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-β-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-β-N-acetylglucosaminidase from rice culture cells and identified the gene encoded (Maeda, M., and Kimura, Y., Trends Glycosci. Glycotech., 17, 205–214 (2005)). Furthermore, we found structural features of free N-glycans in the cells, indicating that high-mannose type species (Man_9-5GlcNAc₁) occur at concentration of several micromolar (μM). 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 Analysis of N-Glycans of Storage Glycoproteins in Soybean (Glycine max. L) Seed

The structures of N-linked sugar chains (N-glycans) of storage glycoproteins in soybean seeds have been identified. Eight pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the storage glycoproteins by reverse-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were first identified by two-dimensional PA-sugar chain mapping and ion-spray mass analysis, considering the results of sugar composition analysis or sequential exoglycosidase digestion. The deduced structures were further analyzed by ion-spray tandem mass spectrometry and 500 MHz ¹H-NMR spectrometry. The eight structures fell into two categories; the major class (96.6%) was a typical high mannose-type, the minor class was a xylose containing-type (Man₃Xyl₁GlcNAc₂, Man₃Fuc₁Xyl₁GlcNAc₂; 3.4%).     

Structural Analysis of Free N-Glycans Occurring in Soybean Seedlings and Dry Seeds

The structures of unconjugated or free N-glycans in stems of soybean seedlings and dry seeds have been identified. The free N-glycans were extracted from the stems of seedlings or defatted dry seeds. After desalting by two kinds of ion-exchange chromatography and a gel filtration, the free N-glycans were coupled with 2-aminopyridine. The resulting fluorescence-labeled (PA-) N-glycans were purified by gel filtration, Con A affinity chromatography, reverse-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains purified were analyzed by the combination of two-dimensional sugar chain mapping, jack bean α-mannosidase digestion, α-1,2-mannosidase digestions, partial acetolysis, and ESI-MS/MS. The free N-glycan structures found showed that two categories of free N-glycans occur in the stems of soybean seedlings. One is a high-mannose type structure having one GlcNAc residue at the reducing end (Man_9~5GlcNAc₁, 93%), that would be derived by endo-GM (Kimura, Y. et al., Biochim. Biophys. Acta, 1381, 27-36 (1998)). The other small component is a xylose-containing type one having two GlcNAc residues at the reducing end (Man₃Xyl₁GlcNAc₂, 7%), which would be derived by PNGase-GM (Kimura, Y. and Ohno, A., Biosci. Biotechnol. Biochem., 62, 412-418 (1998)). The detailed structural analysis of free glycans showed that high-mannose type free N-glycans (Man_9~5GlcNAc₁) in the soybean seedlings have a common core structural unit; Manα1- 6(Man1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc.

Comparing the amount of free N-glycans in the seedling stems and dry seeds, the amount in the stems of seedlings was much higher than that in the dry seeds; approximately 700 pmol per one stem, 8 pmol in one dry seed. This fact suggested that free N-glycans in soybean seedlings could be produced by two kinds of N-glycan releasing enzymes during germination or seedling-development.     

Salt-Adaptation of Tobacco BY2 Cells Induces Change in Glycoform of N-Glycans: Enhancement of Exo- and Endo-Glycosidase Activities by Salt-Adaptation

In this report, we describe that a salt adaptation of plant cells induces glycoform changes in N-glycoproteins. Intracellular and cell-wall glycopeptides were prepared from glycoproteins expressed in wild-type BY2 cells and salt-adapted cells. N-Glycans were liberated from those glycopeptides by hydrazinolysis, and the released oligosaccharides were N-acetylated and pyridylaminated. The structures of pyridylaminated (PA-) N-glycans were analyzed by a combination of two-dimensional sugar-chain mapping, MS analysis, and exoglycosidase digestion. In both wild-type cells and salt-adapted cells, the plant complex type structure was predominant among N-glycans expressed on glycoproteins, but we found that the Man2Xyl1Fuc1GlcNAc2 structure was significantly expressed on intracellular and cell-wall glycoproteins of the salt-adapted cells. Furthermore, enhancement of the specific activities of α-mannosidase and β-N-acetylglucosaminidase was observed in the salt-adapted BY2 cells, suggesting that the glycoform changes are due to changes in glycosidase activities.     

Axonal Glycoproteins with Immunoglobulin- and Fibronectin Type III-Related Domains in Vertebrates: Structural Features, Binding Activities, and Signal Transduction

Abstract: The L1- and F11-like axonal glycoproteins, implicated in neurite outgrowth and fasciculation, are members of the Ig superfamily comprising multiple fibronectin type III-like domains. Their Ig-like and fibronectin type III-related domains are likely to be composed of seven β-strands arranged in two opposing β-sheets of highly similar topology. Whereas the F11-like molecules lack a transmembrane sequence and are anchored in the plasma membrane by a glycosylphosphatidylinositol, the L1 -like molecules comprise cytoplasmic domains with highly conserved sequence motifs. Most of the latter proteins occur in different isoforms generated by alternative pre-mRNA splicing, which has not been documented for molecules of the F11 subgroup. L1 -like proteins undergo heterophils as well as homophilic interactions, whereas only the former mode of binding was observed for F11 -like proteins. Evidence is accumulating that these Ig superfamily molecules with fibronectin type III-like domains are interacting in a complex manner with each other and molecules of the extracellular matrix. Investigations assigning structure to function reveal that their individual extracellular domains serve distinct binding activities. Recent studies also suggest that L1 and NCAM are implicated in the transduction of transmembrane signals.     

与2型糖尿病相关糖复合物中糖链结构变化

2型糖尿病是一种全球性严重危害人类健康的慢性代谢性疾病.在2型糖尿病患者和动物的血液、尿液及受损组织中,糖链的结构均发生了不同程度的变化.本文对近年来有关2型糖尿病发生、发展过程中糖蛋白、蛋白聚糖和糖脂中糖链的结构变化进行综述,为2型糖尿病的诊断及其相关药物的研发提供有用的参考.     

Marine algae from the Gulf of Santa Clara,Sonora, México

Four sites in the Gulf of Santa Clara, northwestern coast of Sonora, Mexico, were sampled seasonally during 1995–1996. A total of 43 species of marine algae were determined, which are recorded for the first time for the area of study. The families with best representation are: Gracilariaceae and Cladophoraceae, each with 5 species; and Corallinaceae, Rhodomelaceae, and Ulvaceae, each with 3. The highest diversity was found in autumn, the lowest in summer. The sites with the low diversity were Piedras del Burro with 7 species and El Tornillal with 18. These places are broad beaches, composed of sand and with few pebbles. The higher diversity was found at Punta Gorda with 34 species and Piedras de La Salina with 26. These localities are characterized by relatively stable rocky substrates, with some intertidal pools. The most common species regardless to distribution and occurrence over time were: Spyridia filamentosa, Dictyota flabellata, Struveopsis robusta, Cladophora microcladioides and Enteromorpha linza. Rosenvingea antillarum and Cladophora vagabunda represent new records for the Gulf of California. Nine epiphytic species were identified, which were frequently observed on Gelidium crinale, Spyridia filamentosa and Dictyota flabellata.     

A Simple 96-Well Microplate Method for Estimation of Total Polyphenol Content in Seaweeds

Seaweed polyphenols are potent antioxidants and have also been shown to have α-glucosidase inhibiting activity. In our continuous efforts to develop new marine-based nutraceuticals and functional food ingredients, we have investigated many algal species collected on the Atlantic coast of Canada. A simple method for estimating the total polyphenol content in seaweeds and their extracts was developed based on the classic Folin-Ciocalteau colorimetric reaction. By using the 96-well microplate and a microplate reader, this new method saves experimental time, significantly reduces the amount of sample required, handles large number of samples in one experiment, and also improves the repeatability of the results.A number of algal samples collected on the seashore of Nova Scotia, Canada, were analyzed for their levels of polyphenol content using this microplate-based method. The antioxidant activity of these samples was also assessed by using DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical scavenging assay. The results showed that there is a strong correlation between the total polyphenol content and the potency of antioxidant effect.     

Using the biogeographical distribution and diversity of seaweed species to test the efficacy of marine protected areas in the warm-temperate Agulhas Marine Province, South Africa

Aim To study the siting of marine protected areas (MPAs) with respect to the biogeographical distribution of seaweeds within the Agulhas Marine Province and to assess the effectiveness of current MPAs in including (conserving) seaweeds of the South African south coast. Location South Africa – the south coast between Cape Agulhas and the Eastern Cape/Kwazulu‐Natal border, and eight MPAs within that area. Methods We used interpolated seaweed distribution records from all available sources, in 50‐km coastal sections. Cluster analysis (Jaccard Average Linkage) of species presence/absence data provided measures of similarity between coastal sections and between MPAs. Complementarity analyses identified the sequence of ‘importance’ of sections/MPAs for conserving seaweed species. Results Species presence/absence data indicated two main groups, representing western (cooler water) and eastern (warmer water) biogeographical divisions, as well as several biogeographical subdivisions within each of these groups. Complementarity analysis yielded a sequence of ‘importance’ of coastal sections (in terms of the highest number of species included) that began with a section just east of central in the Agulhas Marine Province, around Port Alfred, where there is no MPA. This was followed by the easternmost section (warmest water), which contains the Pondoland MPA, and then by the westernmost (coolest water) section, containing the De Hoop MPA. Similar analysis of the actual species collected in MPAs showed a generally similar pattern. Main conclusions Seven current MPAs and one proposed coastal MPA in the Agulhas Marine Province appear to be well distributed and well sited to include (conserve) the full biogeographical range of seaweeds. However, if further MPAs are to be considered, the Port Alfred area is recommended for improved conservation. This study did not examine estuaries, which may require improved conservation efforts. Seaweed distribution data, which are often relatively complete, offer a good tool for planning the siting of coastal MPAs.     

Characteristics of Eucheuma cottonii waste from East Malaysia: physical,thermal and chemical composition

The aim of this paper was to examine the characteristics of Eucheuma cottonii waste in order to analyse its potential as renewable material. The morphology of Eucheuma cottonii (raw and wastes) was investigated through scanning electron microscopy (SEM), the thermal behaviour through thermogravimetric analysis (TGA) and the physical properties through FT-IR, XRD, gas pycnometer, particle size analyser, water absorption and moisture content analysis. The chemical compositions were determined by using acid detergent fibre (ADF), neutral detergent fibre (NDF) and acid detergent lignin (ADL) analysis. It was found that Eucheuma cottonii wastes have better thermal stability, higher crude fibre content, lower moisture content and similar density to the raw Eucheuma cottonii, which suggests that these biomass wastes have good potential as renewable filler material.     

Review: Prospects for the Use of Extracts and Polysaccharides from Marine Algae to Prevent and Treat the Diseases Caused by Helicobacter pylori

Helicobacter pylori possesses a broad spectrum of pathogenic factors that allow it to survive and colonize the gastric mucosa, and thus, the pathogenetic targets, which have the same diversity, require search for and the development of alternative, effective, and innocuous means for the eradication of H. pylori. In recent years, fucoidans have been extensively studied due to the numerous interesting biological activities, including the anti‐adhesive, anti‐oxidative, antitoxic, immunomodulatory, anticoagulant, and anti‐infection effects. This review summarizes the data on the effects of extracts and sulfated polysaccharides of marine algae, mainly fucoidans, on pathogenic targets in Helicobacter infection. The pathogenetic targets for therapeutic agents after H. pylori infection, such as flagellas, urease, and other enzymes, including adhesins, cytotoxin A (VacA), phospholipase, and L‐8, are characterized here. The main target for the sulfated polysaccharides of seaweed is cell receptors of the gastric mucosa. This review presents the published data about the pleiotropic anti‐inflammatory effects of polysaccharides on the gastric mucosa. It is known that fucoidan and other sulfated polysaccharides from algae have anti‐ulcer effects, prevent the adhesion of H. pylori to, and reduce the formation of biofilm. The authors speculate that the effect of sulfated polysaccharides on the infectious process caused by H. pylori is related to their action on innate and adaptive immunity cells, and also anti‐oxidant and antitoxic potential. Presented in the review are materials indicated for the study of extracts and sulfated polysaccharides from seaweed during H. pylori infection, as these compounds are characterized by multimodality actions. Based on the analysis of literary materials in recent years, the authors concluded that fucoidan can be attributed to the generation of new candidates to create drugs intended for the inclusion in the scheme of eradication therapy of H. pylori infection.     

Occurrence of a novel group of hemagglutinins extractable by Pronase treatment in marine algae

Nine species of marine algae have been assessed for the presence of novel hemagglutinins not extractable with buffer, unless the algal tissue was pretreated with Pronase. All species examined contained hemagglutinins, indicating the existence of a novel group of hemagglutinins which differed from those reported previously in marine algae.     

被子植物叶绿体基因组的结构变异研究进展

叶绿体是绿色植物特有的细胞器,其基因组信息被广泛应用于植物系统发育和比较基因组学研究。目前,越来越多的物种有了叶绿体全基因组序列,人们对叶绿体基因组的结构及其变异规律有了更深入的了解。该文对近年来国内外有关被子植物叶绿体基因组插入/缺失、短片段倒位与重复、基因组结构重排以及基因丢失等结构变异式样的研究进展进行综述,并分析了叶绿体基因组结构研究中仍存在的问题以及该领域未来的发展趋势。     

转基因植物转录后基因沉默的特点、机理及应用

自在转基因植物中发现转基因沉默现象以来 ,很多学者对该现象进行了广泛的研究 ,认为其作用机制有三种 :位置依赖性基因沉默、转录水平基因沉默、转录后水平基因沉默。目前主要集中于转录后水平基因沉默的研究 ,通过研究发现它具有广泛性、可传导性及特异性的特点 ,并对其机制提出了一些假说。最近 ,人们还发现转录后水平基因沉默与植物抗病毒能力有联系。本文对转基因植物转录后基因沉默的特点、机理及应用方面的进展作了综述     

Occurrence of Marine Birnavirus Through the Year in Coastal Seawater in the Uwa Sea

This study aims to determine the seasonal occurrence of marine birnavirus (MABV) at a coastal site in the Uwa Sea, Japan, in 1997 and 1998. To detect MABV from seawater, a simple method was developed for concentrating MABV by dialysis and ethanol precipitation. The concentrated virus was used for polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, and virus isolation. Viral genome was detected through the experimental period. The amount of PCR product varied; it was small in summer, but increased from fall to winter. Viral protein was also detected, and the amount in the January sample was equivalent to approximately 10² TCID₅₀ (50% tissue culture infectious dose) of the virus. However, infectious viruses were not isolated. This suggested that MABV was released from hosts to environmental seawater in winter and possibly degraded after release. Received May 7, 1999; accepted September 16, 1999.