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.     

Occurrence of mRNA for storage protein in dry soybean seeds

Poly(A)-containing RNA has been isolated from the cotyledons of soybean seeds by adsorption on a poly(U)-Sepharose column. Approximately 0.15% of the total soybean RNA applied bound to the column. The bound RNA (poly(A)-containing RNA) was shown to be mRNA by its ability to serve as template in a cell-free system derived from wheat germ. Poly(A)-containing RNA was polydisperse, migrating from approximately 50,000 to 700,000 daltons with a mean of 150,000 daltons in polyacrylamide gel electrophoresis. The size of the poly(A) portion of this RNA was in the range of 55 to 290 nucleotides. The adenylic acid content of the presumed poly(A) fragment was about 95%. The radioactive products of translation directed by the poly(A)-containing RNA in the wheat germ cell-free system were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoprecipitation using antisera against beta-conglycinin and glycinin. The results of this investigation show that mRNAs for the subunit proteins of the major components of a soybean storage protein exist in the poly(A)-containing RNA preparation obtained from the cotyledons of dry soybean seeds.     

Proteomic analysis and extensive protein identification from dry, germinating Arabidopsis seeds and young seedlings

Proteins accumulated in dry, stratified Arabidopsis seeds or young seedlings, totaled 1100 to 1300 depending on the time of sampling, were analyzed by using immobilized pH gradient 2-DE gel electrophoresis. The molecular identities of 437 polypeptides, encoded by 355 independent genes, were determined by MALDI-TOF or TOF-TOF mass spectrometry. In the sum, 293 were present at all stages and 95 were accumulated during the time of radicle protrusion while another 18 appeared in later stages. Further analysis showed that 226 of the identified polypeptides could be located in different metabolic pathways. Proteins involved in carbohydrate, energy and amino acid metabolism constituted to about 1/4, and those involved in metabolism of vitamins and cofactors constituted for about 3 % of the total signal intensity in gels prepared from 72 h seedlings. Enzymes related to genetic information processing increased very quickly during early imbibition and reached highest level around 30 h of germination.     

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%).     

Structural analysis of N-glycans of the planarian Dugesia japonica

To investigate the relationship between phylogeny and glycan structures, we analyzed the structure of planarian N-glycans. The planarian Dugesia japonica, a member of the flatworm family, is a lower metazoan. N-glycans were prepared from whole worms by hydrazinolysis, followed by tagging with the fluorophore 2-aminopyridine at their reducing end. The labeled N-glycans were purified, and separated by three HPLC steps. By comparison with standard pyridylaminated N-glycans, it was shown that the N-glycans of planarian include high mannose-type and pauci-mannose-type glycans. However, many of the major N-glycans from planarians have novel structures, as their elution positions did not match those of the standard glycans. The results of mass spectrometry and sugar component analyses indicated that these glycans include methyl mannoses, and that the most probable linkage was 3-O-methylation. Furthermore, the methyl residues on the most abundant glycan may be attached to the non-reducing-end mannose, as the glycans were resistant to α-mannosidase digestion. These results indicate that methylated high-mannose-type glycans are the most abundant structure in planarians.     

Influence of accelerated and natural aging on free radical levels in soybean seeds

Embryonic axes and cotyledons of three soybean ( Glycine max L. cv. Gudzon) seed lots designated as "normal", "naturally aged" and "acceleratedly aged" were analyzed for their organic free radical contents. No signals of free radicals were found in ESR spectra from cotyledonary material of the three samples investigated. High levels of organic free radicals were observed in the embryonic axes. There were significant differences in the free radical contents of the embryonic axes: the relative values of free radicals were 100, 190 and 170% for samples aged normally, naturally and in an accelerated manner, respectively. These results point to the physiological role of the embryonic axes during natural or accelerated aging of seeds, notwithstanding that the axis is a small part as compared to the cotyledons. It is suggested that lipid peroxidation in embryonic axes may play an important role in the seed deterioration during aging.     

EPR imaging and HPLC characterization of the pigment-based organic free radical in black soybean seeds

We investigated the location and distribution of paramagnetic species in dry black, brown, and yellow (normal) soybean seeds using electron paramagnetic resonance (EPR), X-band (9?GHz) EPR imaging (EPRI), and HPLC. EPR primarily detected two paramagnetic species in black soybean. These two different radical species were assigned as stable organic radical and Mn^2+?species based on the g values and hyperfine structures. The signal from the stable radical was noted at g?≈?2.00 and was relatively strong and stable. Subsequent noninvasive two-dimensional (2D) EPRI of the radical present in black soybean revealed that the stable radical was primarily located in the pigmented region of the soybean coat, with very few radicals observed in the soybean cotyledon (interior). Pigments extracted from black soybean were analyzed using HPLC. The major compound was found to be cyanidin-3-glucoside. Multi-EPR and HPLC results indicate that the stable radical was only found within the pigmented region of the soybean coat, and it could be cyanidin-3-glucoside or an oxidative decomposition product.     

Chloroplasts in seeds and dark-grown seedlings of lotus

In most higher plants, mature dry seeds have no chloroplasts but etioplasts. Here we show that in a hydrophyte, lotus (Nelumbo nucifera), young chloroplasts already exist in shoots of mature dry seeds and that they give rise to mature chloroplasts during germination, even in darkness. These shoots contain chlorophyll and chlorophyll-binding proteins CP1 and LHCP. The unique features of chloroplast formation in N. nucifera suggest a unique adaptive strategy for seedling development correlated with the plant's habitat.     

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.     

Quantitative trait loci analysis of individual and total isoflavone contents in soybean seeds

Soybean isoflavones play diverse roles in human health, including cancers, osteoporosis, heart disease, menopausal symptoms and pabulums. The objective of this study was to identify the quantitative trait loci (QTL) associated with the isoflavones daidzein (DC), genistein (GeC), glycitein (GlC) and total isoflavone contents (TIC) in soybean seeds. A population of 184 F_21:0 recombinant inbred lines derived from a ‘Xiaoheidou’ בGR8836’ cross was planted in pot and field conditions to evaluate soybean isoflavones. Twenty-one QTL were detected by composite interval mapping. Several QTL were associated with the traits for DC, GeC, GlC and TIC only. QDGeGlTIC4_1 and QDGlTIC12_1 are reported first in this study and were associated with the DC, GeC, GlC and TIC traits simultaneously. The QTL identified have potential value for marker-assisted selection to develop soybean varieties with desirable isoflavone content.     

Localizations of aluminum in soybean bacteroids and seeds

Aluminum, long known to be detrimental to soybean productivity, was localized in the polyphosphate granules (PPG) of bacteroids in root nodules of soybean plants. By using energy-dispersive X-ray analysis, bacteroids in early infections were shown to have typical PPG constituents. However, in PPG in older infections and after the bacteroids were digested intracellularly, aluminum was also detected. These results indicate that aluminum accumulates in PPG after a period when organisms have been resident in host cells and that high levels of aluminum were present in the bacteroids at the time of their demise. At least some of the aluminum in these laboratory-grown plants could have come from the seeds used.     

Respiratory pathways in aged soybean seeds

Respiratory activities in soybean seeds (Glvcine max (L.) Merr. cv. Chippewa 64) have been examined in the first minutes after water imbibition and after three hours of imbibition, using either particles or intact axes. Cyanide and azide were utilized as inhibitors of the cytochrome oxidase pathway of respiration, and SHAM inhibitions were interpreted as effects on the alternative pathway, since in unaged axes inhibitions by SHAM were obtained only when respiratory activity had either been restricted with inhibitors of the cytochrome oxidase pathway or expanded by an uncoupler (CCCP). From the experiments with these inhibitors, it is suggested that unaged seeds utilize both respiration pathways in the cotyledon but only the cytochrome pathway in the axis. Accelerated aging causes a marked deterioration of respiration, especially that through the cytochrome pathway, and there is an associated engagement of the alternative pathway in the seed axis. It is suggested that the lowering of respiratory activity and the shift in respiratory pathways may play a major role in the decline of germinability and vigor.