii is a red macroalga of economic interest due to its production of kappa carrageenan. In this study, we examined two strains of K. alvarezii (green and red) exposed to ultraviolet B radiation (UVBR) for 3 h per day during 28 days of cultivation in vitro. UVBR caused changes in the ultrastructure of cortical and subcortical cells, which included increased thickness of the cell wall and plastoglobuli, reduced intracellular spaces, changes in the cell contour, and destruction of chloroplast internal organization. While the green strain exposed to photosynthetically active radiation (PAR) showed growth rates of 6.75% day⁻¹, the red strain grew only 6.35% day⁻¹. Upon exposure to PAR + UV-B, a decreasing trend in growth rates was observed for both strains, with the green strain growing 3.0% day⁻¹ and the red strain growing 2.77% day⁻¹. Significant differences in growth rates between control and UV-B-exposed algae were also found in both strains. Furthermore, compared with control algae, phycobiliprotein contents (phycoerythrin, phycocyanin, and allophycocyanin) were observed to decrease in both strains after PAR + UV-B exposure. However, while the chlorophyll a levels increased in both strains, the green strain showed no significant differences in chlorophyll a levels. Taken together, these findings strongly suggested that UVBR negatively affects the ultrastructure, growth rates, and photosynthetic pigments of intertidal macroalgae and, in the long term, their economic viability.     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Effect of ultraviolet-B radiation on growth,photosynthetic pigments,and cell biology of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Rhodophyta,Gigartinales) macroalgae brown strain

Kappaphycus alvarezii is a seaweed of great economic importance for the extraction of kappa carrageenan from its cell walls. The most common strains are dark red, brown, yellow, and different gradations of green. It is known that ultraviolet radiation (UVR) affects macroalgae in many important ways, including reduced growth rate, reduction of primary productivity, and changes in cell biology and ultrastructure. Therefore, we examined the brown strain of K. alvarezii exposed to ultraviolet-B radiaton (UVBR) for 3 h per day during 28 days of cultivation. The control plants showed growth rates of 7.27% d⁻¹, while plants exposed to UVBR grew only 4.0% d⁻¹. Significant differences in growth rates and in phycobiliproteins between control and exposed plants were also found. Compared with control plants, phycobiliprotein contents were observed to decrease after UV-B exposure. Furthermore, the chlorophyll a (Chl a) contents decreased and showed significant differences. UVBR also caused changes in the ultrastructure of cortical and subcortical cells, which included increased thickness of the cell wall and number of plastoglobuli, reduced intracellular spaces, changes in the cell contour, and destruction of chloroplast internal organization. Reaction with Toluidine Blue showed an increase in the thickness of the cell wall, and Periodic Acid-Schiff stain showed a decrease in the number of starch grains. By the significant changes in growth rates, photosynthetic contents and ultrastructual changes observed, it is clear that UVBR negatively affects intertidal macroalgae and, by extension, their economic viability.     

Isolation of a Novel <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-lactosamine Specific Lectin from <Emphasis Type="Italic">Alocasia cucullata</Emphasis> (Schott.)

An N-acetyl-d-lactosamine (LacNAc) specific lectin from tubers of Alocasia cucullata was purified by affinity chromatography on asialofetuin-linked amino activated silica. The pure lectin showed a single band in SDS-PAGE at pH 8.8 and was a homotetramer with a subunit molecular mass of 13.5 kDa and native molecular mass of 53 kDa. It was heat stable up to 55 °C for 15 min and showed optimum hemagglutination activity from pH 2 to 11. The lectin was affected by denaturing agents such as urea (2 m), thiourea (2 m) and guanidine–HCl (0.5 m) and did not require Ca²⁺ and Mn²⁺ for its activity. It was a potent mitogen at 10 μg/ml towards human peripheral blood mononuclear cells with 50% growth inhibitory potential towards SiHa (human cervix ) cancer cell line at 100 μg/ml.     

<Emphasis Type="Italic">KRAS</Emphasis>, <Emphasis Type="Italic">BRAF</Emphasis>, <Emphasis Type="Italic">EGFR</Emphasis> and <Emphasis Type="Italic">HER2</Emphasis> gene status in a Spanish population of colorectal cancer

To evaluate the KRAS, BRAF, EGFR, and HER2 gene status in colorectal cancer by novel techniques and evaluate whether anti-HER2 therapies could be offered in the treatment of these patients. There are conflicting data on the prevalence of BRAF mutations and EGFR and HER2 gene amplification in colorectal KRAS wild type patients. In our study we tried to evaluate these expressions and their relationship to future treatment assays. Clinical–pathological data and paraffin-embedded specimens were collected from 186 patients who underwent colorectal resections at General Yagüe Hospital in Burgos, Spain. KRAS and BRAF status was analyzed by real-time PCR in all patients. EGFR and HER2/NEU gene amplification was detected using fluorescent in situ hybridisation technique (FISH) in 38 KRAS and BRAF wild type patients. KRAS mutations were present in 48% of the colorectal cancer patients. BRAF mutations were present in 6.25% of the KRAS wild type patients. EGFR and HER2 gene amplification was observed in 5.3% and 26.3%, respectively, of KRAS and BRAF wild type colorectal cancer patients. HER2, but not EGFR gene amplification, was frequently observed in KRAS and BRAF wild type colorectal cancer patients. These data indicate that HER2 amplification could be one of the genes to be considered in the therapeutic management of colorectal cancer.     

<Emphasis Type="Italic">Funastrum rupicola</Emphasis> (<Emphasis Type="Italic">Apocynaceae:</Emphasis><Emphasis Type="Italic">Asclepiadoideae</Emphasis>), a new species from Bolivia

Summary   Funastrum rupicola Goyder, a new species of Apocynaceae: Asclepiadoideae from Bolivia, is described and illustrated. The conservation status of this species is assessed.