N-linked oligosaccharides of glycoproteins from Ginkgo biloba pollen, an allergenic pollen.

The pollen of Ginkgo biloba is one of the allergens that cause pollen allergy symptoms. The plant complex type N-glycans bearing beta1-2 xylose and/or alpha1-3 fucose residue(s) linked to glycoallergens have been considered to be critical epitopes in various immune reactions. In this report, the structures of N-glycans of total glycoproteins prepared from Ginkgo biloba pollens were analyzed to confirm whether such plant complex type N-glycans occur in the pollen glycoproteins. The glycoproteins were extracted by SDS-Tris buffer. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine and the resulting pyridylaminated (PA-)N-glycans were purified by a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, IS-MS, and MS/MS. The plant complex type structures (GlcNAc2Man3Xyl1Fuc1GlcNAc2 (31%), GlcNAc2Man3Xyl1GlcNAc2 (5%), Man3Xyl1Fuc1GlcNAc2 (13%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (8%), and GlcNAc1Man3Xyl1GlcNAc2 (17%)) have been found among the N-glycans of the glycoproteins of Ginkgo biloba pollen, which might be candidates for the epitopes involved in Ginkgo pollen allergy. The remaining 26% of the total pollen N-glycans have the typical high-mannose type structures: Man8GlcNAc2 (11%) and Man6GlcNAc2 (15%).     

Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry. The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing beta1-2 xylosyl and/or alpha1-3 fucosyl residue(s) and occurrence of beta1-4GlcNAc residue in the insect glycoproteins. The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man(9 to approximately 4)GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3 GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1 Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3 Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of beta-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a beta1-4 linkage N-acetylglucosaminyl residue.     

Glycoform analysis of N-glycans linked to glycoproteins expressed in rice culture cells: predominant occurrence of complex type N-glycans

Although it has been found that plant endo-beta-N-acetylglucosaminidase shows strong activity towards denatured glycoproteins and glycopeptides with high-mannose type N-glycans and free high-mannose type N-glycans bearing the chitobiosyl unit, the endogenous substrates for plant endoglycosidase have not yet been identified. Recently we purified and characterized an endo-beta-N-acetylglucosaminidase from rice culture cells and identified the gene encoded. Furthermore, we found structural features of free N-glycans in the cells, indicating that high-mannose type species (Man(9-5)GlcNAc(1)) occur at concentration of several micromolar (microM). Hence, in this study we analyzed glycoform of N-glycans linked to glycoproteins expressed in rice culture cells to see whether endogenous glycoproteinous substrate occurs in reasonable amounts. Structural analysis revealed that more than 95% of total N-glycans linked to glycoproteins in the rice cells had the plant complex type structure, including Lewis a epitope-harboring type, although high-mannose type structures account for less than 5% of total N-glycans.     

Structural feature of N-glycans of bamboo shoot glycoproteins: useful source of plant antigenic N-glycans

An effective method to prepare plant complex type (PCT) N-glycans in large amounts has been required to evaluate their immunological activity. In this study, we found that glycoproteins in bamboo shoots predominantly carry PCT N-glycans including the Lewis a epitope-containing ones, suggesting that bamboo shoot is an excellent source for the plant antigenic glycans to synthesize immunoactive neoglycopolymers.     

N-glycans linked to glycoproteins in edible beans (zatsu-mame): natural resources for bioactive oligosaccharides

In this study, we analyzed structural features of the N-glycans linked to glycoproteins expressed in various edible beans to identify excellent sources of useful N-glycans and N-glycopeptides. Structural analysis of N-glycans of the glycopeptides prepared from the pepsin digests of bean powder that the useful high-mannose type N-glycans occur predominantly in Phaseolus and Vigna beans; tora bean for Man?GlcNAc?-Asn, dainagon bean for Man?GlcNAc?-Asn, and azuki bean for Man?GlcNAc?-Asn.     

Structural analysis of N-glycans from human neutrophil azurocidin

N-glycans of human neutrophil azurocidin, enzymatic inactive homolog of serine proteinase playing important and multifunctional roles in antimicrobial defense, endotoxin binding, monocyte, and T-cell activation, were isolated by hydrazinolysis and fluorescence labeled. An ion-exchange chromatography on GlycoSep C column separated neutral, mono-, and disialylated glycans. The glycans from each group were separated subsequently on GlycoSep N and GlycoSep H columns. Sequential exoglycosidase treatment and HPLC mapping allowed determining 21 different glycan structures, majority of them being neutral (79.8%), the rest-mono- (13.1%) and disialylated (1.2%).     

Analysis of allergenic pollen data,focusing on a pollen load threshold statement

Aerobiologia - Airborne allergenic pollen affects a significant part of the population and the information on pollen load is a valuable tool for public health prevention. The messages should be...     

Structural features of carbohydrate moieties in snake venom glycoproteins.

The structures of the carbohydrate moieties of glycoproteins in snake venoms are largely unknown. In the present study, we have analyzed venoms of several species of snakes as well as plasma and tissue glycoproteins from one species of cobra (Naja naja kaouthia) by lectin affinity staining of Western blots. The data demonstrate that glycoproteins in cobra venom invariably contain terminal alpha-galactosyl residues with negligible proportions of sialic acids. Interestingly, however, terminal alpha-galactosyl residues are present in significantly lower proportions in cobra tissues such as brain, liver, lung, kidney, spleen, muscle, and totally absent in cobra plasma glycoproteins. In sharp contrast to cobras, venom glycoproteins of other snakes do not contain terminal alpha-galactosyl residues but do contain terminal 2,3- and/or 2,6-linked sialic acids as well as beta-galactosyl residues. Cobra venom also contains high molecular weight heavily glycosylated proteins bearing poly-N-acetyllactosaminyl oligosaccharides, the majority of which appear to be linked to the protein core via O-glycosidic bonds.     

Structural features of isolated generative cells and their protoplasts from pollen of some liliaceous plants

Large quantities of intact generative cells and their protoplasts were isolated from pollen protoplasts of four liliaceous plants, and their structural features were investigated. The generative cells, liberated from the vegetative cell cytoplasm of the pollen protoplasts, were initially spindle-shaped with two long, oppositely oriented extensions, and were surrounded by two cell membranes, one on each side of a wall of uniform thickness. The generative nuclei, stained with 4′,6-diamidino-2-phenylindole (DAPI), showed ellipsoidal and highly condensed chromatin, whereas the generative cell cytoplasm, whose quantity was widely different from species to species, showed no fluorescence, suggesting the absence of plastid and mitochondria! DNA, although many mitochondria were present. The isolated generative cells, which were spindle-shaped at first, became spherical in shape in vitro. Immunocytochemistry and transmission electron microscopy revealed that this change was associated with the depolymerization of an axial array of microtubules present in generative cells in situ. These results are discussed in relation to the function of the generative cell within the bicellular pollen of angiosperms.     

Reproducibility between counts of airborne allergenic pollen from two cities in the East Midlands,UK

Historically in the East Midlands, UK, airborne pollen has been monitored in two cities, Derby and Leicester, situated 41 km (25 miles) apart. The aim of the present study was to compare aerobiological data from both sites to determine if a forecast based on data from one site would be sufficient for both, and to address the wider issue of reproducibility between geographically separated sites. Pollen types recorded could be split into two groups according to annual abundance, maximum daily concentration and the number of high count days. Six taxa made up the abundant group; ash, birch, grass, oak, nettle-type and yew-type, representing 90 and 88% of the total air spora for Derby and Leicester, respectively. Three consecutive years of grass and nettle pollen data are presented, supported by one year of abundant tree pollen data. There were highly significant positive correlations between the counts obtained. Line charts showing the average number of pollen grains m⁻³ air day⁻¹ show similar trends, and Bland–Altman plots show little discrepancy between the amounts of pollen counted on any given day. Each day was classified according to the UK accepted threshold levels for grass. Weighted kappa statistics showed substantial or almost perfect agreement between the forecast classifications. With the caveat that this would not apply in a region with restrictions to air flow such as a mountain range or with extreme fluctuations such as a coastline site, this study suggests that data from a single site is suitable for forecasting a distance of up to 41 km.     

The pollen dispersal kernel and mating system of an insect-pollinated tropical palm,Oenocarpus bataua

Pollen dispersal shapes the local genetic structure of plant populations and determines the opportunity for local selection and genetic drift, but has been well studied in few animal-pollinated plants in tropical rainforests. Here, we characterise pollen movement for an insect-pollinated Neotropical canopy palm, Oenocarpus bataua, and relate these data to adult mating system and population genetic structure. The study covers a 130-ha parcel in which all adult trees (n=185) were mapped and genotyped at 12 microsatellite loci, allowing us to positively identify the source tree for 90% of pollination events (n=287 of 318 events). Mating system analysis showed O. bataua was effectively outcrossed (t_m=1.02) with little biparental inbreeding (t_m−t_s=−0.005) and an average of 5.4 effective pollen donors (N_ep) per female. Dispersal distances were relatively large for an insect-pollinated species (mean=303 m, max=1263 m), and far exceeded nearest-neighbour distances. Dispersal kernel modelling indicated a thin-tailed Weibull distribution offered the best fit to the genetic data, which contrasts with the fat-tailed kernels typically reported for pollen dispersal in trees. Preliminary analyses suggest that our findings may be explained, at least in part, by a relatively diffuse spatial and temporal distribution of flowering trees. Comparison with previously reported estimates of seed movement for O. bataua suggests that pollen and seed dispersal distances may be similar. These findings add to the growing body of information on dispersal in insect-pollinated trees, but underscore the need for continued research on tropical systems in general, and palms in particular.     

Process integration possibilities for biodiesel production from palm oil using ethanol obtained from lignocellulosic residues of oil palm industry

In this paper, integration possibilities for production of biodiesel and bioethanol using a single source of biomass as a feedstock (oil palm) were explored through process simulation. The oil extracted from Fresh Fruit Bunches was considered as the feedstock for biodiesel production. An extractive reaction process is proposed for transesterification reaction using in situ produced ethanol, which is obtained from two types of lignocellulosic residues of palm industry (Empty Fruit Bunches and Palm Press Fiber). Several ways of integration were analyzed. The integration of material flows between ethanol and biodiesel production lines allowed a reduction in unit energy costs down to 3.4%, whereas the material and energy integration leaded to 39.8% decrease of those costs. The proposed integrated configuration is an important option when the technology for ethanol production from biomass reaches such a degree of maturity that its production costs be comparable with those of grain or cane ethanol.     

Structural characterization of the N-glycans of gpMuc from Mucuna pruriens seeds

Mucuna pruriens seeds are used in some countries as a human prophylactic oral anti-snake remedy. Aqueous extracts of M. pruriens seeds possess in vivo activity against cobra and viper venoms, and protect mice against Echis carinatus venom. It was recently demonstrated that the seed immunogen generating the antibody that cross-reacts with the venom proteins is a multiform glycoprotein (gpMuc), and the immunogenic properties of gpMuc seemed to mainly reside in its glycan chains. In the present study, gpMuc was found to contain only N-glycans. Part of the N-glycans could be released with peptide-(N ⁴-(N-acetyl-β -glucosaminyl)asparagine amidase F (PNGase F-sensitive N-glycans); the PNGase F-resistant N-glycans were PNGase A-sensitive. The oligosaccharides released were analyzed by a combination of MALDI-TOF mass spectrometry, HPLC profiling of 2-aminobenzamide-labelled derivatives and ¹H NMR spectroscopy. The PNGase F-sensitive N-glycans comprised a mixture of oligomannose-type structures ranging from Man₅GlcNAc₂ to Man₉GlcNAc₂, and two xylosylated structures, Xyl₁Man₃GlcNAc₂ and Xyl₁Man₄GlcNAc₂. The PNGase A-sensitive N-glycans, containing (α 1-3)-linked fucose, were identified as Fuc₁Xyl₁Man₂GlcNAc₂ and Fuc₁Xyl₁Man₃GlcNAc₂. In view of the determined N-glycan ensemble, the immunoreactivity of gpMuc was ascribed to the presence of core (β 1-2)-linked xylose- and core α (1-3)-linked fucose-modified N-glycan chains.     

Somatic embryogenesis from immature inflorescences of oil palm

Summary Immature inflorescences of oil palm (Elaeis guineensis) var. Pisifera were inoculated onto modified MS medium containing 0.3% (w/v) activated charcoal and 475 M 2,4-D. After 2—3 months of culture, a hard yellow callus proliferated at the base of the shoot-like structures. The high incidence of phenolic oxidation required the use of increased levels of activated charcoal (0.5% w/v) and 2,4-D (500 M). Development of floral structures from inflorescence expiants was frequently observed during the culture period. After 81 weeks of culture, an embryogenic tissue characterized by compact consistency and pearly white color was observed in tissues derived from very young inflorescences. This compact embryogenic tissue differentiated into normal somatic embryos when transferred onto regeneration medium containing NAA (15 M) and ABA (2 M). Normal plantlets were recovered from these somatic embryos after 8 weeks on regeneration medium.Abbreviations 2, 4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - ABA abscisic acid - PVP-40 polyvinylpyrrolidone     

Airborne pollen from allergenic herbaceous plants in urban and rural areas of Western Pomerania,NW Poland

This paper presents the course of the pollen season of selected allergenic taxa (Rumex spp., Plantago spp., Urtica spp. and Chenopodiaceae) in two towns with different degrees of urbanisation (urban, the city Szczecin, and rural, the village Gudowo in Western Pomerania, northwest Poland). The study was conducted in the years 2012–2014 with the volumetric method. The pollen of allergenic herbaceous plants was present in the air from early May until the end of September. From among the taxa studied, the genus Rumex is characterised by the longest pollination period, while the genus Urtica by the shortest. The highest percentage contribution of pollen grains to the total pollen count in the air reaching over 90% is from nettle. The pollen seasons determined for the majority of taxa are longer in the city, however the mean pollen grain counts in the air are higher for the rural area.     

Development of a method for the efficient release of N-glycans from glycoproteins generating native deglycosylated proteins

In many cases, it is desirable to maintain the native status of the target glycoproteins when they are deglycosylated. However, most conventional deglycosylation process often causes the irreversible denaturation of the target glycoproteins. In the present study, we developed a deglycosylation method that could obtain the native deglycosylated proteins employing Png1p-ΔH1, which was confirmed to tolerate high concentration of dithiothreitol (DTT). To prove this process, ribonuclease B (RNase B) and Yeast carboxypeptidase (CPY) were employed as the targeting glycoproteins. Our results confirmed that both of them could be completely deglycosylated in the presence of high concentration DTT and could be refolded when DTT was removed. The circular dichroism spectroscopy (CD) measurement of refolded CPY and RNase B indicated that the structure of deglycosylated proteins had recovered their native status. This method offers the possibility of efficiently releasing N-linked glycans from glycoproteins and obtaining the native target proteins.