Synthesis of Sucrose-Mimicking Disaccharide by Intramolecular Aglycone Delivery.

Rare sugars are known for their ability to suppress postprandial blood glucose levels. Therefore, oligosaccharides and disaccharides derived from rare sugars could potentially serve as functional sweeteners. A disaccharide [α-d-allopyranosyl-(1→2)-ß-d-psicofuranoside] mimicking sucrose was synthesized from rare monosaccharides D-allose and D-psicose. Glycosylation using the intermolecular aglycon delivery (IAD) method was employed to selectively form 1,2-cis α-glycosidic linkages of the allopyranose residues. Moreover, ß-selective psicofuranosylation was performed using a psicofuranosyl acceptor with 1,3,4,6-tetra-O-benzoyl groups. This is the first report on the synthesis of non-reducing disaccharides comprising only rare d-sugars by IAD using protected ketose as a unique acceptor; additionally, this approach is expected to be applicable to the synthesis of functional sweeteners.

Synthesis of a fluorescent probe for measuring the activity of endo-ß-N-acetylglucosaminidases recognizing hybrid-type N-glycans.

A fluorescence-quenching-based assay system was constructed to determine the hydrolytic activity of endo-ß-N-acetylglucosaminidases (ENGases) interacting with hybrid-type N-glycans. This was achieved using a dual-labeled fluorescent probe with a nonasaccharide structure. We produced the nonasaccharide skeleton by the stepwise glycosylation of the galactose residue on a galactosyl chitobiose derivative. Next, we introduced azido and acetoxy groups into the nonasaccharide derivative in a stepwise manner, which led to stereochemistry inversion at both the C-4 and C-2 hydroxy groups on its galactose residue. The protecting groups of the resulting nonasaccharide derivative were removed, and the derivative was labeled with an N-methylanthraniloyl group to obtain a reporter dye and a 2,4-dinitrophenyl group as a quenching molecule to obtain target probe 1. The use of this probe along with a microplate reader enabled a facile evaluation of the hydrolytic activities of ENGases Endo-H, Endo-M, Endo-F3, Endo-S, and Endo-CC. Furthermore, this probe could also assist in the search for novel ENGases that are specific to hybrid-type N-glycans.

Synthesis of azide-modified glycerophospholipid precursor analogs for detection of enzymatic reactions.

Glycerophospholipids (GPLs) are major cell membrane components. Although various phosphorylated molecules are attached to lipid moieties as their headgroups, GPLs are biosynthesized from phosphatidic acid (PA) via its derivatives, diacylglycerol (DAG) or cytidine diphosphate diacylglycerol (CDP-DAG). A variety of molecular probes capable of introducing detection tags have been developed to investigate biological events involved in GPLs. In this study, we report the design, synthesis, and evaluation of novel analytical tools suitable to monitor the activity of GPL biosynthetic enzymes in vitro. Our synthetic targets, namely, azide-modified PA, azide-modified DAG, and azide-modified CDP-DAG, were successfully obtained from solketal as their common starting material. Moreover, using CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT), an enzyme that catalyzed the final reaction step in synthesizing phosphatidylinositol, we demonstrated that azide-modified CDP-DAG worked as a substrate for CDIPT.

Quantification of Extramyocellular Lipids and Intramuscular Fat from Muscle Echo Intensity in Lower Limb Muscles: A Comparison of Four Ultrasound Devices against Magnetic Resonance Spectroscopy.

This study aimed to compare different ultrasound devices with magnetic resonance spectroscopy (MRS) to quantify muscle lipid content from echo intensity (EI). Four different ultrasound devices were used to measure muscle EI and subcutaneous fat thickness in four lower-limb muscles. Intramuscular fat (IMF), intramyocellular (IMCL) and extramyocellular lipids (EMCL) were measured using MRS. Linear regression was used to compare raw and subcutaneous fat thickness-corrected EI values to IMCL, EMCL and IMF. IMCL had a poor correlation with muscle EI (r = 0.17-0.32, NS), while EMCL (r = 0.41-0.84, p < 0.05-p < 0.001) and IMF (r = 0.49-0.84, p < 0.01-p < 0.001) had moderate to strong correlation with raw EI. All relationships were improved when considering the effect of subcutaneous fat thickness on muscle EI measurements. The slopes of the relationships were similar across devices, but there were some differences in the y-intercepts when raw EI values were used. These differences disappeared when subcutaneous fat thickness-corrected EI values were considered, allowing for the creation of generic prediction equations (r = 0.41-0.68, p < 0.001). These equations can be used to quantify IMF and EMCL within lower limb muscles from corrected-EI values in non-obese subjects, regardless of the ultrasound device used.

Is the muscle-tendon architecture of non-athletic Kenyans different from that of Japanese and French males?

BACKGROUND: In endurance running, elite Kenyan runners are characterized by longer thigh, shank, and Achilles tendon (AT) lengths combined with shorter fascicles and larger medial gastrocnemius (MG) pennation angles than elite Japanese runners. These muscle-tendon characteristics may contribute to the running performance of Kenyans. Furthermore, these specific lower-leg musculoskeletal architectures have been confirmed not only in elite Kenyan runners but also in non-athletic Kenyans since early childhood. However, it remains questionable whether the differences in muscle-tendon architecture between Kenyans and Japanese differ from those of European Caucasians. Therefore, this study aimed to compare anthropometry and muscle-tendon architecture of young non-athletic Kenyan males with their Japanese and French counterparts. METHODS: A total of 235 young non-athletic males, aged 17-22 years, volunteered. The anthropometric measures, thigh, and shank lengths, as well as AT and MG muscle architecture, were measured using ultrasonography and a tape measure. Inter-group differences in anthropometry and muscle-tendon architecture were tested using one-way ANOVA and ANCOVA analyses controlling for shank length and muscle thickness. RESULTS: The anthropometric and muscle-tendon characteristics of the non-athletic French were closer to those of the Kenyans than to those of the Japanese. However, the ultrasonography analysis confirmed that the non-athletic Kenyans had the longest AT as well as the shortest MG fascicles and the largest pennation angle compared to the French and Japanese, even after controlling for shank length and muscle thickness with ANCOVA, respectively. CONCLUSIONS: These results confirmed the specificity of the muscle-tendon architecture of the triceps surae in Kenyans in comparison to their Japanese and French counterparts in non-athletic adults. This study provides additional support to the fact that Kenyans may have musculotendinous advantages in endurance running.

Convergent synthesis of oligomannose-type glycans via step-economical construction of branch structures.

Oligomannose-type glycans on glycoproteins are important signaling molecules in the glycoprotein quality control system in the endoplasmic reticulum. Recently, free oligomannose-type glycans generated by the hydrolysis of glycoproteins or dolichol pyrophosphate-linked oligosaccharides were recognized as important signals for immunogenicity. Hence, there is a high demand for pure oligomannose-type glycans for biochemical experiments; however, the chemical synthesis of glycans to achieve high-concentration products is laborious. In this study, we demonstrate a simple and efficient synthetic strategy for oligomannose-type glycans. Sequential regioselective α-mannosylation at the C-3 and C-6 positions of 2,3,4,6-unprotected galactose residues in galactosylchitobiose derivatives was demonstrated. Subsequently, the inversion of the configuration of the two hydroxy groups at the C-2 and C-4 positions of the galactose moiety was successfully carried out. This synthetic route reduces the number of the protection-deprotection reactions and is suitable for constructing different branching patterns of oligomannose-type glycans, such as M9, M5A, and M5B.

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.

Phenotypic analysis of α1,2-mannosidase-like protein deletion mutants in Saccharomyces cerevisiae.

α1,2­mannosidase-like proteins mediate quality control of glycoproteins in the endoplasmic reticulum. This study explored α1,2­mannosidase-like protein functions in Saccharomyces cerevisiae. Single disruptants in targeted protein-coding genes were found to be viable; however, deletion of MNL2 resulted in declined yeast growth at 37 °C. The normal growth rate was recovered in double-deletion strains where one of the deletions was in MNS1 . We also measured the mannosidase activity of microsomal fractions of deficient strains using artificial glycan. Increased mannose trimming activities were demonstrated by the microsomes of MNL2 -deletion strains compared to levels of activity exhibited by the microsomes of the control strain.

Muscle-tendon architecture in Kenyans and Japanese: Potential role of genetic endowment in the success of elite Kenyan endurance runners.

AIM: The specificity of muscle-tendon and foot architecture of elite Kenyan middle- and long-distance runners has been found to contribute to their superior running performance. To investigate the respective influence of genetic endowment and training on these characteristics, we compared leg and foot segmental lengths as well as muscle-tendon architecture of Kenyans and Japanese males (i) from infancy to adulthood and (ii) non-athletes versus elite runners. METHODS: The 676 participants were divided according to their nationality (Kenyans and Japanese), age (nine different age groups for non-athletes) and performance level in middle- and long-distance races (non-athlete, non-elite and elite adult runners). Shank and Achilles tendon (AT) lengths, medial gastrocnemius (MG) fascicle length, pennation angle and muscle thickness, AT moment arm (MAAT ), and foot lever ratio were measured. RESULTS: Above 8 years old, Kenyans had a longer shank and AT, shorter fascicle, greater pennation angle, thinner MG muscle as well as longer MAAT , with lower foot lever ratio than age-matched Japanese. Among adults of different performance levels and independently of the performance level, Kenyans had longer shank, AT and MAAT , thinner MG muscle thickness, and lower foot lever ratio than Japanese. The decrease in MG fascicle length and increase pennation angle observed for the adult Japanese with the increase in performance level resulted in a lack of difference between elite Kenyans and Japanese. CONCLUSION: The specificity of muscle-tendon and foot architecture of elite Kenyan runners could result from genetic endowment and contribute to the dominance of Kenyans in middle- and long-distance races.

Limb-Specific Features and Asymmetry of Nerve Conduction Velocity and Nerve Trunk Size in Human.

This study aimed to simultaneously examine the differences of human nerve conduction velocity (NCV) and nerve cross-sectional area (nCSA) between the upper and lower limbs and between different regions of the upper and lower limbs. Thirty healthy subjects volunteered for the study. NCV and nCSA of the ulnar and tibial nerves were measured with the dominant and non-dominant arms and the supporting and reacting legs using supramaximal electric stimulation and peripheral nerve ultrasonography at three regions for ulnar and tibial nerves, respectively. Supramaximal electric stimulation was superficially applied to the ulnar and tibial nerves at each point. These action potentials were recorded from the digiti minimi and soleus muscles for the ulnar and tibial nerves, respectively. Our results clearly showed that the NCV, nCSA, and circumference of the ulnar and tibial nerves were higher and greater in the lower limbs than in the upper limbs. The greater the circumference, the greater the nCSA for both the upper and lower limbs. However, unlike the upper limbs, the supporting leg did not have higher NCV than the reacting leg despite its greater circumference. Therefore, nCSA can be related to the circumference but not necessarily function for NCV developments of the lower limbs. These various aspects between the upper and lower limbs suggest that NCV does not depend on the nCSA sizes or upper and lower limb circumference; the results indicate the existence of limb-specific NCV but not nCSA developments.

Efficient synthesis of α(1,2)-linked oligomannoside derivatives through one-pot glycosylation.

An α(1,2)-linked oligomannoside derivative having a free C-2 hydroxyl group and a C-3 pivaloyl group was synthesized from a thiophenyl mannose derivative 1 using a one-pot self-condensation and applying a α-stereoselective procedure. The mannosylation exclusively generated α-mannoside linkages. The observed α-directing effect was rationalized by the remote participation of the pivaloyl group in C-3 position. The polymerization degree was controlled by the promoter amount providing the mannobiose derivative as a major product. Applying this method eliminated many synthetic steps. The α(1,2)-linked oligomannoside derivatives, which are key intermediates for the synthesis of oligomannose type N-glycans for glycoproteins, were easily prepared.

Muscle-Tendon Interaction During Human Dolphin-Kick Swimming.

Without high impact forces, it is not clear how humans can utilize tendon elasticity during low-impact activities. The purpose of the present study was to examine the muscle-tendon behavior together with the electromyographic (EMG) activities of the vastus lateralis (VL) muscle during the human dolphin-kicking. In a swimming pool, each subject (n = 11) swam the 25 m dolphin-kicking at two different speeds (NORMAL and FAST). Surface EMGs were recorded from the VL and biceps femoris (BF) muscles. Simultaneous recordings of the knee joint angle by electro-goniometer and of the VL fascicle length by ultrasonography were used to calculate the muscle-tendon unit and tendinous length of VL (LMTU and LTT, respectively). In the dolphin-kicking, the stretching and shortening amplitudes of VL LMTU did not differ significantly between the two kicking speed conditions. However, both stretching and shortening amplitudes of the VL fascicle length were lower at FAST than at NORMAL speed whereas the opposite was found for the VL LTT values. At FAST, the contribution of the VL tendinous length to the entire VLMTU length changes increased. The EMG analysis revealed at FAST higher agonist VL activation from the late up-beat (MTU stretching) to the early down-beat phases as well as increased muscle co-activation of VL and BF muscles from the late down-beat to early up-beat phases of dolphin-kicking. These results suggest that at increasing kicking speeds, the VL fascicles and tendinous tissues during aquatic movements can utilize tendon elasticity in a similar way than in terrestrial forms of locomotion. However, these activation profiles of VL and BF muscles may differ from their activation pattern in terrestrial locomotion.

A bioactive mammalian disaccharide associated with autoimmunity activates STING-TBK1-dependent immune response.

Glycans from microbial pathogens are well known pathogen-associated molecular patterns that are recognized by the host immunity; however, little is known about whether and how mammalian self-glycans activate the host immune response, especially in the context of autoimmune disease. Using biochemical fractionation and two-dimensional HPLC, we identify an abundant and bioactive free glycan, the Manß1-4GlcNAc disaccharide in TREX1-associated autoimmune diseases. We report that both monosaccharide residues and the ß1-4 linkage are critical for bioactivity of this disaccharide. We also show that Manß1-4GlcNAc is produced by oligosaccharyltransferase hydrolysis of lipid-linked oligosaccharides in the ER lumen, followed by ENGase and mannosidase processing in the cytosol and lysosomes. Furthermore, synthetic Manß1-4GlcNAc disaccharide stimulates a broad immune response in vitro, which is in part dependent on the STING-TBK1 pathway, and enhances antibody response in vivo. Together, our data identify Manß1-4GlcNAc as a novel innate immune modulator associated with chronic autoimmune diseases.

Fluorogenic probe for measuring high-mannose type glycan-specific endo-ß-N-acetylglucosaminidase H activity.

We synthesized a fluorogenic probe with a high-mannose type heptasaccharide structure to detect the hydrolytic activity of endo-ß-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H). The heptasaccharide derivative (1) was labeled with an N-methylanthraniloyl group as a reporter dye at the branching point of the ß-mannoside residue and 2,4-dinitrophenyl group as a quencher molecule at the reducing end, which was hydrolyzed by Endo-H, resulting in increased fluorescence intensity. Thus, Endo-H activities could be evaluated easily and quantitatively by measuring the fluorescence signal. Using both this probe (1) and a previously synthesized pentasaccharide probe, the hydrolysis activity of Endo-H and Endo-M were investigated. The results clearly showed a correlation with the substrate specificity of each enzyme.

Fluorescence Quenching-based Assay for Measuring Golgi endo-α-Mannosidase.

Golgi endo-α-mannosidase (G-EM) catalyzes an alternative deglucosylation process for N-glycans and plays important roles in the post-endoplasmic reticulum (ER) quality control pathway. To understand the post-ER quality control mechanism, we synthesized a tetrasaccharide probe for the detection of the hydrolytic activity of G-EM based on a fluorescence quenching assay. The probe was labeled with an N-methylanthraniloyl group as a reporter dye at the non-reducing end and a 2,4-dinitrophenyl group as a quencher at the reducing end. This probe is hydrolyzed to disaccharide derivatives by G-EM, resulting in increased fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of disaccharide derivative present, allowing the G-EM activity to be evaluated easily and quantitatively.

A New Fluorogenic Probe for the Detection of endo-ß-N-Acetylglucosaminidase.

We developed a fluorescence-quenching-based assay system to determine the hydrolysis activity of endo-ß-N-acetylglucosaminidases (ENGases). The pentasaccharide derivative 1 was labeled with an N-methylanthraniloyl group as a reporter dye at the non-reducing end and with a 2,4-dinitrophenyl group as a quencher molecule at the reducing end. This derivative is hydrolyzed by ENGase, resulting in an increase in fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of the tetrasaccharide derivative, hence allowing ENGase activity to be evaluated easily and quantitatively. Using this system, we succeeded in measuring the hydrolysis activities of ENGases and thus the inhibitory activities of known inhibitors. We confirmed that this assay system is suitable for high-throughput screening for potential inhibitors of human ENGase that might serve as therapeutic agents for the treatment of N-glycanase 1 (NGLY1) deficiency.

Specificity of Donor Structures for endo-ß-N-Acetylglucosaminidase-Catalyzed Transglycosylation Reactions.

To demonstrate the structural specificity of the glycosyl donor for the transglycosylation reaction by using endo-ß-N-acetylglucosaminidase from Mucor hiemalis (endo-M), a series of tetrasaccharide oxazoline derivatives was synthesized. These derivatives correspond to the core structure of an asparagine-linked glycoprotein glycan with a ß-mannose unit of a non-natural-type monosaccharide, including ß-glucose, ß-galactose, and ß-talose in place of the ß-mannose moiety. The transglycosylation activity of wildtype (WT) endo-M and two mutants, N175Q and N175A, was examined by using these tetrasaccharide donors with p-nitrophenyl N-acetylglucosaminide (GlcNAc-pNp). The essential configuration of the hydroxy group for the transglycosylation reaction was determined. On the basis of these results, the transglycosylation reaction was investigated by using chemically modified donors, and transglycosylated products were successfully obtained.

Neuromechanical Modulation of the Achilles Tendon During Bilateral Hopping in Patients with Unilateral Achilles Tendon Rupture, Over 1 Year After Surgical Repair.

BACKGROUND: Patients who have had an Achilles tendon (AT) rupture repaired are potentially at higher risk for re-rupture than those without previous rupture. Little attention has been given to the neuromechanical modulation of muscle-tendon interaction and muscle activation profiles during human dynamic movements after AT rupture repair. OBJECTIVE: The purpose of this study was to examine muscle-tendon behavior and muscle activation during bilateral hopping. METHODS: We enrolled nine subjects who had undergone surgical repair of unilateral AT rupture within the past 1-2 years. Subjects performed bilateral hopping while we took ultrasound, kinematic, and electromyogram recordings and measurements. AT behaviors were also recorded. We then compared responses between values obtained from the ruptured AT leg (LEGATR) and non-ruptured AT leg (LEGNOR). RESULTS: During hopping, the AT stretching amplitudes were greater in the LEGATR than in the LEGNOR, although the peak AT force and stiffness were smaller in the LEGATR than in the LEGNOR. The AT negative mechanical work did not show any significant differences between both legs. However, positive works were significantly lower in the LEGATR than in the LEGNOR. Electromyogram patterns in both soleus and tibialis anterior muscles clearly differed after ground contact for the LEGATR and the LEGNOR. CONCLUSIONS: These results suggest that the repaired ruptured AT can be compliant and have insufficient Young's modulus, which can influence mechanical responses in muscle activities. The modulation of agonist-antagonist muscle activities corresponding to the different levels of stiffness between the LEGATR and the LEGNOR may not be fully functioning during the pre-activation phase.

Can measures of muscle-tendon interaction improve our understanding of the superiority of Kenyan endurance runners?

PURPOSE: Leg muscle activation profiles and muscle-tendon interaction were studied with eleven internationally high-level Kenyan and eleven national level Japanese distance runners. METHODS: Ultrasonography and kinematics were applied together with surface electromyography (EMG) recordings of leg muscles when subjects ran on treadmill at 9.0 (SLOW) and 13.9 km h(-1) (MEDIUM). RESULTS: At each speed, both groups presented similar contact and flight times. The kinematic and ultrasound analyses revealed that, in contrast to the Japanese runners, the Kenyans demonstrated during contact smaller stretching and shortening amplitudes (p < 0.01) of the tendinous tissue of medial gastrocnemius (MG), but greater tendon contribution to the muscle-tendon unit shortening (p < 0.05). The MG fascicles of the Kenyans were shorter not only at the resting standing position, but also during the contact phase at both running speeds (p < 0.01). The EMG profiles of the Kenyans showed lower braking/preactivation ratio in both MG and tibialis anterior (p < 0.05) muscles. They were also characterized by negative relationships between the Achilles tendon moment arm and the MG fascicle shortening during contact (r = -0.54, p < 0.01). In contrast, the Japanese presented the classical stretch-shortening cycle muscle activation profile of relatively high MG EMG activity during the braking phase. CONCLUSION: These findings provide new suggestions that the Kenyans have unique structural characteristics which can result in the reduction of muscle and tendinous stretch-shortening loading together with smaller muscle activation during contact at submaximal running speed.

Modulation of pK(a) of Brooker's merocyanine by DNA hybridization.

Brooker's merocyanine (BM), which changes its emission and absorption maxima upon protonation, was introduced into oligodeoxyribonucleotide (ODN) via d-threoninol by postsynthetic modification on a CPG (controlled-pore glass) support. The pK(a) of BM in the modified ODN increased from 9.5 to 10.1 upon hybridization. As a result, absorption maxima shifted from 492 to 432 nm at pH 10.0 by the presence of its complementary strand. This spectral shift was sufficiently large so that DNA hybridization could easily be discriminated even by the naked eye; the color of the solution changed from orange to yellow upon hybridization. In addition, the fluorescence emission was strongly quenched upon hybridization, demonstrating that this probe can also detect the target DNA by the fluorescence change. Ratiometric detection of hybridization was also possible by simultaneous excitation of both protonated and deprotonated BMs. Furthermore, we could also modulate its pK(a) by the antiparallel stacking of two BM molecules in the duplex; the pK(a) of BM decreased from 10.1 to 9.7 by the stacking of two BMs in an antiparallel manner. Thus, control of the microenvironment around the BM molecule allowed modulation of its pK(a), which is applicable to the sequence-specific recognition of target DNA.