The legacy of terrestrial plant evolution on cell wall fine structure.

The evolution of land flora was an epochal event in the history of planet Earth. The success of plants, and especially flowering plants, in colonizing all but the most hostile environments required multiple mechanisms of adaptation. The mainly polysaccharide-based cell walls of flowering plants, which are indispensable for water transport and structural support, are one of the most important adaptations to life on land. Thus, development of vasculature is regarded as a seminal event in cell wall evolution, but the impact of further refinements and diversification of cell wall compositions and architectures on radiation of flowering plant families is less well understood. We approached this from a glyco-profiling perspective and, using carbohydrate microarrays and monoclonal antibodies, studied the cell walls of 287 plant species selected to represent important evolutionary dichotomies and adaptation to a variety of habitats. The results support the conclusion that radiation of flowering plant families was indeed accompanied by changes in cell wall fine structure and that these changes can obscure earlier evolutionary events. Convergent cell wall adaptations identified by our analyses do not appear to be associated with plants with similar lifestyles but that are taxonomically distantly related. We conclude that cell wall structure is linked to phylogeny more strongly than to habitat or lifestyle and propose that there are many approaches of adaptation to any given ecological niche.

Structural Characterization and Cytotoxic Activity Evaluation of Ulvan Polysaccharides Extracted from the Green Algae Ulva papenfussii.

Ulvan, a sulfated heteropolysaccharide with structural and functional properties of interest for various uses, was extracted from the green seaweed Ulva papenfussii. U. papenfussii is an unexplored Ulva species found in the South China Sea along the central coast of Vietnam. Based on dry weight, the ulvan yield was ~15% (w/w) and the ulvan had a sulfate content of 13.4 wt%. The compositional constitution encompassed L-Rhamnose (Rhap), D-Xylose (Xylp), D-Glucuronic acid (GlcAp), L-Iduronic acid (IdoAp), D-Galactose (Galp), and D-Glucose (Glcp) with a molar ratio of 1:0.19:0.35:0.52:0.05:0.11, respectively. The structure of ulvan was determined using High-Performance Liquid Chromatography (HPLC), Fourier Transform Infrared Spectroscopy (FT-IR), and Nuclear Magnetic Resonance spectroscopy (NMR) methods. The results showed that the extracted ulvan comprised a mixture of two different structural forms, namely ("A3s") with the repeating disaccharide [→4)-ß-D-GlcAp-(1→4)-α-L-Rhap 3S-(1→]n, and ("B3s") with the repeating disaccharide [→4)-α-L-IdoAp-(1→4)-α-L-Rhap 3S(1→]n. The relative abundance of A3s, and B3s was 1:1.5, respectively. The potential anticarcinogenic attributes of ulvan were evaluated against a trilogy of human cancer cell lineages. Concomitantly, Quantitative Structure-Activity Relationship (QSAR) modeling was also conducted to predict potential adverse reactions stemming from pharmacological interactions. The ulvan showed significant antitumor growth activity against hepatocellular carcinoma (IC50 ≈ 90 µg/mL), human breast cancer cells (IC50 ≈ 85 µg/mL), and cervical cancer cells (IC50 ≈ 67 µg/mL). The QSAR models demonstrated acceptable predictive power, and seven toxicity indications confirmed the safety of ulvan, warranting its candidacy for further in vivo testing and applications as a biologically active pharmaceutical source for human disease treatment.

Structural and functional characterization of the novel endo-α(1,4)-fucoidanase Mef1 from the marine bacterium Muricauda eckloniae.

Fucoidanases (EC 3.2.1.-) catalyze the hydrolysis of glycosidic bonds between fucose residues in fucoidans. Fucoidans are a compositionally and structurally diverse class of fucose-containing sulfated polysaccharides that are primarily found in brown seaweeds. Here, the structural characterization of a novel endo-α(1,4)-fucoidanase, Mef1, from the marine bacterium Muricauda eckloniae is presented, showing sequence similarity to members of glycoside hydrolase family 107. Using carbohydrate polyacrylamide gel electrophoresis and nuclear magnetic resonance analyses, it is shown that the fucoidanase Mef1 catalyzes the cleavage of α(1,4)-linkages between fucose residues sulfated on C2 in the structure [-3)-α-L-Fucp2S-(1,4)-α-L-Fucp2S-(1-]n in fucoidan from Fucus evanescens. Kinetic analysis of Mef1 activity by Fourier transform infrared spectroscopy revealed that the specific Mef1 fucoidanase activity (Uf) on F. evanescens fucoidan was 0.1 × 10-3 Uf µM-1. By crystal structure determination of Mef1 at 1.8 Šresolution, a single-domain organization comprising a (ß/α)8-barrel domain was determined. The active site was in an extended, positively charged groove that is likely to be designed to accommodate the binding of the negatively charged, sulfated fucoidan substrate. The active site of Mef1 comprises the amino acids His270 and Asp187, providing acid/base and nucleophile groups, respectively, for the hydrolysis of glycosidic bonds in the fucoidan backbone. Electron densities were identified for two possible Ca2+ ions in the enzyme, one of which is partially exposed to the active-site groove, while the other is very tightly coordinated. A water wire was discovered leading from the exterior of the Mef1 enzyme into the active site, passing the tightly coordinated Ca2+ site.

Impact of Enzymatically Extracted High Molecular Weight Fucoidan on Lipopolysaccharide-Induced Endothelial Activation and Leukocyte Adhesion.

The endothelial cell lining creates an interface between circulating blood and adjoining tissue and forms one of the most critical barriers and targets for therapeutical intervention. Recent studies suggest that fucoidans, sulfated and fucose-rich polysaccharides from brown seaweed, show multiple promising biological effects, including anti-inflammatory properties. However, their biological activity is determined by chemical characteristics such as molecular weight, sulfation degree, and molecular structure, which vary depending on the source, species, and harvesting and isolation method. In this study, we investigated the impact of high molecular weight (HMW) fucoidan extract on endothelial cell activation and interaction with primary monocytes (MNCs) in lipopolysaccharide (LPS)-induced inflammation. Gentle enzyme-assisted extraction combined with fractionation by ion exchange chromatography resulted in well-defined and pure fucoidan fractions. FE_F3, with a molecular weight ranging from 110 to 800 kDa and a sulfate content of 39%, was chosen for further investigation of its anti-inflammatory potential. We observed that along with higher purity of fucoidan fractions, the inflammatory response in endothelial mono- and co-cultures with MNCs was reduced in a dose-dependent manner when testing two different concentrations. This was demonstrated by a decrease in IL-6 and ICAM-1 on gene and protein levels and a reduced gene expression of TLR-4, GSK3ß and NF-kB. Expression of selectins and, consequently, the adhesion of monocytes to the endothelial monolayer was reduced after fucoidan treatment. These data indicate that the anti-inflammatory effect of fucoidans increases with their purity and suggest that fucoidans might be useful in limiting the inflammatory response of endothelial cells in cases of LPS-induced bacterial infection.

Establishment of specific age-related macular degeneration relevant gene expression panels using porcine retinal pigment epithelium for assessing fucoidan bioactivity.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of severe vision loss in industrialized nations. Important factors in pathogenesis are oxidative stress, inflammation, and, in the wet form of AMD, angiogenesis. Fucoidans, sulfated polysaccharides from brown algae, may have antioxidant, anti-inflammatory, and antiangiogenic effects. In this study, we established specific gene expression panels for inflammation, oxidative stress and angiogenesis in porcine retinal pigment epithelium (RPE), and investigated the effect of fucoidans on gene expression under different noxious agents. METHODS: Primary porcine RPE cells cultured for at least 14 days were used. Using viability assays with tetrazolium bromide and real-time polymerase chain reaction of marker genes, positive controls were established for appropriate concentrations and exposure times of selected noxious agents (lipopolysaccharide (LPS), H2O2, CoCl2). Three different AMD relevant gene panels specific for porcine RPE for inflammation, oxidative stress, and angiogenesis were established, and the influence of fucoidans (mainly Fucus vesiculosus; FV) on gene expression was investigated. RESULTS: The following was shown by gene expression analyses: (1) Inflammation panel: Expression of 18 genes was affected under LPS (three days). Among them, LPS increased genes for interleukin 1 receptor 2, interleukin 8, cyclooxygenase-2 and vascular cell adhesion protein 1 expression which were diminished when FV was present. (2) Oxidative stress panel: Under stimulation of H2O2 (one day) and LPS (one day), expression of a total of 15 genes was affected. LPS induced increase in genes for superoxide dismutase-1, C-X-C motif chemokine 10, and CC chemokine ligand-5 expression was not detected when FV was present. (3) Angiogenesis panel: Under stimulation with CoCl2 (three days) expression of six genes was affected, with the increase of genes for angiopoietin 2, vascular endothelial growth factor receptor-1, and follistatin being diminished when FV was present. CONCLUSION: Three specific gene expression panels for porcine RPE that map genes for three of the major pathological factors of AMD, inflammation, oxidative stress, and angiogenesis, were established. Further, we demonstrated that fucoidans can reduce stress related gene activation in all of these three major pathogenic pathways. This study is another indication that fucoidans can act on different pathomechanisms of AMD simultaneously, which provides further evidence for fucoidans as a possible drug for treatment and prevention of AMD.

The Endo-α(1,3)-Fucoidanase Mef2 Releases Uniquely Branched Oligosaccharides from Saccharina latissima Fucoidans.

Fucoidans are complex bioactive sulfated fucosyl-polysaccharides primarily found in brown macroalgae. Endo-fucoidanases catalyze the specific hydrolysis of α-L-fucosyl linkages in fucoidans and can be utilized to tailor-make fucoidan oligosaccharides and elucidate new structural details of fucoidans. In this study, an endo-α(1,3)-fucoidanase encoding gene, Mef2, from the marine bacterium Muricauda eckloniae, was cloned, and the Mef2 protein was functionally characterized. Based on the primary sequence, Mef2 was suggested to belong to the glycosyl hydrolase family 107 (GH107) in the Carbohydrate Active enZyme database (CAZy). The Mef2 fucoidanase showed maximal activity at pH 8 and 35 °C, although it could tolerate temperatures up to 50 °C. Ca2+ was shown to increase the melting temperature from 38 to 44 °C and was furthermore required for optimal activity of Mef2. The substrate specificity of Mef2 was investigated, and Fourier transform infrared spectroscopy (FTIR) was used to determine the enzymatic activity (Units per µM enzyme: Uf/µM) of Mef2 on two structurally different fucoidans, showing an activity of 1.2 × 10-3 Uf/µM and 3.6 × 10-3 Uf/µM on fucoidans from Fucus evanescens and Saccharina latissima, respectively. Interestingly, Mef2 was identified as the first described fucoidanase active on fucoidans from S. latissima. The fucoidan oligosaccharides released by Mef2 consisted of a backbone of α(1,3)-linked fucosyl residues with unique and novel α(1,4)-linked fucosyl branches, not previously identified in fucoidans from S. latissima.

A new FTIR assay for quantitative measurement of endo-fucoidanase activity.

Endo-fucoidanases, including EC 3.2.1.211 endo-α-1,3-L-fucanase and EC 3.2.1.212 endo-α-1,4-L-fucanase activities, catalyze depolymerization of fucoidans - a group of bioactive, sulfated fucosyl-polysaccharides found primarily in brown macroalgae (brown seaweeds). Quantitative assessment of endo-fucoidanase activity is critical for characterizing endo-fucoidanase kinetics and for comparing the action of different endo-fucoidanases on different types of fucoidans. However, the current state-of-the-art endo-fucoidanase assay consists of a qualitative assessment based on Carbohydrate-Polyacrylamide Gel Electrophoresis. Here, we report a new quantitative endo-fucoidanase assay based on real time spectral evolution profiling of changes in substrate and product during endo-fucoidanase action using Fourier Transform InfraRed spectroscopy (FTIR) combined with Parallel Factor Analysis (PARAFAC). The FTIR-PARAFAC assay was validated by monitoring the reaction progress of three different microbial endo-fucoidanase enzymes, FcnAΔ229, FFA2 and Fhf1Δ470, on two different fucoidan substrates. The substrates were purified from the brown macroalgae Fucus evanescens and Fucus vesiculosus, respectively. The evolution profiling showed that the strongest spectral change of the fucoidans during enzymatic depolymerization occurred in the spectral range 1220-1260 cm-1, but the profiles differed depending on the substrate and the enzyme used. Spectral changes within 1220-1260 cm-1 are in agreement with the enzymatic depolymerization inducing signature changes in the mid-infrared absorption of sulfated fucosyls as sulfate ester bonds and C-O stretching vibrations absorb in this spectral region. Based on the data obtained, we also introduce an activity unit for endo-fucoidanases: One endo-fucoidanase Unit, Uf, is the amount of enzyme able to catalyze a change in the FTIR-PARAFAC score by 0.01 during 498 s of reaction (8.3 min) on 20 g/L pure fucoidan from F. evanescens at 42 °C, pH 7.4, 100 mM NaCl and 10 mM CaCl2. This new quantitative endo-fucoidanase assay can pave the way for better kinetic characterizations as well as novel explorations of endo-fucoidanases.

Depolymerization of fucoidan with endo-fucoidanase changes bioactivity in processes relevant for bone regeneration.

Fucoidans are polysaccharides from brown macroalgae, showing multiple bioactivities important for bone regeneration and bone health. However, the use of fucoidans in medical applications remains sparse due to the heterogeneity in their chemical properties and unclear structure-function relationships. Innovations in extraction techniques and post processing steps are needed to produce homogeneous fucoidan molecules with tailorable bioactivities. Here, we applied enzyme-assisted extraction coupled with enzymatic hydrolysis by Fhf1 fucoidanase to generate low (LMW) and medium molecular weight (MMW) fucoidans from Fucus evanescens. In contrast to the anti-angiogenic properties of the high molecular weight fucoidan, LMW and MMW no longer suppressed the production of pro-angiogenic molecules by bone stem cells, nor impaired the formation of prevascular structures in vitro. In contrast to LMW, a pro-inflammatory response of OEC was observed after treatment with high concentrations of MMW. Thus, fucoidanase hydrolysis could be a useful tool to tailor the bioactivity of fucoidans.

The Endo-α(1,4) Specific Fucoidanase Fhf2 From Formosa haliotis Releases Highly Sulfated Fucoidan Oligosaccharides.

Fucoidanases are endo-fucoidanases (also known as endo-fucanases) that catalyze hydrolysis of α-glycosidic linkages in fucoidans, a family of sulfated fucose-rich polysaccharides primarily found in the cell walls of brown seaweeds. Fucoidanases are promising tools for producing bioactive fucoidan oligosaccharides for a range of biomedical applications. High sulfation degree has been linked to high bioactivity of fucoidans. In this study, a novel fucoidanase, Fhf2, was identified in the genome of the aerobic, Gram-negative marine bacterium Formosa haliotis. Fhf2 was found to share sequence similarity to known endo-α(1,4)-fucoidanases (EC 3.2.1.212) from glycoside hydrolase family 107. A C-terminal deletion mutant Fhf2∆484, devoid of 484 amino acids at the C-terminus, with a molecular weight of approximately 46 kDa, was constructed and found to be more stable than the full-length Fhf2 protein. Fhf2∆484 showed endo-fucoidanase activity on fucoidans from different seaweed species including Fucus evanescens, Fucus vesiculosus, Sargassum mcclurei, and Sargassum polycystum. The highest activity was observed on fucoidan from F. evanescens. The Fhf2∆484 enzyme was active at 20-45°C and at pH 6-9 and had optimal activity at 37°C and pH 8. Additionally, Fhf2∆484 was found to be calcium-dependent. NMR analysis showed that Fhf2∆484 catalyzed hydrolysis of α(1,4) linkages between L-fucosyl moieties sulfated on C2 (similar to Fhf1 from Formosa haliotis), but Fhf2∆484 in addition released oligosaccharides containing a substantial amount of 2,4-disulfated fucose residues. The data thus suggest that the Fhf2∆484 enzyme could be a valuable candidate for producing highly sulfated oligosaccharides applicable for fucoidan bioactivity investigations.

Efficacy of marine bioactive compound fucoidan for bone regeneration and implant fixation in sheep.

The need for a substitute for allograft and autograft is rising as bone graft surgeries exceed available supplies. We investigated the efficacy of the low-molecular weight marine bioactive compound fucoidan (FUC) on bone regeneration and implant fixation in seven female sheep, as FUC has shown great promise as a bone substitute. Titanium implants were inserted bilaterally in the distal femurs to test three hydroxyapatite/fucoidan (HA/FUC) groups and compared to allograft. The HA was coated with either 500 or 1500 µg of FUC, obtained by microwave-assisted chemical extraction, or 500 µg of FUC obtained by an enzyme-assisted extraction method. The concentric 2-mm gap around the implant was filled with either one of the HA/FUCs or allograft from the donor sheep. After 12 weeks, implant-bone blocks were harvested and divided into three parts for mechanical push-out testing, immunohistochemistry, and micro-CT and histomorphometry. Pronounced bone formations were observed by micro-CT and histomorphometry in all groups, but higher bone volume fractions were seen in the allograft group compared to the three HA/FUC groups. The trabecular thickness, trabecular separation, and architectural anisotropy were all significantly higher in the allograft group compared to the three HA/FUC groups. In conclusion, adequate bone formation was observed in all groups, although the bone formation was significantly greater in the allograft group. Also, no significant differences existed in the shear mechanical properties between groups, suggesting that the combination of HA and FUC can achieve a similar fixation strength to allograft in this model.

A novel thermostable prokaryotic fucoidan active sulfatase PsFucS1 with an unusual quaternary hexameric structure.

Fucoidans are sulfated, fucose-rich marine polysaccharides primarily found in cell walls of brown seaweeds (macroalgae). Fucoidans are known to possess beneficial bioactivities depending on their structure and sulfation degree. Here, we report the first functional characterization and the first crystal structure of a prokaryotic sulfatase, PsFucS1, belonging to sulfatase subfamily S1_13, able to release sulfate from fucoidan oligosaccharides. PsFucS1 was identified in the genome of a Pseudoalteromonas sp. isolated from sea cucumber gut. PsFucS1 (57 kDa) is Ca2+ dependent and has an unusually high optimal temperature (68 °C) and thermostability. Further, the PsFucS1 displays a unique quaternary hexameric structure comprising a tight trimeric dimer complex. The structural data imply that this hexamer formation results from an uncommon interaction of each PsFucS1 monomer that is oriented perpendicular to the common dimer interface (~ 1500 Å2) that can be found in analogous sulfatases. The uncommon interaction involves interfacing (1246 Å2) through a bundle of α-helices in the N-terminal domain to form a trimeric ring structure. The high thermostability may be related to this unusual quaternary hexameric structure formation that is suggested to represent a novel protein thermostabilization mechanism.

Ancient origin of fucosylated xyloglucan in charophycean green algae.

The charophycean green algae (CGA or basal streptophytes) are of particular evolutionary significance because their ancestors gave rise to land plants. One outstanding feature of these algae is that their cell walls exhibit remarkable similarities to those of land plants. Xyloglucan (XyG) is a major structural component of the cell walls of most land plants and was originally thought to be absent in CGA. This study presents evidence that XyG evolved in the CGA. This is based on a) the identification of orthologs of the genetic machinery to produce XyG, b) the identification of XyG in a range of CGA and, c) the structural elucidation of XyG, including uronic acid-containing XyG, in selected CGA. Most notably, XyG fucosylation, a feature considered as a late evolutionary elaboration of the basic XyG structure and orthologs to the corresponding biosynthetic enzymes are shown to be present in Mesotaenium caldariorum.

Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis.

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37-40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

Effect of Enzymatically Extracted Fucoidans on Angiogenesis and Osteogenesis in Primary Cell Culture Systems Mimicking Bone Tissue Environment.

Angiogenesis, the formation of new blood vessels from existing ones, is an essential process for successful bone regeneration. Further, angiogenesis is a key factor for the development of bone-related disorders like osteosarcoma or arthritis. Fucoidans, sulfated polysaccharides from brown algae, have been shown to affect angiogenesis as well as a series of other physiological processes including inflammation or infection. However, the chemical properties of fucoidan which define the biological activity vary tremendously, making a prediction of the bioactivity or the corresponding therapeutic effect difficult. In this study, we compare the effect of four chemically characterized high molecular weight fucoidan extracts from Fucus distichus subsp. evanescens (FE_crude and fractions F1, F2, F3) on angiogenic and osteogenic processes in bone-related primary mono- and co-culture cell systems. By determining the gene expression and protein levels of the regulatory molecules vascular endothelial growth factor (VEGF), angiopoietin-1 (ANG-1), ANG-2 and stromal-derived factor 1 (SDF-1), we show that the extracted fucoidans negatively influence angiogenic and osteogenic processes in both the mono- and co-culture systems. We demonstrate that purer fucoidan extracts with a high fucose and sulfate content show stronger effects on these processes. Immunocytochemistry of the co-culture system revealed that treatment with FE_F3, containing the highest fucose and sulfate content, impaired the formation of angiogenic tube-like structures, indicating the anti-angiogenic properties of the tested fucoidans. This study highlights how chemical properties of fucoidan influence its bioactivity in a bone-related context and discusses how the observed phenotypes can be explained on a molecular level-knowledge that is indispensable for future therapies based on fucoidans.

Enzyme-Assisted Fucoidan Extraction from Brown Macroalgae Fucus distichus subsp. evanescens and Saccharina latissima.

Fucoidans from brown macroalgae (brown seaweeds) have different structures and many interesting bioactivities. Fucoidans are classically extracted from brown seaweeds by hot acidic extraction. Here, we report a new targeted enzyme-assisted methodology for fucoidan extraction from brown seaweeds. This enzyme-assisted extraction protocol involves a one-step combined use of a commercial cellulase preparation (Cellic®CTec2) and an alginate lyase from Sphingomonas sp. (SALy), reaction at pH 6.0, 40 °C, removal of non-fucoidan polysaccharides by Ca2+ precipitation, and ethanol-precipitation of crude fucoidan. The workability of this method is demonstrated for fucoidan extraction from Fucus distichus subsp. evanescens (basionym Fucus evanescens) and Saccharina latissima as compared with mild acidic extraction. The crude fucoidans resulting directly from the enzyme-assisted method contained considerable amounts of low molecular weight alginate, but this residual alginate was effectively removed by an additional ion-exchange chromatographic step to yield pure fucoidans (as confirmed by 1H NMR). The fucoidan yields that were obtained by the enzymatic method were comparable to the chemically extracted yields for both F. evanescens and S. latissima, but the molecular sizes of the fucoidans were significantly larger with enzyme-assisted extraction. The molecular weight distribution of the fucoidan fractions was 400 to 800 kDa for F. evanescens and 300 to 800 kDa for S. latissima, whereas the molecular weights of the corresponding chemically extracted fucoidans from these seaweeds were 10-100 kDa and 50-100 kDa, respectively. Enzyme-assisted extraction represents a new gentle strategy for fucoidan extraction and it provides new opportunities for obtaining high yields of native fucoidan structures from brown macroalgae.

Effects of a Newly Developed Enzyme-Assisted Extraction Method on the Biological Activities of Fucoidans in Ocular Cells.

Fucoidans from brown seaweeds are promising substances as potential drugs against age-related macular degeneration (AMD). The heterogeneity of fucoidans requires intensive research in order to find suitable species and extraction methods. Ten different fucoidan samples extracted enzymatically from Laminaria digitata (LD), Saccharina latissima (SL) and Fucus distichus subsp. evanescens (FE) were tested for toxicity, oxidative stress protection and VEGF (vascular endothelial growth factor) inhibition. For this study crude fucoidans were extracted from seaweeds using different enzymes and SL fucoidans were further separated into three fractions (SL_F1-F3) by ion-exchange chromatography (IEX). Fucoidan composition was analyzed by high performance anion exchange chromatography (HPAEC) after acid hydrolysis. The crude extracts contained alginate, while two of the fractionated SL fucoidans SL_F2 and SL_F3 were highly pure. Cell viability was assessed with an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay in OMM-1 and ARPE-19. Protective effects were investigated after 24 h of stress insult in OMM-1 and ARPE-19. Secreted VEGF was analyzed via ELISA (enzyme-linked immunosorbent assay) in ARPE-19 cells. Fucoidans showed no toxic effects. In OMM-1 SL_F2 and several FE fucoidans were protective. LD_SiAT2 (Cellic®CTec2 + Sigma-Aldrich alginate lyase), FE_SiAT3 (Cellic® CTec3 + Sigma-Aldrich alginate lyase), SL_F2 and SL_F3 inhibited VEGF with the latter two as the most effective. We could show that enzyme treated fucoidans in general and the fractionated SL fucoidans SL_F2 and SL_F3 are very promising for beneficial AMD relevant biological activities.

Pea Border Cell Maturation and Release Involve Complex Cell Wall Structural Dynamics.

The adhesion of plant cells is vital for support and protection of the plant body and is maintained by a variety of molecular associations between cell wall components. In some specialized cases, though, plant cells are programmed to detach, and root cap-derived border cells are examples of this. Border cells (in some species known as border-like cells) provide an expendable barrier between roots and the environment. Their maturation and release is an important but poorly characterized cell separation event. To gain a deeper insight into the complex cellular dynamics underlying this process, we undertook a systematic, detailed analysis of pea (Pisum sativum) root tip cell walls. Our study included immunocarbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy, quantitative reverse transcription-PCR of cell wall biosynthetic genes, analysis of hydrolytic activities, transmission electron microscopy, and immunolocalization of cell wall components. Using this integrated glycobiology approach, we identified multiple novel modes of cell wall structural and compositional rearrangement during root cap growth and the release of border cells. Our findings provide a new level of detail about border cell maturation and enable us to develop a model of the separation process. We propose that loss of adhesion by the dissolution of homogalacturonan in the middle lamellae is augmented by an active biophysical process of cell curvature driven by the polarized distribution of xyloglucan and extensin epitopes.

High throughput screening of starch structures using carbohydrate microarrays.

In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers the potential for rapidly analysing resistant and slowly digested dietary starches.

Penium margaritaceum as a model organism for cell wall analysis of expanding plant cells.

The growth of a plant cell encompasses a complex set of subcellular components interacting in a highly coordinated fashion. Ultimately, these activities create specific cell wall structural domains that regulate the prime force of expansion, internally generated turgor pressure. The precise organization of the polymeric networks of the cell wall around the protoplast also contributes to the direction of growth, the shape of the cell, and the proper positioning of the cell in a tissue. In essence, plant cell expansion represents the foundation of development. Most studies of plant cell expansion have focused primarily upon late divergent multicellular land plants and specialized cell types (e.g., pollen tubes, root hairs). Here, we describe a unicellular green alga, Penium margaritaceum (Penium), which can serve as a valuable model organism for understanding cell expansion and the underlying mechanics of the cell wall in a single plant cell.

Barley HvHMA1 is a heavy metal pump involved in mobilizing organellar Zn and Cu and plays a role in metal loading into grains.

Heavy metal transporters belonging to the P(1B)-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. Heavy metal transporters belonging to the P(1B)-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. In this study we investigated the properties of HvHMA1, which is a barley orthologue of Arabidopsis thaliana AtHMA1 localized to the chloroplast envelope. HvHMA1 was localized to the periphery of chloroplast of leaves and in intracellular compartments of grain aleurone cells. HvHMA1 expression was significantly higher in grains compared to leaves. In leaves, HvHMA1 expression was moderately induced by Zn deficiency, but reduced by toxic levels of Zn, Cu and Cd. Isolated barley chloroplasts exported Zn and Cu when supplied with Mg-ATP and this transport was inhibited by the AtHMA1 inhibitor thapsigargin. Down-regulation of HvHMA1 by RNA interference did not have an effect on foliar Zn and Cu contents but resulted in a significant increase in grain Zn and Cu content. Heterologous expression of HvHMA1 in heavy metal-sensitive yeast strains increased their sensitivity to Zn, but also to Cu, Co, Cd, Ca, Mn, and Fe. Based on these results, we suggest that HvHMA1 is a broad-specificity exporter of metals from chloroplasts and serve as a scavenging mechanism for mobilizing plastid Zn and Cu when cells become deficient in these elements. In grains, HvHMA1 might be involved in mobilizing Zn and Cu from the aleurone cells during grain filling and germination.