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.     

Age-related changes of dietary intake and blood eicosapentaenoic acid, docosahexaenoic acid, and arachidonic acid levels in Japanese men and women

We studied the relationship between dietary intake and the blood compositions of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (ARA) in four study groups with different ages and sexes. One hundred and four subjects were recruited. Dietary records together with photographic records from 28 consecutive days were amassed and the fatty acid composition in erythrocyte membranes and plasma lipid fractions was analyzed. Fish intake in the elderly group was significantly higher than that in the young group in both men and women. The compositions of ARA in erythrocytes and plasma phospholipids in the elderly were lower than those in the young, but the ARA intake was nearly identical. In the elderly group, the percentage of dietary ARA consumed at the same time as EPA and DHA derived from fish was high. We considered that these fatty acids markedly inhibited the incorporation of dietary ARA into blood phospholipids.     

Purification and characterization of a co(II)-sensitive alpha-mannosidase from Ginkgo biloba seeds

An alpha-mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified alpha-mannosidase was estimated to be 120 kDa by SDS-PAGE in the presence of 2-mercaptoethanol, and 340 kDa by gel filtration, indicating that Ginkgo alpha-mannosidase may function in oligomeric structures in the plant cell. The N-terminal amino acid sequence of the purified enzyme was Ala-Phe-Met-Lys-Tyr-X-Thr-Thr-Gly-Gly-Pro-Val-Ala-Gly-Lys-Ile-Asn-Val-His-Leu-. The alpha-mannosidase activity for Man(5)GlcNAc(1) was enhanced by the addition of Co(2+), but the addition of Zn(2+), Ca(2+), or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man(6)GlcNAc(1) was significantly faster than that for pyridylaminated Man(6)GlcNAc(2), suggesting the possibility that this enzyme is involved in the degradation of free N-glycans occurring in developing plant cells (Kimura, Y., and Matsuo, S., J. Biochem., 127, 1013-1019 (2000)). To our knowledge, this is the first report showing that plant cells contain an alpha-mannosidase, which is activated by Co(2+) and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.     

Molecular identification of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors

Platelets play an important role in hemostasis, thrombosis, and antimicrobial host defense and are also involved in the induction of inflammation, tissue repair, and tumor metastasis. We have previously characterized the platelet aggregation-inducing sialoglycoprotein (Aggrus/gp44) overexpressed on the surface of tumor cells. Because a platelet aggregation-neutralizing 8F11 monoclonal antibody that could specifically recognize Aggrus suppressed tumor-induced platelet aggregation, we have previously purified Aggrus by 8F11-affinity chromatography and found that purified Aggrus possessed the ability to induce aggregation of platelets. Here we show that Aggrus is identical to the T1alpha/gp38P/OTS-8 antigen, the function of which in tumors is unknown. Expression of mouse Aggrus and its human homologue (also known as T1alpha-2/gp36) induced platelet aggregation without requiring plasma components. Using the 8F11 antibody, we identified the highly conserved platelet aggregation-stimulating domain with putative O-glycosylated threonine residues as the critical determinant for exhibiting platelet aggregation-inducing capabilities. We compared the expression level of human aggrus mRNA using an array containing 160 cDNA pair samples derived from multiple human tumorigenic and corresponding normal tissues from individual patients. We found that expression level of aggrus was enhanced in most colorectal tumor patients. To confirm the protein expression, we generated anti-human Aggrus polyclonal antibodies. Immunohistochemical analysis revealed that Aggrus expression was frequently up-regulated in colorectal tumors. These results suggest that Aggrus/T1alpha is a newly identified, platelet aggregation-inducing factor expressed in colorectal tumors.     

Differential secretion of cytokines and adhesion molecules by HUVEC stimulated with low concentrations of bleomycin

Bleomycin (BLM) is known to induce lung inflammation and subsequent fibrosis. Endothelial cells have been reported to play an important role, producing cytokines and adhesion molecules during the inflammatory process in pulmonary fibrosis. To examine the effects of BLM on endothelial cells, we investigated the expression profiles of various cytokines and adhesion molecules produced by endothelial cells stimulated with BLM. Increased expressions of interleukin-8 and monocyte chemoattractant protein-1 measured as protein as well as mRNA by human umbilical vein endothelial cells (HUVECs) were detected after exposure to BLM. Similarly, increased expressions of E-selectin and intercellular adhesion molecule-3 were detected both at the protein and mRNA levels. Under these conditions, a small but significant decrease of [3H]thymidine uptake was detected. These findings indicate that HUVEC were stimulated to secrete cytokines and express adhesion molecules in the presence of low concentrations of BLM which have a mildly inhibitory effect on cellular proliferation.     

Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3

Modification of the cytoplasmic tails of the integrin alpha(IIb)beta(3) plays an important role in the signal transduction in platelets. We searched for proteins that bind to the alpha(IIb) cytoplasmic tail using the yeast two-hybrid assay with a cDNA library of the megakaryocyte-derived cell line and identified a protein, ancient ubiquitous protein 1 (Aup1), that is ubiquitously expressed in human cells. Observation of UT7/TPO cells expressing a red fluorescent protein-tagged Aup1 indicated its localization in the cytoplasm. Immunoprecipitation of UT7/TPO cells by an antibody for Aup1 revealed that approximately 40% of alpha(IIb) is complexed with Aup1. Binding study with an alpha(IIb) cytoplasmic tail peptide and glutathione S-transferase-Aup1 fusion protein revealed a low affinity (K(d) = 90 microm). Subsequent yeast two-hybrid assay indicated binding of Aup1 to cytoplasmic tails of other integrin alpha subunits. Binding study with the purified Aup1 and various glutathione S-transferase-alpha(IIb) cytoplasmic tail peptides revealed specific binding of Aup1 to the membrane-proximal sequence (KVGFFKR) that is conserved among the integrin alpha subunits and plays a crucial role in the alpha(IIb)beta(3) inside-out signaling. As Aup1 possesses domains related to signal transduction, these results suggest involvement of Aup1 in the integrin signaling.     

Structural change of ribosomes during apoptosis: degradation and externalization of ribosomal proteins in doxorubicin-treated Jurkat cells

Changes in the amount and localization of human ribosomal proteins during apoptosis were determined. When total lysates of Jurkat cells undergoing apoptosis induced by doxorubicin were analyzed by Western blotting, degradation of three ribosomal proteins, S18, L5, and L14, was detected at 48 h after the induction of apoptosis. Decreases in the amounts of these three ribosomal proteins were also observed in ribosome-enriched fractions. These changes were partly abolished by the addition of the pan-caspase inhibitor z-VAD-fmk. Moreover, formation of the 80S ribosome complex appeared to be inhibited at 48 h after apoptosis induction. On the other hand, the rate of protein synthesis, assessed by measuring the incorporation of [35S]Met into bulk proteins, decreased as early as 12 h after the addition of doxorubicin. These results indicate that changes in the amount of ribosomal proteins and the overall structure of ribosomes in apoptosing cells occur after protein synthesis declines. Finally, analyses by flow cytometry, immunofluorescence, and Western blotting showed that six ribosomal proteins, S15, P0, L5, L6, L36a, and L41, were relocalized and expressed at the cell surface during apoptosis. The above results collectively indicate that ribosomes are structurally altered in apoptotic cells following inactivation of protein synthesis.     

Preventive effect of a chicken extract on the development of hypertension in stroke-prone spontaneously hypertensive rats

The antihypertensive effect of Brand's Essence of Chicken (BEC), a popular chicken extract used as a traditional health food, was examined with stroke-prone spontaneously hypertensive rats (SHRSPs). The animals were maintained from 6 to 25 weeks of age on drinking water with or without BEC. The BEC-fed group showed a significant reduction in the development of hypertension when compared with the control animals. The levels of blood urea nitrogen and plasma creatinine in the BEC-fed group were significantly lower than those in the control group, suggesting that the renal glomerular function had been improved by the daily administration of BEC. It thus seems likely that BEC would be useful as a prophylactic treatment against the development of hypertension and renal injury.