Rapid preparation of a glycan oxazoline and a homogeneously glycosylated antibody with an enzyme-immobilized monolithic column.

Chemo-enzymatic glycan engineering is considered to be one of the most promising strategies to enhance efficiency in pharmaceutical research. However, it is assumed that this technology has limited industrial application for the production of biological therapeutics because of the high cost of the process. In this study, we developed a scheme for rapidly preparing a glycan oxazoline and a homogeneously glycosylated antibody. The enzyme-immobilized monolith and the flow chemistry-based approach enabled a glycan oxazoline and a homogeneously glycosylated antibody to be obtained at the gram scale from starting materials (sialylglycopeptide and heterogeneously glycosylated protein) within 2.5 h. This cost-effective scheme for obtaining a large amount of glycan donors and homogeneously glycosylated proteins in a short time will be helpful to implement glycan engineering technology for industrial purposes such as pharmaceutical production.

Isolation and evaluation of erythroid progenitors in the livers of larval, froglet, and adult Xenopus tropicalis.

Xenopus liver maintains erythropoietic activity from the larval to the adult stage. During metamorphosis, thyroid hormone mediates apoptosis of larval-type erythroid progenitors and proliferation of adult-type erythroid progenitors, and a globin switch occurs during this time. In addition, the whole-body mass and the liver also change; however, whether there is a change in the absolute number of erythroid progenitors is unclear. To isolate and evaluate erythroid progenitors in the Xenopus liver, we developed monoclonal ER9 antibodies against the erythropoietin receptor (EPOR) of Xenopus. ER9 recognized erythrocytes, but not white blood cells or thrombocytes. The specificity of ER9 for EPOR manifested as its inhibitory effect on the proliferation of a Xenopus EPOR-expressing cell line. Furthermore, ER9 recognition was consistent with epor gene expression. ER9 staining with Acridine orange (AO) allowed erythrocyte fractionation through fluorescence-activated cell sorting. The ER9+ and AO-red (AOr)high fractions were highly enriched in erythroid progenitors and primarily localized to the liver. The method developed using ER9 and AO was also applied to larvae and froglets with different progenitor populations from adult frogs. The liver to body weight and the number of ER9+ AOrhigh cells per unit body weight were significantly higher in adults than in larvae and froglets, and the number of ER9+ AOrhigh cells per unit liver weight was the highest in froglets. Collectively, our results show increased erythropoiesis in the froglet liver and demonstrate growth-dependent changes in erythropoiesis patterns in specific organs of Xenopus.

A fluorogenic probe for core-fucosylated glycan-preferred ENGase.

A fluorescence-quenching-based assay system to determine the hydrolytic activity of endo-ß-N-acetylglucosaminidases (ENGases), which act on the innermost N-acetylglucosamine (GlcNAc) residue of the chitobiose segment of core-fucosylated N-glycans, was constructed using a dual-labeled fluorescent probe with a hexasaccharide structure. The fluorogenic probe was evaluated using a variety of ENGases, including Endo-M W251N mutant, Endo-F3, and Endo-S, which recognize core fucosylated N-glycans. The occurrence of a hydrolysis reaction was detected by observing an increased fluorescence intensity, ultimately allowing the ENGase activities to be easily and quantitatively evaluated, with the exception of Endo-S. The obtained results clearly indicated the substrate specificities of the examined ENGases.

Development of Erythroid Progenitors under Erythropoietin Stimulation in Xenopus laevis Larval Liver.

Erythroid progenitors that respond to erythropoietin (Epo) are present in the liver of adult Xenopus laevis. However, cells responding to Epo in the larval liver and through the metamorphosis period under hepatic remodeling have not been characterized. In this study, tadpoles were staged using the tables of Nieuwkoop and Faber (NF). Liver cells from pre- (NF56) or post- (NF66) metamorphic stage were cultured in the presence of Epo. ß2-globin mRNA expression peaked at day 7 after the start of culture. Larval ß2-globin was highly expressed in NF56-derived cells, while adult ß2-globinwas detected in those of NF66. In both NF56- and NF66-derived cells, mRNA expression of eporand gata2 peaked at day 5 and days 3-4, respectively. In contrast, gata1 expression peaked at day 6 in NF56 cells and at day 5 in NF66 cells. Half maximal proliferation of erythrocytic blast cells derived from the liver at NF66 was observed at day 3, which was earlier than that of NF56. These results indicate that erythroid progenitors that respond to Xenopus laevis Epo are maintained in pre- and post-metamorphic liver, although the tissue architecture changes dramatically during metamorphosis. Additionally, the globin switching occurred, and/or the erythroid progenitors for larval erythrocytes were replaced by those for adult erythrocytes in the metamorphic liver.

Dominant structural factors for complexation and denaturation of proteins using carboxylic acid receptors.

Complexation accompanied by denaturation of protein with synthetic carboxylic acid receptors was investigated, to evaluate the key factors for recognition of proteins. The synthetic receptors used were tetraphenylporphyrin (TPP) derivatives and receptors bearing multiple (2-8) carboxylic acid groups. The complexation behavior was quantified from the absorption in the far UV CD spectrum attributed to the secondary structure of the protein. TPP derivatives bearing multiple carboxylic acid groups in the side chains exhibited higher affinity than other receptors that were smaller and had fewer carboxylic acid groups. As the degree of complexation was influenced by the pH and ionic strength in aqueous solution, electrostatic interaction was one of the most important factors for the recognition of proteins. Complexation was also estimated by observation of fluorescence quenching of the TPP derivatives. The stoichiometry of the complexes between lysozyme and the porphyrins was investigated by quantitative analysis of the denaturation using CD spectra. From the results of Job plots and slope analysis for the amount of denatured protein, formation of 1:1 complexes was confirmed. The equilibrium association constants (K(ass)) for lysozyme and the TPP receptors ranged from 0.6×10(6) to 1.1×10(6)M(-1). The lytic activity of lysozyme was partially lost in the presence of anionic TPP derivatives, due to complexation and denaturation.

Filmless versus film-based systems in radiographic examination costs: an activity-based costing method.

BACKGROUND: Since the shift from a radiographic film-based system to that of a filmless system, the change in radiographic examination costs and costs structure have been undetermined. The activity-based costing (ABC) method measures the cost and performance of activities, resources, and cost objects. The purpose of this study is to identify the cost structure of a radiographic examination comparing a filmless system to that of a film-based system using the ABC method. METHODS: We calculated the costs of radiographic examinations for both a filmless and a film-based system, and assessed the costs or cost components by simulating radiographic examinations in a health clinic. The cost objects of the radiographic examinations included lumbar (six views), knee (three views), wrist (two views), and other. Indirect costs were allocated to cost objects using the ABC method. RESULTS: The costs of a radiographic examination using a filmless system are as follows: lumbar 2,085 yen; knee 1,599 yen; wrist 1,165 yen; and other 1,641 yen. The costs for a film-based system are: lumbar 3,407 yen; knee 2,257 yen; wrist 1,602 yen; and other 2,521 yen. The primary activities were "calling patient," "explanation of scan," "take photographs," and "aftercare" for both filmless and film-based systems. The cost of these activities cost represented 36.0% of the total cost for a filmless system and 23.6% of a film-based system. CONCLUSIONS: The costs of radiographic examinations using a filmless system and a film-based system were calculated using the ABC method. Our results provide clear evidence that the filmless system is more effective than the film-based system in providing greater value services directly to patients.

Structure determination of Pb/Ge(111)-ß-(√3 × âˆš3)R30° by dynamical low-energy electron diffraction analysis and first-principles calculation.

We have determined the atomic structure of the Pb/Ge(111)-ß-(√3 × âˆš3)R30° surface, which was shown to exhibit a large Rashba spin splitting in a metallic surface state by dynamical low-energy electron diffraction analysis. The Pb coverage for the optimized atomic structure is 4/3 with one Pb atom located at every third H(3) site of the bulk-truncated Ge(111) surface and the other three near the T(1) sites but slightly displaced towards the T(4) sites. The determined atomic structure agrees well with the energetically optimized one obtained from the first-principles calculation. The calculation also revealed that the potential for the Pb atoms on the H(3) sites is very soft along the surface normal, suggesting that their vertical position is distributed within a range of about 0.2-0.3 Å. The previously proposed phase transition associated with the surface melting is discussed.

Willingness to pay for municipality hospital services in rural Japan: a contingent valuation study.

BACKGROUND: The Japanese healthcare system has undergone reforms to address the struggles that municipality hospitals face. Reform guidelines clearly define criteria for administrative improvement. However, criteria to evaluate the demand for healthcare provisions in rural Japan, including the needs of rural residents for municipality hospitals in particular have not been specified. The purpose of this paper is to measure residents' willingness to pay (WTP) for municipality hospital services using the contingent valuation method, and to evaluate municipality hospital valuation on the basis of WTP. K town, located in the Hokkaido prefecture of Japan, was selected as the location for this study. Participants were recruited by a town hall healthcare administrator, hospital and clinic staff, and a local dentist. Participants were asked what amount they would be willing to pay as taxes to continue accessing the services of the municipality hospital for one year by using open-ended questions in face-to-face interviews. FINDINGS: Forty-eight residents were initially recruited, and 40 participants were selected for the study (response rate 83%). As compared to K town's population, this data slanted toward the elderly, although there was no significant difference in frequency among the characteristics. The median WTP was estimated at 39,484 yen ($438.71), with a 95% confidence interval 27,806-55,437 yen ($308.95-615.96). Logistic regression revealed no significant factors affecting WTP. CONCLUSIONS: If the total amount of residents' WTP for the municipality hospital were to be estimated by this result, it would calculate with 129,586,000 yen ($1,439,844). This is approximately equal to the amount of money to be transferred from the general account of the government of K town, more than one-half of the town tax of K town, and about two-fold in comparison to Japan as a whole. This showed that K town's residents placed a high valuation on the municipality hospital, which nearly equalled the amount that the K town government provided to the municipality hospital to cover its annual deficit. K town residents had come to expect not only general clinical practice, but also emergency medical services and night practice provided by their own town's municipality hospital. WTP can be used as a measure of hospital evaluation because it reflects the importance of the hospital to the residents in its region.

Development of terminology for mammographic techniques for radiological technologists.

We are developing a mammographic ontology to share knowledge of the mammographic domain for radiologic technologists, with the aim of improving mammographic techniques. As a first step in constructing the ontology, we used mammography reference books to establish mammographic terminology for identifying currently available knowledge. This study proceeded in three steps: (1) determination of the domain and scope of the terminology, (2) lexical extraction, and (3) construction of hierarchical structures. We extracted terms mainly from three reference books and constructed the hierarchical structures manually. We compared features of the terms extracted from the three reference books. We constructed a terminology consisting of 440 subclasses grouped into 19 top-level classes: anatomic entity, image quality factor, findings, material, risk, breast, histological classification of breast tumors, role, foreign body, mammographic technique, physics, purpose of mammography examination, explanation of mammography examination, image development, abbreviation, quality control, equipment, interpretation, and evaluation of clinical imaging. The number of terms that occurred in the subclasses varied depending on which reference book was used. We developed a terminology of mammographic techniques for radiologic technologists consisting of 440 terms.

Rebamipide and mosapride enhance pilocarpine-induced salivation.

BACKGROUND: During esophageal acid clearance, salivation plays an important role in defending the esophageal mucosa. Mosapride, an agent used in chronic, long-term therapy of gastro-esophageal reflux disease (GERD) was regarded as mediating its efficacy through prokinetic properties. Rebamipide is also widely used as an anti-gastritis and anti-ulcer agent in GERD patients with chronic gastritis. The aim of this study is to investigate the effects of rebamipide, mosapride, and risperidone on the salivation induced by pilocarpine. MATERIALS AND METHODS: The experiments were conducted on 4-week male SD rats (120-150g). The salivation was induced by intraperitoneally administrated pilocarpine and saliva was collected using preweighted small cotton balls inserted into the animal's mouth every 30 min for 180 min. Thirteen minutes before intraperitoneal administration of pilocarpine, rebamipide, mosapride, and risperidone were administered intraduodenally. Control rats were conducted by intraperitoneal administration of saline and intraduodenal administration of 0.5% methylcellulose solution. RESULTS: The saliva weight at 0-30 min was significantly (p<0.01) increased after administration of pilocarpine, compared to control rats. An additional administration of mosapride and rebamipide increased the saliva weight at 0-30 min. The total volume of saliva for 150 min after administration of pilocarpine was the highest after preadministration of rebamipide, followed by mosapride, and risperidone. CONCLUSIONS: Increase in salivation produced by i.p. pilocarpine was enhanced by preadministration of rebamipide and mosapride.

Ammonia-induced apoptosis is accelerated at higher pH in gastric surface mucous cells.

Gastric luminal ammonia produced by Helicobacter pylori has been shown to cause gastric mucosal injury. This study was conducted to examine the mechanisms by which gastric luminal ammonia causes apoptosis of gastric epithelial cells. Monolayers of GSM06 cells, developed from murine gastric surface mucous cells, were cultured in the absence or presence of 10-30 mM NH(4)Cl at ambient pH of 5.0, 6.0, and 7.0. In the presence of luminal NH(4)Cl, GSM06 cells showed 1) cell shrinkage and nuclear chromatin condensation, 2) DNA fragmentation into oligonucleosomes, 3) leakage of cytochrome c into cytosolic fraction without affecting bax expression, and 4) increases in activity of caspases-3 and -9. These changes were accentuated when the cells were cultured at pH 7.0. In the absence of NH(4)Cl, none of these changes was detected at any pH examined. These results suggest that gastric luminal ammonia, at concentrations detected in H. pylori-infected subjects, induces apoptosis of gastric epithelial cells by release of cytochrome c from mitochondria, followed by activation of caspases-9 and -3, especially at higher ambient pH.