Human Erythrocyte Receptor of l-Fucose-specific Lectin Produced by Streptomyces sp.

L-Fucose-specific lectin produced by Streptomyces no. 16-3 (SFL 16-3) was labeled with N- succinimidyl-[2, 3-³H]-propionate to quantitatively investigate its binding to human erythrocytes. The binding inhibition by sugars was competitive, and 5mM L-fucose or 20 mM d-mannose completely inhibited the binding. Among plant lectins, Lotus tetragonolobus, Ulex europeus I, soybean and wheat germ lectin showed competitive inhibition. The association constant and the average number of binding sites for human blood group O erythrocytes were approximately 3 × 10⁷ M^-1 and 1 × 10⁶ cell^-1, respectively. Trypsinization of erythrocytes preferentially increased the number of binding sites for human A and B erythrocytes but not for O erythrocytes.

Membrane components were extracted from human B and O erythrocytes and their binding activity for SFL 16-3 was tested using the hemagglutination-inhibition assay. Poly(glycosyl)-ceramide was the predominant receptor and its fucosyl residue was essential for binding. The crude glycoprotein fraction showed only slight inhibition activity.     

The Flavonoid Apigenin Downregulates CDK1 by Directly Targeting Ribosomal Protein S9

Flavonoids have been reported to inhibit tumor growth by causing cell cycle arrest. However, little is known about the direct targets of flavonoids in tumor growth inhibition. In the present study, we developed a novel method using magnetic FG beads to purify flavonoid-binding proteins, and identified ribosomal protein S9 (RPS9) as a binding partner of the flavonoid apigenin. Similar to treatment with apigenin, knockdown of RPS9 inhibited the growth of human colon cancer cells at the G2/M phase by downregulating cyclin-dependent kinase 1 (CDK1) expression at the promoter level. Furthermore, knockdown of RPS9 suppressed G2/M arrest caused by apigenin. These results suggest that apigenin induces G2/M arrest at least partially by directly binding and inhibiting RPS9 which enhances CDK1 expression. We therefore raise the possibility that identification of the direct targets of flavonoids may contribute to the discovery of novel molecular mechanisms governing tumor growth.     

Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation

Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.     

A new analysis of the depolymerized fragments of lignin polymer using ToF-SIMS

Lignin in plant cell walls is a complex, irregular polymer built from phenylpropanoid C6-C3 units that are connected via various C-C and C-O linkages. A recent study using time-of-flight secondary ion mass spectrometry (ToF-SIMS) with Ga primary ion bombardment showed that lignin polymers can be characterized by specific positive ions possessing a substituted aromatic ring (so-called guaiacyl or syringyl rings), which are the basic building units of lignin. To study the relationship between the characteristic ions of lignin and the common interunit linkages, various lignin dimer model compounds were investigated using ToF-SIMS. The resulting dimer spectra showed that the characteristic ions with a guaiacyl ring at m/z 137 and 151 result from rupture of most common interunit linkages, not only 8-O-4' linkages, which are the most abundant in lignin, but also 8-1', 8-5', and 8-8'. There was no evidence of rupture of 5-5' linkages. These results show that ToF-SIMS offers a new tool for the direct analysis of the depolymerized fragments of lignin polymers. The mechanisms for the fragmentation of lignin dimer models in ToF-SIMS were proposed that allow ToF-SIMS fragmentation rules to be deduced. Adduct ions such as [M + 13]+ ([M + CH]+) were also produced in fragmentation of the dimers and are thought to arise from the combination of the molecules with their stable fragments.     

Effects of nitric oxide synthase inhibitor on the digestive system measured by simultaneous monitoring of gastric motility, gastric emptying activity and postprandial pancreaticobiliary secretion in dogs.

Relationships between the NO synthase inhibitor and gastric and pancreaticobiliary functions measured simultaneously in the digestive state have been little studied. The aim of this study was to estimate the effect of NO synthase inhibitor on integrated digestive function in conscious dogs. A strain gauge force transducer was implanted on the gastric antrum of 6 mongrel dogs to measure gastric contractile activity and two duodenal cannulas were inserted into the proximal and distal sites to measure the gastric emptying rate and the pancreaticobiliary output into the duodenum using our novel method. Postprandial pancreatic and biliary secretion were presented as amylase and bile acid activity, respectively. Furthermore, a cervical cannula was placed into the superior vena cava as a route for the administration of NO synthase inhibitor, N omega-nitro-L-arginine (L-NNA), at a dose of 2.5 mg/kg-h. In a group given L-NNA, gastric contractile activity after ingestion was significantly enhanced, but the emptying rates of gastric solids and liquids were significantly suppressed in comparison with the control. The mean 0-1 h amylase integrated output was significantly (P < 0.05) decreased in comparison with the control, and the mean bile acid integration of 0-1 h output was also significantly (P < 0.01) decreased. A possible explanation for this observation is that smaller volumes of nutrient are delivered into the duodenum; however, it could also be that postprandial pancreaticobiliary secretion is inhibited by an alteration of blood flow or by a change in contractions of the sphincter of Oddi after the administration of L-NNA.     

Nitric oxide inhibits IFN-alpha production of human plasmacytoid dendritic cells partly via a guanosine 3',5'-cyclic monophosphate-dependent pathway

NO, a free radical gas, is known to be critically involved not only in vascular relaxation but also in host defense. Besides direct bactericidal effects, NO has been shown to inhibit Th1 responses and modulate immune responses in vivo, although the precise mechanism is unclear. In this study, we examined the effect of NO on human plasmacytoid dendritic cells (pDCs) to explore the possibility that NO might affect innate as well as adaptive immunity through pDCs. We found that NO suppressed IFN-alpha production of pDCs partly via a cGMP-dependent mechanism, which was accompanied by down-regulation of IFN regulatory factor 7 expression. Furthermore, treatment of pDCs with NO decreased production of IL-6 and TNF-alpha and up-regulated OX40 ligand expression. In accordance with these changes, pDCs treated with NO plus CpG-oligodeoxynucleotide AAC-30 promoted differentiation of naive CD4(+) T cells into a Th2 phenotype. Moreover, pDCs did not express inducible NO synthase even after treatment with AAC-30, LPS, and several cytokines. These results suggest that exogenous NO and its second messenger, cGMP, alter innate as well as adaptive immune response through modulating the functions of pDCs and may be involved in the pathogenesis of certain Th2-dominant allergic diseases.     

Development of a microbioreactor for glycoconjugate synthesis

A microbioreactor immobilized with a synthase-type mutant enzyme, Endo-M-N175Q (glycosynthase) of endo-β-N-acetylglucosaminidase derived from Mucor hiemalis (Endo-M), was constructed and used for glycoconjugate synthesis. The transglycosylation was performed with a reaction mixture containing an oxazoline derivative of sialo complex-type glycoside (SG), which was prepared from a sialo complex-type glycopeptide SGP derived from hen egg yolk, as a glycosyl donor and N-Fmoc-N-acetylglucosaminyl-l-asparagine [Fmoc-Asn(GlcNAc)-OH] as an acceptor. The reaction mixture was injected into a glycosynthase microbioreactor at a constant flow rate. Highly efficient and nearly stoichiometric transglycosylation occurred in the microbioreactor, and the transglycosylation product was eluted from the other end of the reactor. The glycosynthase microbioreactor was stable and could be used repeatedly for a long time.     

Homogeneity of Borrelia japonica and heterogeneity of Borrelia afzelii and 'Borrelia tanukii' isolated in Japan, determined from ospC gene sequences

Borrelia afzelii, B. japonica, and `B. tanukii' isolated from various sources and geographical origins in Japan were characterized by restriction fragment length polymorphism (RFLP) analysis and sequencing analysis of the outer surface protein C (OspC) amplicon. B. afzelii and `B. tanukii' generated variable RFLP patterns and differences in ospC gene sequence were confirmed. In contrast, 26 isolates of B. japonica generated one OspC RFLP type, and sequence similarity between B. japonica ranged from 96.4 to 99.7%. These finding suggests that B. japonica is unique in comparison with other members of B. burgdorferi sensu lato species with respect to homogeneity of the ospC gene.