A quantitative description of glyco-alteration/differences in diseases can lead to the development of a diagnostic agent for use in vitro to monitor the degree of change in target glycoproteins. ...Analytical systems have been developed along with the progress of omics-oriented technologies. For clinical implementation, their full automation is required with an apparatus that is simple to operate. Here, we report an automatic analysis system for quantitative characterization of glyco-alteration/differences that depends on the unique strategy of “bead arrays in a single tip.” The alternative lectin array can obtain a minimum characterization of the glycan profile for nanogram quantities of an endogenous glycoprotein. A simple autopipetting robot produces the precise chemiluminescence detection of glycan–lectin interactions with a wide dynamic range that is superior to fluorescence-based lectin arrays. The tip-based array format enables automatic glycan profiling from sample pretreatment to detection with low variation and linear detection, which may facilitate the use of this lectin array in clinical practice.
Nearly all secreted proteins are glycosylated, and serum glycoproteins that exhibit disease-associated glycosylation changes have potential to be biomarkers. In rheumatoid arthritis (RA), C-reactive ...protein (CRP), and matrix metalloproteinase-3 (MMP-3) are widely used as serologic biomarkers, but they lack sufficient specificity or precision. We performed comparative glycosylation profiling of MMP-3 using a recently developed antibody-overlay lectin microarray technology that allows semicomprehensive and quantitative analysis of specific protein glycosylation to develop an RA-specific disease activity biomarker.
Serum was taken from patients with RA (n = 24) whose disease activity was scored using composite measures, and MMP-3 was immunoprecipitated and subjected to lectin microarray analysis. A disease activity index (DAI) based on lectin signal was developed and validated using another cohort (n = 60). Synovial fluid MMP-3 in patients with RA and patients with osteoarthritis (OA) was also analyzed.
Intense signals were observed on a sialic acid-binding lectin (Agrocybe cylindracea galectin ACG) and O-glycan-binding lectins (Jacalin, Agaricus bisporus agglutinin ABA, and Amaranthus caudatus agglutinin ACA) by applying subnanogram levels of serum MMP-3. ACG, ABA, and ACA revealed differences in MMP-3 quantity, and Jacalin revealed differences in MMP-3 quality. The resultant index, ACG/Jacalin, correlated well with disease activity. Further validation using another cohort confirmed that this index correlated well with several DAIs and their components, and reflected DAI changes following RA treatment, with correlations greater than those for MMP-3 and CRP. Furthermore, MMP-3, which generated a high ACG/Jacalin score, accumulated in synovial fluid of patients with RA but not in that of patients with OA. Sialidase digestion revealed that the difference in quality was derived from O-glycan α-2,6-sialylation.
This is the first report of a glycoprotein biomarker using glycan change at a local lesion to assess disease activity in autoimmune diseases. Differences in the degree of serum MMP-3 α-2,6-sialylation may be a useful index for estimating disease activity.
Purpose
Wisteria floribunda agglutinin positive human Mac‐2‐binding protein (WFA+‐hM2BP) was recently validated as a liver fibrosis glycobiomarker with a fully automated lectin–antibody sandwich ...immunoassay. In this study, we supplied recombinant WFA+‐hM2BP as the standard glycoprotein and the overlaid antibody to enhance the robustness of WFA+‐hM2BP quantification.
Experimental design
The optimum conditions for producing recombinant WFA+‐hM2BP were selected by cell glycome analysis based on a lectin microarray. Interlot variability of recombinant WFA+‐hM2BP was determined using an antibody‐overlay lectin microarray. Screening of anti‐M2BP mAb was completed by incorporating a WFA–antibody sandwich ELISA and an antibody‐overlay lectin microarray.
Results
The lectin microarray analysis revealed that human embryonic kidney 293 cells efficiently and stably produced WFA+‐hM2BP in DMEM containing 10% FCS without any variation in the M2BP glycosylation level. A spiking experiment with recombinant WFA+‐hM2BP was mostly effective for antibody screening. The reconstituted sandwich immunoassay was useful for the continuous quantification and cutoff index expression of serum WFA+‐hM2BP.
Conclusions and clinical relevance
The multiple use of lectin‐assisted glycan profiling enabled us to construct a reliable sandwich assay kit for monitoring liver fibrosis in patients with viral hepatitis. This will assist in the development pipeline for other glycodiagnostic agents.
Glucokinase (GK) plays a key role in the control of blood glucose homeostasis. We identified a small molecule GK activator, compound A, that increased the glucose affinity and maximal velocity (Vmax) ...of GK. Compound A augmented insulin secretion from isolated rat islets and enhanced glucose utilization in primary cultured rat hepatocytes. In rat oral glucose tolerance tests, orally administrated compound A lowered plasma glucose elevation with a concomitant increase in plasma insulin and hepatic glycogen. In liver, GK activity is acutely controlled by its association to the glucokinase regulatory protein (GKRP). In order to decipher the molecular aspects of how GK activator affects the shuttling of GK between nucleus and cytoplasm, the effect of compound A on GK-GKRP interaction was further investigated. Compound A increased the level of cytoplasmic GK in both isolated rat primary hepatocytes and the liver tissues from rats. Experiments in a cell-free system revealed that compound A interacted with glucose-bound free GK, thereby impairing the association of GK and GKRP. On the other hand, compound A did not bind to glucose-unbound GK or GKRP-associated GK. Furthermore, we found that glucose-dependent GK-GKRP interaction also required ATP. Given the combined prominent role of GK on insulin secretion and hepatic glucose metabolism where the GK-GKRP mechanism is involved, activation of GK has a new therapeutic potential in the treatment of type 2 diabetes.
Glucokinase activators (GKAs) are small-molecule agents that enhance glucose sensing by pancreatic β cells and glucose metabolism by hepatocytes. There is strong interest in these agents as potential ...therapies for type 2 diabetes. Here, we report key pharmacokinetic and pharmacodynamic findings from preclinical studies of the GKA 3-6-(ethylsulfonyl)-3-pyridinyloxy-5-(1S)-2-hydroxy-1-methylethoxy-N-(1-methyl-1H-pyrazol-3-yl)benzamide (MK-0941). Incubated in vitro with recombinant human glucokinase, 1 μM MK-0941 lowered the S(0.5) of this enzyme for glucose from 6.9 to 1.4 mM and increased the maximum velocity of glucose phosphorylation by 1.5-fold. In 2.5 and 10 mM glucose, the EC(50) values for activation of GK by MK-0941 were 0.240 and 0.065 μM, respectively. Treatment of isolated rat islets of Langerhans and hepatocytes with 10 μM MK-0941 increased insulin secretion by 17-fold and glucose uptake up to 18-fold, respectively. MK-0941 exhibited strong glucose-lowering activity in C57BL/6J mice maintained on a high-fat diet (HFD), db/db mice, HFD plus low-dose streptozotocin-treated mice, and nondiabetic dogs. In both mice and dogs, oral doses of MK-0941 were rapidly absorbed and rapidly cleared from the blood; plasma levels reached maximum within 1 h and fell thereafter with a half-life of ~2 h. During oral glucose tolerance testing in dogs, MK-0941 reduced total area-under-the-curve postchallenge (0-2 h) plasma glucose levels by up to 48% compared with vehicle-treated controls. When administered twice daily to mice for 16 days, and once daily to the dog for 4 days, MK-0941 remained efficacious on successive days. These findings support further investigation of MK-0941 as a potential therapeutic agent for treatment of type 2 diabetes.
Protein glycosylation is a crucial factor that must be evaluated in biological pharmaceuticals. The glycoform profile of a protein can vary depending on the conditions of the cultivation, ...purification process, and the selection of a host cell. Lectin microarrays are reliable bioanalytical methods used in the early phases of bioprocesses for the detection of glycosylation. The concept of a fully automated glycan detection with a bead array has been previously reported; however, no simple system has been constructed on fluorescence-based detection using a microarray. Here, we present a fully automated detection system equipped with a novel fluorescence detector for a 13-lectin bead array with a single tip. The lattice-like arrangement of a set of fibers proximate to the tip of the light emitting diode and photomultiplier tube detector minimized the noise caused by the reflection of incident light on the plastic capillary tip and bead. A unique rolling-circle fiber unit with quadruple lattices stacked in two layers realizes the 8-parallel automeasurement with a drastic reduction in scanning time and machine size. The 8-glycan profiles obtained automatically within 25 min were identical with those obtained with the conventional lectin microarray after overnight incubation. The signals obtained were represented as lectin dotcodes. Therefore, autolectin dotcoding assisted by the twin 8 legs named as “detection and irradiation octopuses” may be a rapid glyco-evaluation system during the production and development of biopharmaceuticals.
We investigated the effect of glucokinase activator (GKA) on glucose metabolism and β-cell mass. We analyzed four mouse groups: wild-type mice and β-cell-specific haploinsufficiency of glucokinase ...gene (Gck+/−) mice on a high-fat (HF) diet. Each genotype was also treated with GKA mixed in the HF diet. Rodent insulinoma cells and isolated islets were used to evaluate β-cell proliferation by GKA. After 20 wk on the above diets, there were no differences in body weight, lipid profiles, and liver triglyceride content among the four groups. Glucose tolerance was improved shortly after the GKA treatment in both genotypes of mice. β-Cell mass increased in wild-type mice compared with Gck+/− mice, but a further increase was not observed after the administration of GKA in both genotypes. Interestingly, GKA was able to up-regulate insulin receptor substrate-2 (Irs-2) expression in insulinoma cells and isolated islets. The administration of GKA increased 5-bromo-2-deoxyuridine (BrdU) incorporation in insulinoma cells, and 3 d administration of GKA markedly increased BrdU incorporation in mice treated with GKA in both genotypes, compared with those without GKA. In conclusion, GKA was able to chronically improve glucose metabolism for mice on the HF diet. Although chronic GKA administration failed to cause a further increase in β-cell mass in vivo, GKA was able to increase beta cell proliferation in vitro and with a 3-d administration in vivo. This apparent discrepancy can be explained by a chronic reduction in ambient blood glucose levels by GKA treatment.
Glucokinase activator is able to improve glucose metabolism and has an effect on β cell proliferation.
Abstract
Experimental autoimmune myocarditis (EAM) is mediated by myocardial infiltration by myosin-specific T-cells secreting inflammatory cytokines. In this study, rat models of EAM were prepared ...by injection with porcine cardiac myosin. One week after immunization, edaravone was administered intraperitoneally at 3 or 10 mg/kg/day to rats for 2 weeks. Cardiac function was measured by haemodynamic and echocardiographic studies and TUNEL assay was performed. Left ventricular (LV) expression of NADPH oxidase sub-units (p47phox and p67phox), pro-inflammatory cytokines (TNF-α), endoplasmic reticulum (ER) stress signalling proteins (GRP78, caspase-12 and GADD153) and mitogen-activated protein kinase (MAPK) family proteins (phospho-p38 MAPK and phospho-JNK) were measured by western blotting. Edaravone improved LV function in a dose-dependent manner. Central venous pressure was significantly low and LV ejection fraction and fractional shortening was significantly high in edaravone groups compared with those in the vehicle group. In addition, edaravone treatment down-regulated LV expressions of p47phox, TNF-α, GADD153, phospho-p38 MAPK and phospho-JNK. Furthermore, the LV expressions of p67phox, GRP78, caspase-12 and TUNEL-positive cells of rats with EAM treated with edaravone were significantly low compared with those of the vehicle group. These findings suggest that edaravone ameliorated the progression of EAM by inhibiting oxidative and ER stress and, subsequently, cardiac apoptosis.
•In 60ZnCl2–20KCl–20BaCl2–xTbCl3 glasses, no energy transfer occurred for 0<x≤0.34.•For 0.34<x≤1.25, the cross relaxation, (5D3→5D4)→(7F0←7F6) occurred between Tb3+ ions.•For 0<x≤0.34, the 72% of ...excited electrons were radiatively relaxed by the 5D3→7FJ transitions.•The lifetime of the 5D3 initial level was long and 1.1ms.
60ZnCl2–20KCl–20BaCl2–xTbCl3 glasses (x=0.10, 0.25, 0.50, 0.75, 1.00, and 1.25) were prepared by melt-quenching method, and Tb3+ fluorescence properties were investigated under 355nm excitation. Regardless of x values, the electrons that were relaxed from the 5D3 to 5D4 level of Tb3+ ions by the multiphonon relaxation, were repressed to 28% of all the excited electrons because the ZnCl2-based glass had much lower phonon energy than oxide glasses. For 0<x≤0.34, the cross relaxation, (5D3→5D4)→(7F0←7F6), was repressed, and consequently 72% and 28% of all the excited electrons were radiatively relaxed by the 5D3→7FJ (J=6, 5, 4, 3, and 2) and 5D4→7FJ (J=6, 5, 4, and 3) transitions, respectively. The lifetimes of the 5D3 and 5D4 initial levels were obtained to be 1.1 and 2.1ms, respectively.
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