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Cellulose nanosphere (CNS) is a novel material isolated through delignification, bleaching, acid hydrolysis, dialysis, and sonication of cellulose fibers from agricultural wastes such as corn husk and rice straw. Comparative examination through multiple characterizations of CNS from these cellulose sources was conducted to investigate its potential applications. Corn husk cellulose nanospheres (CHCNS) and rice straw cellulose nanospheres (RSCNS) were characterized by its chemical characteristics, crystallinity, surface morphology and thermal stability. FTIR showed the same transmittance patterns for both CNS samples. The peaks for CHCNS and RSCNS in the NMR analysis were comparable, and the presence of toluene enabled the CNS dissolve better in DMSO-d6. Higher crystallinity index was calculated in CHCNS than in RSCNS according to XRD analysis. TEM analysis demonstrated that CHCNS has shorter diameter than RSCNS. Lower onset degradation temperature relative to their corresponding bleached cellulose fiber was determined through TG Analysis. Biomedical applications such as drug delivery, wound dressings and tissue engineering are the most suitable applications based on the profiles of both CNS samples.

Glucose is the most important energy source in all organisms; however, our understanding of the pathways and mechanisms underlying glucose transportation and localization in living cells is incomplete. Here, we prepared two glucose analogs labeled with a dansylamino group at the C-1 (1-Dansyl) or C-2 (2-Dansyl) position; the dansyl group is a highly fluorescent moiety that is characterized by a large Stokes shift between its excitation and emission wavelengths. We then examined the cytotoxicity of the two glucose analogs in mammalian fibroblast cells and in the ciliated protozoan Tetrahymena thermophila. In both cell types, 2-Dansyl had no negative effects on cell growth. The specificity of cellular uptake of glucose analogs was confirmed using an inhibitor of glucose transporter in NIH3T3 cells. In NIH3T3 cells and T. thermophila, fluorescence microscopy revealed that the glucose analogs localized throughout the cytoplasm, but especially at the periphery of the nucleus. In T. thermophila, we also found that swimming speed was comparable in media containing non-labeled glucose or one of the glucose analogs, which provided more evidence not only that the analogs were not cytotoxic in these cells but also that the analogs had no negative effect on the ciliary motion. Together, the present results suggest that the glucose analogs have low toxicity and will be useful for bioimaging of glucose-related systems.

Triplet regimens, such as lenalidomide, bortezomib, and dexamethasone (RVd) or thalidomide, bortezomib, and dexamethasone (VTd), are standard induction therapies for transplant-eligible patients with newly diagnosed multiple myeloma (NDMM). The addition of daratumumab to RVd and VTd has been investigated in the GRIFFIN and CASSIOPEIA trials, respectively, resulting in improvement in the rate of minimal residual disease (MRD) negativity. In this study, we conducted a cost-effectiveness analysis with a 10-year time horizon to compare first-line and second-line use of daratumumab for transplant-eligible patients with NDMM. Because long-term follow-up data for these clinical trials are not yet available, we developed a Markov model that uses MRD status to predict progression-free survival. Daratumumab was used either in the first-line setting in combination with RVd or VTd or in the second-line setting with carfilzomib plus dexamethasone (Kd). Quality-adjusted life-years (QALYs) and incremental cost-effectiveness ratios were calculated from a Japanese and US payer perspective. In the Japanese analysis, D-RVd showed higher QALYs (5.43 vs 5.18) and lower costs (¥64 479,793 vs ¥71  287 569) compared with RVd, and D-VTd showed higher QALYs (5.67 vs 5.42) and lower costs (¥43  600 310 vs ¥49 471,941) compared with VTd. Similarly, the US analysis demonstrated dominance of a strategy incorporating daratumumab in first-line treatment regimens. Given that overall costs are reduced and outcomes are improved when daratumumab is used as part of a first-line regimen, the economic analysis indicates that addition of daratumumab to first-line RVd and VTd regimens is a dominant strategy compared with reserving its use for the second-line setting.

Gastrointestinal acute graft-versus-host disease (aGVHD) is a life-threatening complication that requires urgent and appropriate treatment. An endoscopic examination is considered the gold-standard for the diagnosis of gastrointestinal aGVHD. However, the prognostic value of endoscopy remains controversial. This study aimed to investigate the usefulness of pre-treatment macroscopic and histopathologic findings of upper and lower endoscopy with respect to predicting steroid-resistant gastrointestinal aGVHD. This retrospective study included 44 patients with gastrointestinal aGVHD who underwent endoscopy at the time of diagnosis and received systemic steroid treatment at our hospital. We graded the macroscopic and histopathologic findings using a previously validated 4-point scale. Univariate analyses of endoscopic grading revealed that a higher macroscopic grade in the ileum and higher histopathologic grades in the ileum and colon predicted a poor response to systemic steroids. In a multivariate analysis, macroscopic and histopathologic severity in the ileum were identified as significant prognostic factors that indicated resistance to steroid therapy. The presence of granulation tissue was also a strong independent predictor of resistance to steroid therapy. These findings suggest that both macroscopic and histopathologic findings in the ileum may be useful predictors of steroid-refractory gastrointestinal aGVHD and can indicate an immediate need to develop a second-line strategy.

The cost of tyrosine kinase inhibitors (TKIs) in the treatment of chronic myeloid leukemia (CML) is a substantial economic burden. In Japan, imatinib, dasatinib, and nilotinib are now approved as first-line treatment of CML in chronic phase. Recent “stop TKI” trials have shown that TKIs can be safely discontinued in nearly one-half of patients with sustained deep molecular response (DMR). In this study, we analyzed the cost-effectiveness of a simulated 10 years of CML treatment including stop TKI in both the United States and Japan. We constructed Markov models to compare 4 strategies in which treatment was initiated with imatinib, dasatinib, nilotinib, or any of these TKIs at the physician’s discretion. Treatment was switched to another TKI in the case of intolerance or resistance to the initial TKI, and TKIs were discontinued if DMR persisted for 2 years. “Imatinib first” offered 7.34 quality-adjusted life years (QALYs) at the cost of $1 022 148 in the United States (US dollars) and ¥32 526 785 in Japan (Japanese yen). In comparison with imatinib first, the incremental cost-effectiveness ratio per QALY of “dasatinib first” (7.68 QALY, $1 236 052, ¥51 506 254), “nilotinib first” (7.64 QALY, $1 245 667, ¥39 635 598), and “physician’s choice” (7.55 QALY, $1 167 818, ¥41 187 740) was $641 324, $696 717, and $666 634 in the United States and ¥54 456 325, ¥23 154 465, and ¥39 635 615 in Japan, respectively. None of the 3 strategies met the willingness-to-pay threshold. The results were robust to univariate and multivariate sensitivity analyses. Imatinib first was shown to be the most cost-effective approach even with the incorporation of stop TKI.

The cost of tyrosine kinase inhibitors (TKIs) in the treatment of chronic myeloid leukemia (CML) is a substantial economic burden. In Japan, imatinib, dasatinib, and nilotinib are now approved as first-line treatment of CML in chronic phase. Recent “stop TKI” trials have shown that TKIs can be safely discontinued in nearly one-half of patients with sustained deep molecular response (DMR). In this study, we analyzed the cost-effectiveness of a simulated 10 years of CML treatment including stop TKI in both the United States and Japan. We constructed Markov models to compare 4 strategies in which treatment was initiated with imatinib, dasatinib, nilotinib, or any of these TKIs at the physician's discretion. Treatment was switched to another TKI in the case of intolerance or resistance to the initial TKI, and TKIs were discontinued if DMR persisted for 2 years. “Imatinib first” offered 7.34 quality-adjusted life years (QALYs) at the cost of $1 022 148 in the United States (US dollars) and ¥32 526 785 in Japan (Japanese yen). In comparison with imatinib first, the incremental cost-effectiveness ratio per QALY of “dasatinib first” (7.68 QALY, $1 236 052, ¥51 506 254), “nilotinib first” (7.64 QALY, $1 245 667, ¥39 635 598), and “physician's choice” (7.55 QALY, $1 167 818, ¥41 187 740) was $641 324, $696 717, and $666 634 in the United States and ¥54 456 325, ¥23 154 465, and ¥39 635 615 in Japan, respectively. None of the 3 strategies met the willingness-to-pay threshold. The results were robust to univariate and multivariate sensitivity analyses. Imatinib first was shown to be the most cost-effective approach even with the incorporation of stop TKI.

Protein kinases are attractive targets for both biological research and drug development. Several assay kits, especially for the detection of adenosine diphosphate (ADP), which is universally produced by kinases, are commercially available for high-throughput screening (HTS) of kinase inhibitors, but their cost is quite high for large-scale screening. Here, we report a new enzyme-coupled fluorescence assay for ADP detection, which uses just 10 inexpensive, commercially available components. The assay protocol is very simple, requiring only the mixing of test solutions with ADP detection solution and reading the fluorescence intensity of resorufin produced by coupling reaction. To validate the assay, we focused on CDC2-like kinase 1 (CLK1), a dual-specificity kinase that plays an important role in alternative splicing, and we used the optimized assay to screen an in-house chemical library of about 215,000 compounds for CLK1 inhibitors. We identified and validated 12 potent inhibitors of CLK1, including a novel inhibitory scaffold. The results demonstrate that this assay platform is not only simple and cost-effective, but also sufficiently robust, showing good reproducibility and giving similar results to those obtained with the widely used ADP-Glo bioluminescent assay.

Protein kinases are attractive targets for both biological research and drug development. Several assay kits, especially for the detection of adenosine diphosphate (ADP), which is universally produced by kinases, are commercially available for high-throughput screening (HTS) of kinase inhibitors, but their cost is quite high for large-scale screening. Here, we report a new enzyme-coupled fluorescence assay for ADP detection, which uses just 10 inexpensive, commercially available components. The assay protocol is very simple, requiring only the mixing of test solutions with ADP detection solution and reading the fluorescence intensity of resorufin produced by coupling reaction. To validate the assay, we focused on CDC2-like kinase 1 (CLK1), a dual-specificity kinase that plays an important role in alternative splicing, and we used the optimized assay to screen an in-house chemical library of about 215,000 compounds for CLK1 inhibitors. We identified and validated 12 potent inhibitors of CLK1, including a novel inhibitory scaffold. The results demonstrate that this assay platform is not only simple and cost-effective, but also sufficiently robust, showing good reproducibility and giving similar results to those obtained with the widely used ADP-Glo bioluminescent assay.

Significant activity changes due to small structural changes (i.e., activity cliffs) of serine/threonine kinase Pim1 inhibitors were studied theoretically using the fragment molecular orbital method with molecular mechanics Poisson–Boltzmann surface area (FMO+MM-PBSA) approach. This methodology enables quantum-chemical calculations for large biomolecules with solvation. In the course of drug discovery targeting Pim1, six benzofuranone-class inhibitors were found to differ only in the position of the indole-ring nitrogen atom. By comparing the various qualities of complex structures based on X-ray, classical molecular mechanics (MM)-optimized, and quantum/molecular mechanics (QM/MM)-optimized structures, we found that the QM/MM-optimized structures provided the best correlation (R2= 0.85) between pIC50and the calculated FMO+MM-PBSA binding energy. Combining the classical solvation energy with the QM binding energy was important to increase the correlation. In addition, decomposition of the interaction energy into various physicochemical components by pair interaction energy decomposition analysis suggested that CH−π and electrostatic interactions mainly caused the activity differences.

Lactobionic acid was produced by acetic acid bacteria to oxidize lactose. Gluconobacterspp. and Gluconacetobacterspp. showed higher lactose-oxidizing activities than Acetobacterspp. Gluconobacter frateuriiNBRC3285 produced the highest amount of lactobionic acid per cell, among the strains tested. This bacterium assimilated neither lactose nor lactobionic acid. At high lactose concentration (30%), resting cells of the bacterium showed sufficient oxidizing activity for efficient production of lactobionic acid. These properties may contribute to industrial production of lactobionic acid by the bacterium. The bacterium showed higher oxidizing activity on cellobiose than that on lactose and produced cellobionic acid.

Elderly patients with non-Hodgkin lymphoma NHL have a poor prognosis. Owing to treatment-related toxicities, there is no standard chemotherapy for the elderly patients, especially those aged 70 years or older. In this study, we retrospectively evaluated the efficacy and toxicity of reduced-dose two-thirds R-CHOP chemotherapy as an initial chemotherapy for 45 patients aged 70 years or older with B-cell NHL. The WHO classification of NHL included diffuse large B-cell lymphoma DLBCL 31, mantle cell lymphoma 5, follicular lymphoma 4, extranodal marginal zone lymphoma 1, Burkitt lymphoma 1, and B-cell lymphoma whose further types were unclassified 3. The incidences of grade 4 neutropenia and febrile neutropenia FN were 51·1 and 15·6, respectively. Efficacy was evaluated in patients with DLBCL. The overall and complete response CR rates were 96·7 and 90·0, respectively. Two-year event-free survival EFS and overall survival OS were 84·4 and 89·2, respectively. There was no treatment-related mortality. In conclusion, two-thirds R-CHOP chemotherapy is a promising treatment for elderly patients with B-cell NHL in terms of its efficacy and toxicity.

The most remarkable difference between a chemical reaction and a lipase reaction is the fact that lipase possesses several substrate specificities, in particular, fatty acid specificity on hydrolysis, esterification, or transesterification reaction of the ester bond. Several lipases act weakly on polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and arachidonic acid. Through fatty acid specificity of lipases, a DHA-rich oil (acylglycerols) has been industrially produced from tuna oil. Lipase specificity also facilitates purification of PUFAs, conjugated linoleic acid isomers, tocopherols, and sterol esters and production of triacylglycerol that is DHA– rich at the sn- 1(3) position. Microorganism-mediated processes using Aeromonas hydrophila N- 6 are effective for production of rare unsaturated fatty acids and rare unsaturated fatty alcohols from several vegetable oils. These lipids are rarely observed in natural oils. We also demonstrate that Acinetobacter sp. N- 476- 2 effectively converts functional arachidonic acid to functional arachidonyl alcohol, which can be used as a substrate for medicine.

Gold nanorods with aspect ratios of 2, 4, and 6 were generated by a seed-mediated growth method developed by Jana et al. and improved by Nikoobakht and El-Sayed. Nanorod formation during synthesis was investigated by time-resolved measurements of small-angle X-ray scattering (SAXS) and in situ UV−vis−NIR absorption. A sample holder made entirely of titanium was newly constructed for the simultaneous measurements. Distance distribution functions were calculated from the SAXS profiles, and the growth in nanorod length as a function of reaction time was determined without having to approximate or simplify the actual nanorod shape. Structural anisotropy, which corresponds to aspect ratio of nanorods, was also evaluated from the functions. For formed nanorods with aspect ratios in the range of 2−4, the time dependence of anisotropy shows maxima. In contrast, for formed aspect-ratio 6 nanorods, a maximum is not observed, and anisotropy continuously increases until an early reaction-equilibrium is reached. Growth kinetics for the variation in the aspect ratios of the formed nanorods is evaluated based on the length growth rate and the width growth rate, each considered individually. Finally, structural changes of nanorod end-caps during synthesis are discussed.

AbstractBackground/Aims:The application of electrolyzed water (EW) at the cathode side to manufacture reverse osmosis (RO) water and hemodialysis (HD) solution can actually lead to less oxidative capacity in chemical terms. The present study examined the biological actions of this water on human polymorphonuclear leukocytes (PMNs), and the clinical feasibility of applying this technology to HD treatment. Methods:RO water using EW (e-RO) exhibited less chemiluminescence in luminol-hydrogen peroxide and higher dissolved hydrogen levels (–99.0 ppb) compared with control RO water. The effects of e-RO on PMN viability were tested. HD using e-RO was performed for 12 consecutive sessions in 8 patients for the feasibility test. Results:Basal cellular viability and function to generate superoxide radicals of PMNs were better preserved by e-RO application. In the clinical trial, reductions of blood pressure were noted, but no adverse events were observed. There were no changes in the blood dialysis parameters, although methylguanidine levels were significantly decreased at the end of study. Conclusion:The present study demonstrated the capacity of e-RO to preserve the viability of PMNs, and the clinical feasibility of applying this water for HD treatment. The clinical application of this technology may improve the bio-compatibility of HD treatment.Copyright © 2009 S. Karger AG, Basel

A carbohydrate:acceptor oxidoreductase from Paraconiothyriumsp. was purified and characterized. The enzyme efficiently oxidized β-(1→4) linked sugars, such as lactose, xylobiose, and cellooligosaccharides. The enzyme also oxidized maltooligosaccharides, D-glucose, D-xylose, D-galactose, L-arabinose, and 6-deoxy-D-glucose. It specifically oxidized the β-anomer of lactose. Molecular oxygen and 2,6-dichlorophenol indophenol were reduced by the enzyme as electron acceptors. The Paraconiothyriumenzyme was identified as a carbohydrate:acceptor oxidoreductase according to its specificity for electron donors and acceptors, and its molecular properties, as well as the N-terminal amino acid sequence. Further comparison of the amino acid sequences of lactose oxidizing enzymes indicated that carbohydrate:acceptor oxidoreductases belong to the same group as glucooligosaccharide oxidase, while they differ from cellobiose dehydrogenases and cellobiose:quinone oxidoreductases.

Background Transluminal replacement of a displaced peritoneal catheter (TRC) using a metal guidewire is a noninvasive treatment of catheter displacement with acute malfunction. The “alpha-replacer” is a special guidewire developed specifically for TRC. This wire is usually flexible but becomes harder if coiled in the hand. Application of this unique guidewire might improve the effectiveness of TRC.Methods TRC using an alpha-replacer was performed in 10 cases for urgent correction of peritoneal catheter displacement. All patients had received a double cuffed, straight-end swan-neck catheter.Results The primary success rate of TRC using the alpha-replacer was 90% (9 of 10 cases). No patients developed complications such as injury of the peritoneum or abdominal organs. Relapse of catheter displacement was observed in 2 cases (20%) within 1 week, requiring surgical intervention to prevent relapse.Conclusion TRC using the alpha-replacer as described herein offers effective and safe treatment of peritoneal catheter displacement.

Kanda: Daiichi Sankyo: Honoraria; Shire: Honoraria; Alexion Pharmaceuticals: Honoraria; Takeda Pharmaceuticals: Honoraria; Novartis: Honoraria; Kyowa Kirin: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Celgene: Honoraria; Otsuka: Honoraria, Research Funding; Chugai Pharma: Honoraria, Research Funding; Janssen: Honoraria; Astellas Pharma: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Merck Sharp & Dohme: Honoraria; Mochida Pharmaceutical: Honoraria; Mundipharma: Honoraria; Sanofi: Honoraria, Research Funding; Meiji Seika Kaisha: Honoraria; Bristol-Myers Squibb: Honoraria; Shionogi: Research Funding; Ono Pharmaceutical: Honoraria; Nippon Shinyaku: Honoraria, Research Funding.

Background

From a mixture of N-acetylglucosaminyl-β-cyclodextrin (GlcNAc-βCD) and lactose, β-D-galactosyl-GlcNAc-βCD (Gal-GlcNAc-βCD) was synthesized by the transfer action of β-galactosidase. GlcNAc-maltotriose (Glc3) and Gal-GlcNAc-Glc3were produced with hydrolysis of GlcNAc-βCD by cyclodextrin glycosyltransferase, and Gal-GlcNAc-βCD by bacterial saccharifying α-amylase respectively. Finally, GlcNAc-Glc3-βCD and Gal-GlcNAc-Glc3-βCD were synthesized in 5.2% and 3.5% yield when Klebsiella pneumoniaepullulanase was incubated with the mixture of GlcNAc-Glc3and βCD, or Gal-GlcNAc-Glc3and βCD respectively. The structures of GlcNAc-Glc3-βCD and Gal-GlcNAc-Glc3-βCD were analyzed by FAB-MS and NMR spectroscopy and identified as 6-O-α-(63-O-β-D-N-acetylglucosaminyl-maltotriosyl)-βCD, and 6-O-α-(4-O-β-D-galactopyranosyl-63-O-β-D-N-acetylglucosaminyl-maltotriosyl)-βCD respectively.

A new α-glucuronidase that specifically hydrolyzed O-α-D-glucosyluronic acid α-D-glucosiduronic acid (trehalose dicarboxylate, TreDC) was purified from a commercial enzyme preparation from Aspergillus niger, and its properties were examined. The enzyme did not degrade O-α-D-glucosyluronic acid α-D-glucoside, O-α-D-glucosyluronic acid β-D-glucosiduronic acid, O-α-D-glucosyluronic acid-(1→2)-β-D-fructosiduronic acid, p-nitrophenyl-O-α-D-glucosiduronic acid, methyl-O-α-D-glucosiduronic acid, or 6-O-α-(4-O-α-D-glucosyluronic acid)-D-glucosyl-β-cyclodextrine. Furthermore, it showed no activity on α-glucuronyl linkages of 4-O-methyl-D-glucosyluronic acid-α-(1→2)-xylooligosaccharides, derived from xylan, a supposed substrate of α-glucuronidases.The molecular mass of the enzyme was estimated to be 120 kDa by gel filtration and 58 kDa by SDS–PAGE suggesting, the enzyme is composed of two identical subunits. It was most active at pH 3.0–3.5 and at 40 °C. It was stable in pH 2.0–4.5 and below 30 °C. It hydrolyzed O-α-D-glucosyluronic acid α-D-glucosiduronic acid to produce α- and β-anomers of D-glucuronic acid in an equimolar ratio. This result suggests that inversion of the anomeric configuration of the substrate is involved in the hydrolysis mechanism.

Kawasaki disease (KD) in children takes the form of acute systemic vasculitis, which causes coronary artery dilation and aneurysm formation in 10% to 15% of the patients. We have recently shown that matrix metalloproteinases (MMPs) are intimately involved in coronary arterial wall destruction and the resultant formation of coronary artery lesions (CALs) in this disease. Plasminogen activators (PAs) are known to be a major pathway of MMP activation, and this suggests that their inhibitor, plasminogen activator inhibitor-1 (PAI-1), also plays important roles in the development of CALs in KD. The present study was conducted to test the hypothesis that circulating levels of PAI-I are related to CAL formation in KD. Plasma levels of PAI-1 were measured by enzyme-linked immunoassay in 37 KD patients without CALs (group 1) and 7 KD patients with CALs (group 2). Blood samples were obtained before and after i.v. gammaglobulin therapy (IVGG), and in the convalescent stage. Levels of PAI-1 were significantly higher in KD patients before IVGG than in 18 age-matched healthy control subjects (p < 0.01). More importantly, both pre-IVGG and post-IVGG levels of PAI-1 were significantly higher in group 2 than in group 1 (p < 0.01). Furthermore, PAI-1 levels of 9 patients from group 1 who showed pre-IVGG PAI-1 levels higher than the minimum PAI-1 level in group 2 significantly decreased after IVGG, whereas PAI-1 levels of group 2 patients remained persistently elevated, further suggesting a close association between PAI-1 and CAL development in KD. Thus, PAI-1 may be useful as a predictive marker for CAL development in KD. Studies of the effects of PA inhibition on coronary outcome may provide evidence that PA is a viable therapeutic target for the prevention of KD-related CALs.

A fungus producing an α-glucosidase that synthesizes α-1,3- and α-1,2-linked glucooligosaccharides by transglucosylation was isolated and identified as Paecilomyces lilacinus. The cell-bound enzyme responsible for the synthesis was extracted by suspension of mycelia with 0.1 Mphosphate buffer (pH 8.0), and the extract was purified. The molecular weight and the isoelectric point were estimated to be 54,000 and 9.1, respectively. The enzyme was most active at pH 5.0 and 65°C. The enzyme hydrolyzed maltose, nigerose, and kojibiose. The enzyme also hydrolyzed soluble starch and amylose with the rate toward maltose. p-Nitrophenyl α-glucoside and isomaltose were not good substrates. The enzyme had high transglucosylation activity to synthesize oligosaccharides containing α-1,3- and α-1,2-linkages. At an early stage of the reaction, considerable maltotriose, 4-O-α-nigerosyl-D-glucose, and 4-O-α-kojibiosyl-D-glucose were synthesized. Afterwards, nigerose and kojibiose were accumulated gradually with glucose as an acceptor.

A synthetic β-thio-fructofuranoside of mercaptoethanol inhibited not only β-fructofuranosidases but also α-glucosidases. The compound was hardly hydrolyzed by the glycosidases. The thio-fructoside competitively inhibited β-fructofuranosidases from Aspergillus niger, Candidasp., and Saccharomyces cerevisiae, but not Arthrobacterβ-fructofuranosidase at all. Sucrase activity of rat intestinal sucrase/isomaltase complex was also suppressed in the presence of the thio-fructoside. The thio-fructoside showed noncompetitive inhibition toward maltase activity of the rat intestinal enzyme complex and Saccharomycessp. α-glucosidase. Inhibition against the Bacillus stearothermophilusα-glucosidase, Rhizopusglucoamylase, and porcine kidney trehalase were more slight than that against these two α-glucosidases.

We have previously cloned chondroitin-4-sulfotransferase (C4ST) cDNA from mouse brain. In this paper, we report cloning and characterization of GalNAc 4-sulfotransferase (GalNAc4ST), which transfers sulfate to position 4 of the nonreducing terminal GalNAc residue. The obtained cDNA contains a single open reading frame that predicts a type II transmembrane protein composed of 424 amino acid residues. Identity of the amino acid sequence between GalNAc4ST and human C4ST was 30%. When the cDNA was transfected in COS-7 cells, sulfotransferase activity toward carbonic anhydrase VI was overexpressed but no sulfotransferase activity toward chondroitin or desulfated dermatan sulfate was increased over the control. Sulfation of carbonic anhydrase VI by the recombinant GalNAc4ST occurred at position 4 of the GalNAc residue ofN-linked oligosaccharides. The recombinant GalNAc4ST transferred sulfate to position 4 of GalNAc residue ofp-nitrophenyl GalNAc, indicating that this sulfotransferase transfers sulfate to position 4 at the nonreducing terminal GalNAc residue. Dot blot analysis showed that the message of GalNAc4ST was expressed strongly in the human pituitary, suggesting that the cloned GalNAc4ST may be involved in the synthesis of the nonreducing terminal GalNAc 4-sulfate residues found in the N-linked oligosaccharides of pituitary glycoprotein hormones.

Oral pulsed-dose calcitriol administration has been shown to be effective therapy for patients with secondary hyperparathyroidism. However, this effect is not consistently observed in the clinical setting. This study was undertaken to examine whether enlarged parathyroid glands can serve as a clinical marker that predicts the suppressive effect of calcitriol. Thirty-five patients undergoing chronic hemodialysis (HD) were examined (age, 51.9 ± 14.9 years; duration of HD, 72.0 ± 56.0 months). Based on the volume of parathyroid glands measured using an ultrasonographic scanner, patients were divided into two groups: 15 patients with undetectable parathyroid glands (ND group) and 20 patients with detectable parathyroid glands (D group; mean volume of parathyroid glands, 261.9 ± 347.5 mm3). No significant differences were found in serum ionized calcium (Ca++) or parathyroid hormone (PTH) levels before calcitriol administration between the two groups. For each patient, 8 μg of calcitriol was administered orally at the end of the HD session, and the changes in serum PTH levels were determined 44 hours after dosing. No significant differences were found between the two groups in serum Ca++levels. However, a significant decrease in serum PTH levels was observed in the ND group, whereas no significant changes were found in the D group. The results of the study show that the suppressive effect on PTH through calcitriol therapy was reduced in patients with detectable enlarged parathyroid glands. This may indicate that the size of parathyroid glands is one factor determining the therapeutic potential of pulsed-dose calcitriol administration for secondary hyperparathyroidism.

Abstract:: In the present study, we investigated the kinetics of bone metabolism by determining serum bone metabolic markers and quantifying bone mineral density by dual-energy X-ray absorptiometry to clarify the effect of long-term use of low-calcium hemodialysate on bone metabolism. After changing the calcium concentration in the dialysate from 3.0 mEq/l to 2.5 mEq/l, serum intact parathyroid hormone level, serum highly sensitive parathyroid hormone level, and serum bone metabolic markers were determined in ten patients with chronic nondiabetic renal insufficiency during 1 year. The doses of an oral phosphate binder and activated vitamin D were carefully regulated to control serum ionized calcium levels and serum inorganic phosphorus levels. Bone mineral density was determined at the distal 1/3 and 1/6 of the radius on the nonshunt side. As a result, the required amount of oral phosphate binder was increased; however, there was no need to significantly increase the amount of activated vitamin D. Intact parathyroid hormone showed no significant variation, but the highly sensitive parathyroid hormone was significantly increased. There were no significant changes in any bone metabolic markers or in bone mineral density. From these study results, it was found that it was difficult to increase the dose of activated vitamin D even if low-calcium hemodialysate was used, and that during use of the low-calcium hemodialysate the serum level of parathyroid hormone tended to increase but led to neither acceleration of bone turnover nor a decrease in bone mineral density.

Flavobacterium johnsonaewas isolated as a microorganism that produced a β-glucosidase with hydrolytic activity of β-glucosyl ester linkages in steviol glycosides. The enzyme was purified to homogeneity from a cell-free extract by streptomycin treatment, ammonium sulfate fractionation, and column chromatographies on S-Sepharose and phenyl-Toyopearl. The molecular mass of the purified enzyme was about 72 kDa by SDS-PAGE. An isoelectric point of pI8.8 was estimated by isoelectric focusing. The enzyme was most active at pH 7.0, and was stable between pH 3.0 and 9.0. The optimum temperature was 45°C, and the enzyme was stable below 35°C. The enzyme hydrolyzed glucosyl ester linkages at site 19 of rebaudioside A, stevioside, and rubusoside, although it could not degrad β-glucosidic linkages at site 13 of rebaudioside B or steviol bioside. The enzyme acted on aryl β-glucosides such as p-nitrophenyl β-glucoside, phenyl β-glucoside, and salicin, and glucobioses such as sophorose and laminaribiose. The enzyme activity on Rub was inactivated completely by Hg2+, and reduced by Fe3+, Cu2+, p-chloromercuric benzoate, and phenylmethylsulfonyl fluoride (residual activity; 67.9-84.8%). The pNPG hydrolysis was also inactivated to almost the same degrees. Kinetic behaviors in the mixed substrate reactions of rebaudioside A and steviol monoside, and of steviol monoglucosyl ester and phenyl β-glucoside suggested the glucosidic and glucosyl ester linkages were hydrolyzed at a single active site of the enzyme.

β-Galactosidase catalyzed β-galactosylation not only of a hydroxyl group but also of a thiol group in the condensation reaction of D-galactose and 2-mercaptoethanol. The thio-galactosylation product was confirmed as 2-hydroxyethyl S-β-D-galactoside on the bases of fast atom bombardment mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectorometry. Aspergillus oryzaeβ-galactosidase hydrolyzed p-nitrophenyl S-β-D-galactoside most rapidly among several β-galactosidases and produced the thio-galactosylation product most efficiently. The Penicillim multicolorenzyme was as effective as the A. oryzaeenzyme. However the enzymes from Escherichia coli, Saccharomyces fragilis, Kluyveromyces lactis, and Bacillus circulansgalactosylated hydroxyl groups predominantly to produce O-galactoside. The thio-galactoside was synthesized most effectively at a 2-mercaptoethanol concentration of about 1.25 M. Galactose concentration at 0.8-2.8 Mdid not affect the synthetic yield of the thio-galactoside so greatly.

From the mixture of 42-O-β-D-galactosyl-maltose (Gal-G2) and β-cyclodextrin (βCD), novel heterobranched βCDs, (Gal-G2)-βCD and (Gal-G2)2-βCDs, were synthesized by the reverse action of debranching enzyme. The optimum conditions for the production of (Gal-G2)2-βCDs were examined. A mixture of (Gal-G2)2- βCDs was produced in about 4% yield when Aerobacter aerogenespullulanase (64 units per 1 g of Gal-G2) was incubated with 1.6 MGal-G2 and 0.16 MβCD at 50°C for 4 days. The reaction products, (Gal-G2)2-βCDs, were separated into three peaks by HPLC analysis on a Hypercarb S column. Their structures were analyzed by fast atom bombardment mass spectroscopy and NMR spectroscopies, and confirmed by comparison of their hydrolyzates by β-galactosidase with the authentic (G2)2-βCDs. The structures of (Gal-G2)-βCD and three components of (Gal-G2)2-βCDs were identified as 6-O-(Gal-G2)-βCD, 61,62-, 61,63- and 61,64-di-O-(Gal-G2)2-βCD, respectively.

β-Fructofuranosidase fructosylated not only the hydroxyl group but also the thiol group of 2-mercaptoethanol in a transfer reaction using sucrose as a donor substrate. The enzymes from Candida utilisand Saccharomyces cerevisiae(bakers’ yeast) were effective catalysts for the thio-fructofuranosylation. The thio-fructosylation product was isolated by activated carbon chromatography and its structure was confirmed by Fab-mass spectrometry and NMR spectroscopy. The thio-fructofuranoside was synthesized effectively at around 3.0 Mfor the acceptor concentration. The product increased with the sucrose concentration at least up to 1.9 M. O-Fructofuranoside was simultaneously synthesized at an early stage of the reaction, although it was hydrolyzed on further incubation. On the contrary, the thio-furctofuranoside accumulated efficiently after synthesis, indicating it was very stable against the hydrolytic action of the β-fructofranosidase.

p-Hydroxybenzoyl β-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of β-galactosyl ester linkage by β-galactosidases. The enzyme from Penicillium multicolorhydrolyzed the substrate as fast as p-nitrophenyl β-galactoside (pNP-Gal), a usual substrate with a β-galactosidic linkage. The enzymes from Escherichia coliand Aspergillus oryzaehydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that β-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicilliumenzyme hydrolyzed pHB-Gal in the presence of H218O to liberate galactose containing 18O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.

α-D-Mannosyl-maltotriose (Man-G3) were synthesized from methyl α-mannoside and maltotriose by the transfer action of α-mannosidase. (Man-G3)-βCD and (Man-G3)2-βCD were produced in about 20% and 4% yield, respectively when Aerobacter aerogenespullulanase (160 units per 1 g of Man-G3) was incubated with the mixture of 1.6 MMan-G3 and 0.16 MβCD at 50°C for 4 days. The reaction products, (Man-G3)-βCD were separated to three peaks by HPLC analysis on a YMC-PACK A-323-3 column and (Man-G3)2-βCD were separated to several peaks by HPLC analysis on a Daisopak ODS column. The major product of (Man-G3)-βCDs was identified as 6-O-α-(63-O-α-D-mannosyl-maltotriosyl)-βCD by FAB-MS and NMR spectroscopies. The structures of (Man-G3)2-βCDs were analyzed by TOF-MS and NMR spectroscopies, and confirmed by comparison of elution profiles of their hydrolyzates by α-mannosidase and glucoamylase on a graphitized carbon column with those of the authentic di-glucosyl-βCDs. The structures of three main components of (Man-G3)2-βCDs were identified as 61,62-, 61,63- and 61,64-di-O-(63-O-α-D-mannosyl-maltotriosyl)-βCD.

Triplet regimens such as lenalidomide, bortezomib, and dexamethasone (RVd) or thalidomide, bortezomib, and dexamethasone (VTd) are standard induction therapies for transplant-eligible newly diagnosed multiple myeloma (NDMM) patients. The addition of daratumumab to RVd and VTd has been investigated in the GRIFFIN and CASSIOPEIA trials, respectively, resulting in improvement in the rate of minimal residual disease (MRD) negativity. In this study, we conducted a cost-effectiveness analysis with a 10-year time horizon to compare first-line and second-line use of daratumumab for transplant-eligible NDMM patients. Since long-term follow-up data for these clinical trials are not yet available, we developed a Markov model that uses MRD status to predict PFS. Daratumumab was used either in the first-line setting in combination with RVd or VTd, or in the second-line setting with carfilzomib plus dexamethasone (Kd). Quality-adjusted life years (QALYs) and incremental cost-effectiveness ratios (ICERs) were calculated from a Japanese and US payer perspective. In the Japanese analysis, D-RVd showed higher QALYs (5.43 vs 5.18) and lower costs (¥ 64,479,793 vs 71,287,569) compared to RVd, and D-VTd showed higher QALYs (5.67 vs 5.42) and lower costs (¥ 43,600,310 vs \ 49,471,941) compared to VTd. Similarly, the US analysis demonstrated dominance of a strategy incorporating daratumumab in first-line treatment regimens. Given that overall costs are reduced and outcomes are improved when daratumumab is used as part of a first-line regimen, the economic analysis indicates that addition of daratumumab to first-line RVd and VTd regimens is a dominant strategy compared to reserving its use for the second-line setting.

The preparation of three new types of phenylboronic acid derivatives and their evaluation as enhancers on the luminol–H2O2–horseradish peroxidase (HRP) chemiluminescence (CL) reaction are described. After optimizing the CL reaction conditions, the CL system was applied to the HRP determination. Among the three phenylboronic acid derivatives, i.e. 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phenylboronic acid (DPA), 4‐[4(or 5)‐(4‐dimethylaminophenyl)‐5(or 4)‐phenyl‐1H‐imidazol‐2‐yl]phenylboronic acid (DAPA) and 4‐[4,5‐di(2‐pyridyl)‐1H‐imidazol‐2‐yl]phenylboronic acid (DPPA), DPPA was found to be the most potent enhancer. The sensitivity obtained with DPPA was about 180 times higher than that without an enhancer. The detection limit of HRP obtained with DPPA was 0.15 ng/assay (ca. 3.5 fmol), which is comparable to that with 4‐iodophenol under the conditions examined. All the phenylboronic acid derivatives examined had the effect of prolonging light emission compared to 4‐iodophenol. Copyright © 1999 John Wiley & Sons, Ltd.