Direct Dehydrative Glycosylation Catalyzed by Diphenylammonium Triflate.

Methods for direct dehydrative glycosylations of carbohydrate hemiacetals catalyzed by diphenylammonium triflate under microwave irradiation are described. Both armed and disarmed glycosyl-C1-hemiacetal donors were efficiently glycosylated in moderate to excellent yields without the need for any drying agents and stoichiometric additives. This method has been successfully applied to a solid-phase glycosylation.

1H/13C chemical shifts and cation binding dataset of the corticosteroid Prednisolone titrated with metal cations.

We here reported the 1H/13C chemical shifts, binding affinity and binding free energy of 1,4-pregnadiene-11ß,17α,21-triol-3,20-dione (Prednisolone; Prd) interacting with metal cations. Six different Prd/Ni or Co mixtures were examined at different molar ratios (1:0, 1:0.1, 1:0.2, 1:0.3, 1:0.4 and 1:0.5). In this analysis, the 1H and 13C chemical shifts were measured for the Prd/cation mixtures using a Bruker AV 500 MHz spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany), equipped with a 5 mm z-gradient Prodigy BBO 500 MHz probehead at 298 K, and simulation of the 1H spectra were determined from the Daisy software package (Bruker BioSpin GmbH). Binding affinity and free energy values were deduced from the 13C NMR peak intensities involved in the cation interaction, for more insight on the steroid/cation interactions please see Magnesium and Calcium Reveal Different Chelating Effects in a Steroid Compound: A Model Study of Prednisolone Using NMR Spectroscopy [1].

Establishment of Guidelines for the Control of Glycosylation Reactions and Intermediates by Quantitative Assessment of Reactivity.

Stereocontrolled chemical glycosylation remains a major challenge despite vast efforts reported over many decades and so far still mainly relies on trial and error. Now it is shown that the relative reactivity value (RRV) of thioglycosides is an indicator for revealing stereoselectivities according to four types of acceptors. Mechanistic studies show that the reaction is dominated by two distinct intermediates: glycosyl triflates and glycosyl halides from N-halosuccinimide (NXS)/TfOH. The formation of glycosyl halide is highly correlated with the production of α-glycoside. These findings enable glycosylation reactions to be foreseen by using RRVs as an α/ß-selectivity indicator and guidelines and rules to be developed for stereocontrolled glycosylation.

Magnesium and calcium reveal different chelating effects in a steroid compound: A model study of prednisolone using NMR spectroscopy.

In this work, we used high resolution NMR spectroscopy to investigate metal cation chelation by the steroidal drug 1,4-pregnadiene-11ß,17α,21-triol-3,20-dione (Prednisolone; abbreviated as Prd). Prd/MgCl2 and Prd/CaCl2 mixtures were prepared at eight different molar ratios. Using two-dimensional 1H/13C heteronuclear correlation spectroscopy, we were able to resolve most of the 1H signals, except those at 1.4-1.55 ppm, where signals for H15ß, H16α and Me-19 are superimposed. The chelation sites were determined by the cation concentration-dependent 13C signals. Both ring A and ring D of Prd were found to be involved in Mg2+ chelation, whereas only ring A was involved in Ca2+ chelation. The dihedral angles deduced from the 3JH-H coupling constants indicated that ring D of Prd might undergo rather small, but different, distortions in the presence of Mg2+ and Ca2+. Additionally, using the continuous variation method, we deduced that the stoichiometric ratios of the Prd/Mg2+ and Prd/Ca2+ complexes were 1:1 and 2:1, respectively. All of the evidence led us to conclude that the Prd/Mg2+ and Prd/Ca2+ complexes are mediated by different chelating mechanisms.

TMSBr-mediated solvent- and work-up-free synthesis of α-2-deoxyglycosides from glycals.

The thio-additions of glycals were efficiently promoted by a stoichiometric amount of trimethylsilyl bromide (TMSBr) to produce S-2-deoxyglycosides under solvent-free conditions in good to excellent yields. In addition, with triphenylphosphine oxide as an additive, the TMSBr-mediated direct glycosylations of glycals with a large range of alcohols were highly α-selective.

NMR investigation of magnesium chelation and cation-induced signal shift effect of testosterone.

We have previously reported that testosterone (Tes) is able to interact with magnesium chloride dissolved in methanol. In this study, we have applied 1H and 13C NMR spectroscopies to a series of Tes solutions containing Mg2+ at various concentrations. High-resolution 13C NMR spectra of Tes/Mg2+ revealed well-resolved 13C signals, and the intensities of those arising from C3, C5, C16, and C17 decreased linearly with increasing Mg2+ concentration. The magnitude of the chelation affinity could be deduced from the slopes of the 13C intensity variations; typically, the greater the slope the higher the chelation affinity. The results revealed Tes/Mg2+ chelation to be mediated by the oxygen atom attached to C3 in ring A, and the hydroxyl group attached to C17 in ring D. With regard to the chelation specificity, we showed that Tes chelates Mg2+, but not Ca2+ or Zn2+. We also explored the cation-induced signal shift effects of Tes in the presence of Mg2+, Ca2+, or Zn2+. We demonstrate that high-resolution 13C NMR spectroscopy provides a better probe than 1H NMR for the detection of cation chelation and cation-induced signal shift effects for steroid compounds such as Tes.

Educational efficacy of objective structured clinical examination on clinical training of traditional Chinese medicine - A qualitative study.

OBJECTIVE: We propose applying objective structured clinical examinations (OSCEs) and discuss the educational efficacy of such examinations regarding the clinical competence of traditional Chinese medicine (TCM) practitioners. DESIGN: TCM OSCEs were implemented for evaluation and instruction from 2011 to 2013. Trainees received feedback from clinician-educators and standardized patients. Trainees' survey data were extracted from post-OSCE questionnaires and interviews to analyze TCM OSCEs. RESULTS: Five TCM OSCEs were administered, and the educational backgrounds of the 37 participants were analyzed. According to analysis of the questionnaires, all trainees agreed OSCEs were beneficial. In interviews, trainees expressed appreciation for the direct, real-time feedback during the OSCE and felt it closely resembled actual clinical work. However, the simulation models of OSCEs must be upgraded. CONCLUSIONS: OSCEs can be used in evaluating, teaching, and certifying TCM clinical competencies to improve the quality of TCM practices. The patient-centered training aspect of TCM OSCEs is particularly helpful for participants.

Vaccinia viral protein A27 is anchored to the viral membrane via a cooperative interaction with viral membrane protein A17.

The vaccinia viral protein A27 in mature viruses specifically interacts with heparan sulfate for cell surface attachment. In addition, A27 associates with the viral membrane protein A17 to anchor to the viral membrane; however, the specific interaction between A27 and A17 remains largely unclear. To uncover the active binding sites and the underlying binding mechanism, we expressed and purified the N-terminal (18-50 residues) and C-terminal (162-203 residues) fragments of A17, which are denoted A17-N and A17-C. Through surface plasmon resonance, the binding affinity of A27/A17-N (KA = 3.40 × 10(8) m(-1)) was determined to be approximately 3 orders of magnitude stronger than that of A27/A17-C (KA = 3.40 × 10(5) m(-1)), indicating that A27 prefers to interact with A17-N rather than A17-C. Despite the disordered nature of A17-N, the A27-A17 interaction is mediated by a specific and cooperative binding mechanism that includes two active binding sites, namely (32)SFMPK(36) (denoted as F1 binding) and (20)LDKDLFTEEQ(29) (F2). Further analysis showed that F1 has stronger binding affinity and is more resistant to acidic conditions than is F2. Furthermore, A27 mutant proteins that retained partial activity to interact with the F1 and F2 sites of the A17 protein were packaged into mature virus particles at a reduced level, demonstrating that the F1/F2 interaction plays a critical role in vivo. Using these results in combination with site-directed mutagenesis data, we established a computer model to explain the specific A27-A17 binding mechanism.

Solid-state NMR study of fluorinated steroids.

Solid-state {(1)H}(13)C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy was performed to analyze two fluorinated steroids, i.e., betamethasone (BMS) and fludrocortisone acetate (FCA), that have fluorine attached to C9, as well as two non-fluorinated analogs, i.e., prednisolone (PRD) and hydrocortisone 21-acetate (HCA). The (13)C signals of BMS revealed multiplet patterns with splittings of 16-215Hz, indicating multiple ring conformations, whereas the (13)C signals of FCA, HCA, and PRD exhibited only singlet patterns, implying a unique conformation. In addition, BMS and FCA exhibited substantial deviation (>3.5ppm) in approximately half of the (13)C signals and significant deviation (>45ppm) in the (13)C9 signal compared to PRD and HCA, respectively. In this study, we demonstrate that fluorinated steroids, such as BMS and FCA, have steroidal ring conformation(s) that are distinct from non-fluorinated analogs, such as PRD and HCA.

Does supplementation with green tea extract improve insulin resistance in obese type 2 diabetics? A randomized, double-blind, and placebo-controlled clinical trial.

BACKGROUND: Green tea is one of the most popular beverages in the world. It is believed to have beneficial effects in the prevention and treatment of many diseases, one of which is type 2 diabetes. The aim of the study is to examine the effect of a decaffeinated green tea extract (GTE) providing a daily dose of 856 mg of epigallocatechin gallate (EGCG) on obese individuals with type 2 diabetes. MATERIALS AND METHODS: The clinical trial was a randomized, double-blind, placebo-controlled clinical trial conducted from December 2007 through November 2008. The subjects were randomly assigned to either receive 1,500 mg of a decaffeinated GTE or placebo daily for 16 weeks. Sixty-eight of 80 subjects, ages 20-65 years with BMI > 25 kg/m2 and type 2 diabetes for more than one year, completed this study. Homeostasis model assessment for insulin resistance (HOMA-IR) was used as the major outcome measurement. At baseline and after 16 weeks of treatment, anthropometric measurements, fasting glucose, hemoglobin A1C percent (HbA1C), hormone peptides, and plasma lipoproteins were measured from both groups. RESULTS: No statistically significant differences were detected between the decaffeinated GTE and placebo groups in any measured variable. A statistically significant within-group 0.4-percent reduction in HbA1C (from 8.4 to 8.0%) was observed after GTE treatment compared to baseline. Within-group comparison also revealed that the GTE group had significant reductions in waist circumference (WC), HOMA-IR index, and insulin level, and a significant increase in the level of ghrelin. Within-group comparison of those in the placebo group showed a significant increase in the level of ghrelin. CONCLUSIONS: This study found no statistical difference in any measured variable between the decaffeinated GTE and placebo groups; however, there were some statistically significant within-group changes detected. More research is required to determine whether a decaffeinated GTE standardized for EGCG content will provide any clinical benefits in obese individuals with type 2 diabetes. Clinical Trial Registration NO: NCT00567905.

A gene delivery system based on the N-terminal domain of human topoisomerase I.

The N-terminal 200 amino acid residues of topoisomerase I (TopoN) is highly positive in charge and has DNA binding activity, without DNA sequence and topological specificity. Here, a fusion protein (6 x His-PTD-TopoN) containing a hexahistidine (6 x His) tag, a membrane penetration domain and TopoN (amino acid 3-200) was designed and developed. The protein can bind to different sizes (3.0-8.0 kb) and forms (circular and linear) of DNA and translocates the bound DNA to the nucleus. The protein also showed low cytotoxicity to GF-1 grouper fish fin cells that were previously very sensitive and difficult to transfect in vitro. Maintaining the hexahistidine tag increased the protein's transfection efficiency in COS7 African green monkey kidney cells and simplified the purification process. The plasmid pEGFP-N1 was delivered into COS7 cells by the protein in ATP- and temperature-dependent manners. The results indicate that the binding ability of TopoN is very useful for DNA delivery and the carrier protein can be expressed in Escherichia coli without removal of the hexahistidine tag.

A turn-like structure "KKPE" segment mediates the specific binding of viral protein A27 to heparin and heparan sulfate on cell surfaces.

Vaccinia viral envelope protein A27 (110 amino acids) specifically interacts with heparin (HP) or heparan sulfate (HS) proteoglycans for cell surface attachment. To examine the binding mechanism, a truncated soluble form of A27 (sA27-aa; residues 21-84 of A27) with Cys(71) and Cys(72) mutated to Ala was used as the parent molecule. sA27-aa consists of two structurally distinct domains, a flexible Arg/Lys-rich heparin-binding site (HBS) (residues 21-32; (21)STKAAKKPEAKR(32)) and a rigid coiled-coil domain (residues 43-84), both essential for the specific binding. As shown by surface plasmon resonance (SPR), the binding affinity of sA27-aa for HP (K(A) = 1.25 x 10(8) m(-1)) was approximately 3 orders of magnitude stronger than that for nonspecific binding, such as to chondroitin sulfate (K(A) = 1.65 x 10(5) m(-1)). Using site-directed mutagenesis of HBS and solution NMR, we identified a "KKPE" segment with a turn-like conformation that mediates specific HP binding. In addition, a double mutant T22K/A25K in which the KKPE segment remained intact showed an extremely high affinity for HP (K(A) = 1.9 x 10(11) m(-1)). Importantly, T22K/A25K retained the binding specificity for HP and HS but not chondroitin sulfate, as shown by in vitro SPR and in vivo cell adhesion and competitive binding assays. Molecular modeling of the HBS was performed by dynamics simulations and provides an explanation of the specific binding mechanism in good agreement with the site-directed mutagenesis and SPR results. We conclude that a turn-like structure introduced by the KKPE segment in vaccinia viral envelope protein A27 is responsible for its specific binding to HP and to HS on cell surfaces.

Induction of apoptosis by the retinoid inducible growth regulator RIG1 depends on the NC motif in HtTA cervical cancer cells.

BACKGROUND: Retinoid-inducible gene 1 (RIG1), also known as tazarotene-induced gene 3 or retinoic-acid receptor responder 3, is a growth regulator, which induces apoptosis and differentiation. RIG1 is classified into the NC protein family. This study investigated functional domains and critical amino acids associated with RIG1-mediated cell death and apoptosis. RESULTS: Using enhanced green fluorescence protein (EGFP)-tagged RIG1 variants, RIG1 proteins with deletion at the NC domain significantly decreased cell death induced by RIG1, and fusion variants containing only the NC domain significantly induced apoptosis of HtTA cervical cancer cells. The EGFP-RIG1-induced apoptosis was significantly decreased in cells expressing N(112)C(113) motif double- (NC-->FG) or triple- (NCR-->FGE) mutated RIG1 variants. Using dodecapeptides, nuclear localization and profound cell death was observed in HtTA cells expressing wild type RIG1(111-123) or Leu121-mutated RIG1(111-123):L--> C peptide, but peptides double- or triple-mutated at the NC motif alone, RIG1(111-123):NC-->FG or RIG1(111-123):NCR-->FGE, were cytoplasmically localized and did not induce apoptosis. The RIG1(111-123) also induced apoptosis of A2058 melanoma cells but not normal human fibroblasts. CONCLUSION: The NC domain, especially the NC motif, plays the major role in RIG1-mediated pro-apoptotic activity. The RIG1(111-123) dodecapeptide exhibited strong pro-apoptotic activity and has potential as an anticancer drug.

New chromogenic and fluorescent probes for anion detection: formation of a [2 + 2] supramolecular complex on addition of fluoride with positive homotropic cooperativity.

Two new chromogenic and fluorescent probes for anions have been designed, synthesized, and characterized. These probes contain multiple hydrogen bonding donors including hydrazine, hydrazone, and hydroxyl functional groups for potential anion interacting sites. Despite the possible flexible structural framework due to the presence of sp3 carbon linkage, X-ray structure analysis of probe 2 displayed an essentially planar conformation in the solid state owing to strong crystal packing interactions comprising a combination of favorable pi-pi stacking effect and hydrogen bonding to cocrystallized CH3OH molecules. Both probes 1 and 2 display orange color in DMSO solution and show fairly weak fluorescence at room temperature. Binding studies revealed that both probes 1 and 2 show noticeable colorimetric and fluorescent responses only to F-, OAc-, and H2PO4- among the nine anions tested (F-, Cl-, Br-, I-, OAc-, H2PO4-, HSO4-, ClO4-, and NO3-). The general trend of the sensitivity to anions follows the order of F- > OAc- > H2PO4- > Cl- > Br- approximately I- approximately HSO4- approximately ClO4- approximately NO3-. A 1:2 (probe to anion) binding stoichiometry was found for probe 1 with OAc- and H2PO4- and probe 2 with F-, OAc-, and H2PO4-. The binding isotherm of probe 1 to F- was found to be complicated with apparent multiple equilibria occurring in solution. The formation of an aggregated supramolecular complex upon addition of fluoride is proposed to rationalize the observed optical responses and is supported by ESI mass spectrometry and pulsed-field gradient NMR spectroscopy. Data analysis suggests that the binding of probe 1 to F- shows positive homotropic cooperativity.

The C-terminal aqueous-exposed domain of the 45 kDa subunit of the particulate methane monooxygenase in Methylococcus capsulatus (Bath) is a Cu(I) sponge.

The crystal structure of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) has been reported recently [Lieberman, R. L., and Rosenzweig, A. C. (2005) Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane, Nature 434, 177-182]. Subsequent work has shown that the preparation on which the X-ray analysis is based might be missing many of the important metal cofactors, including the putative trinuclear copper cluster at the active site as well as ca. 10 copper ions (E-clusters) that have been proposed to serve as a buffer of reducing equivalents to re-reduce the copper atoms at the active site following the catalytic chemistry [Chan, S. I., Wang, V. C.-C., Lai, J. C.-H., Yu, S. S.-F., Chen, P. P.-Y., Chen, K. H.-C., Chen, C.-L., and Chan, M. K. (2007) Redox potentiometry studies of particulate methane monooxygenase: Support for a trinuclear copper cluster active site, Angew. Chem., Int. Ed. 46, 1992-1994]. Since the aqueous-exposed domains of the 45 kDa subunit (PmoB) have been suggested to be the putative binding domains for the E-cluster copper ions, we have cloned and overexpressed in Escherichia coli the two aqueous-exposed subdomains toward the N- and C-termini of the subunit: the N-terminal subdomain (residues 54-178) and the C-terminal subdomain (residues 257-394 and 282-414). The recombinant C-terminal water-exposed subdomain is shown to behave like a Cu(I) sponge, taking up to ca. 10 Cu(I) ions cooperatively when cupric ions are added to the protein fragment in the presence of dithiothreitol or ascorbate. In addition, circular dichroism measurements reveal that the C-terminal subdomain folds into a beta-sheet structure in the presence of Cu(I). The propensity for the C-terminal subdomain to bind Cu(I) is consistent with the high redox potential(s) determined for the E-cluster copper ions in the pMMO. These properties of the E-clusters are in accordance with the function proposed for these copper ions in the turnover cycle of the enzyme.

The mushroom Agaricus Blazei Murill in combination with metformin and gliclazide improves insulin resistance in type 2 diabetes: a randomized, double-blinded, and placebo-controlled clinical trial.

BACKGROUND: Complementary and alternative medicine use in adults with type 2 diabetes is popular. Although most of the herbs and supplements appear to be safe, there is still insufficient evidence that demonstrates their definitive beneficial effects. This study was done to determine whether the supplement of Agaricus blazei Murill extract improves insulin resistance in type 2 diabetes. MATERIALS AND METHODS: This study was a clinical randomized, double-blind, placebo-controlled trial. Of a population of 536 registered diabetes patients with 72 subjects (1) aged between 20 and 75 years, (2) being Chinese, (3) having type 2 diabetes for more than 1 year, and (4) having been taking gliclazide and metformin for more than 6 months were enrolled in this study. The enrolled patients were randomly assigned to either receiving supplement of Agaricus blazei Murill (ABM) extract or placebo (cellulose) 1500 mg daily for 12 weeks. Homeostasis model assessment for insulin resistance (HOMA-IR) was used as the major outcome measurement. RESULTS: At the end of the study, subjects who received supplement of ABM extract (n = 29) showed significantly lower HOMA-IR index (3.6[standard deviation, 2.5] versus 6.6[standard deviation, 7.4], p = 0.04) than the control group (n = 31). The plasma adiponectin concentration increased 20.0(standard deviation, 40.7)% in the ABM group after 12 weeks of treatment, but decreased 12.0(20.0)% among those taking the placebo (p < 0.001). CONCLUSIONS: Supplement of ABM extract improves insulin resistance among subjects with type 2 diabetes. The increase in adiponectin concentration after taking AMB extract for 12 weeks might be the mechanism that brings the beneficial effect. Studies with longer periods of follow-up should be conducted in the future.

Photophysical studies of anion-induced colorimetric response and amplified fluorescence quenching in dipyrrolylquinoxaline-containing conjugated polymers.

The dipyrrolylquinoxaline (DPQ)-containing monomer and polymers were synthesized and employed as chromogenic and fluorescent chemosensors for inorganic anions. We have found that in the presence of fluoride or pyrophosphate, the receptors do not form hydrogen bonds between the pyrrole protons and anions. The colorimetric responses and fluorescence quenching in these chemosensors are indeed the result of deprotonation of the N-H proton. The anion selectivity is primarily determined by the relative basicity of anions. The sensitivity of DPQ-based chemosensor was found to display a 34-fold enhancement by incorporation into the conjugated polymer. The anion-induced deprotonation generates low-energy, non-fluorescent trapping sites and is responsible for the signal amplification where the quenching of the excited state occurs from the deprotonated DPQ site in the network by rapid exciton migration along the polymeric backbone.

One O-linked sugar can affect the coil-to-beta structural transition of the prion peptide.

It has been known that the structural transition from PrP(C) to PrP(Sc) leads to the prion formation. This putative conformational change challenges the central dogma of the protein folding theory-"one sequence, one structure." Generally, scientists believe that there must be either a posttranslational modification or environmental factors involved in this event. However, all of the efforts to solve the mystery of the PrP(C) to PrP(Sc) transition have ended in vain so far. Here we provide evidence linking O-linked glycosylation to the structural transition based on prion peptide studies. We find that the O-linked alpha-GalNAc at Ser-135 suppresses the formation of amyloid fibril formation of the prion peptide at physiological salt concentrations, whereas the peptide with the same sugar at Ser-132 shows the opposite effect. Moreover, this effect is sugar specific. Replacing alpha-GalNAc with beta-GlcNAc does not yield the same effect.