Functional and structural analysis of a cyclization domain in a cyclic ß-1,2-glucan synthase.

Cyclic ß-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic ß-1,2-glucans (CßGs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that ß-glucosidase-resistant compounds are produced by TiCGSCy with linear ß-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are CßGs. Next, action pattern analyses using ß-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide being the minimum length of the substrate. These analyses also showed that longer substrate ß-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce CßGs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of ß-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear differences in the reaction pathways between these enzymes. Overall, we propose a novel reaction mechanism of TiCGSCy. Thus, the present group of CGSs defines a new glycoside hydrolase family, GH189. KEY POINTS: • It was clearly evidenced that cyclization domain alone produces cyclic ß-1,2-glucans. • The domain exclusively catalyzes transglycosylation without hydrolysis. • The present catalytic domain defines as a new glycoside hydrolase family 189.

Direct identification of the rotary angle of ATP cleavage in F1-ATPase from Bacillus PS3.

F1-ATPase is the world's smallest biological rotary motor driven by ATP hydrolysis at three catalytic ß subunits. The 120° rotational step of the central shaft γ consists of 80° substep driven by ATP binding and a subsequent 40° substep. In order to correlate timing of ATP cleavage at a specific catalytic site with a rotary angle, we designed a new F1-ATPase (F1) from thermophilic Bacillus PS3 carrying ß(E190D/F414E/F420E) mutations, which cause extremely slow rates of both ATP cleavage and ATP binding. We produced an F1 molecule that consists of one mutant ß and two wild-type ßs (hybrid F1). As a result, the new hybrid F1 showed two pausing angles that are separated by 200°. They are attributable to two slowed reaction steps in the mutated ß, thus providing the direct evidence that ATP cleavage occurs at 200° rather than 80° subsequent to ATP binding at 0°. This scenario resolves the long-standing unclarified issue in the chemomechanical coupling scheme and gives insights into the mechanism of driving unidirectional rotation.

Characterization and structural analyses of a novel glycosyltransferase acting on the ß-1,2-glucosidic linkages.

The IALB_1185 protein, which is encoded in the gene cluster for endo-ß-1,2-glucanase homologs in the genome of Ignavibacterium album, is a glycoside hydrolase family (GH) 35 protein. However, most known GH35 enzymes are ß-galactosidases, which is inconsistent with the components of this gene cluster. Thus, IALB_1185 is expected to possess novel enzymatic properties. Here, we showed using recombinant IALB_1185 that this protein has glycosyltransferase activity toward ß-1,2-glucooligosaccharides, and that the kinetic parameters for ß-1,2-glucooligosaccharides are not within the ranges for general GH enzymes. When various aryl- and alkyl-glucosides were used as acceptors, glycosyltransfer products derived from these acceptors were subsequently detected. Kinetic analysis further revealed that the enzyme has wide aglycone specificity regardless of the anomer, and that the ß-1,2-linked glucose dimer sophorose is an appropriate donor. In the complex of wild-type IALB_1185 with sophorose, the electron density of sophorose was clearly observed at subsites -1 and +1, whereas in the E343Q mutant-sophorose complex, the electron density of sophorose was clearly observed at subsites +1 and +2. This observation suggests that binding at subsites -1 and +2 competes through Glu102, which is consistent with the preference for sophorose as a donor and unsuitability of ß-1,2-glucooligosaccharides as acceptors. A pliable hydrophobic pocket that can accommodate various aglycone moieties was also observed in the complex structures with various glucosides. Overall, our biochemical and structural data are indicative of a novel enzymatic reaction. We propose that IALB_1185 be redefined ß-1,2-glucooligosaccharide:d-glucoside ß-d-glucosyltransferase as a systematic name and ß-1,2-glucosyltransferase as an accepted name.

Enzymatic control and evaluation of degrees of polymerization of ß-(1→2)-glucans.

ß-(1 â†’ 2)-Glucans can be synthesized by 1,2-ß-oligoglucan phosphorylase using ß-(1 â†’ 2)-glucooligosaccharides as acceptors and α-d-glucose 1-phosphate as a donor. Using phosphorolysis of sucrose as a source of α-d-glucose 1-phosphate, we generated ß-(1 â†’ 2)-glucans with degrees of polymerization (DPs) up to approximately 280. Average DPs up to approximately 1000 were obtained using ß-(1 â†’ 2)-glucan with average DP of 160 as an acceptor and pure α-d-glucose 1-phosphate as a donor. A colorimetric assay of the ß-glucosidase activity against the ß-(1 â†’ 2)-glucan products was used to determine their DPs.

Clinical Effects of Early and Short-Term Tolvaptan Administration in Patients Hospitalized for Acute Decompensated Heart Failure.

INTRODUCTION: Previous trials showed that tolvaptan improves acute heart failure (HF). However, the optimal timing for administering tolvaptan to achieve the best outcome remains unclear. Therefore, the current study investigated the relationship between the timing of tolvaptan treatment initiation and clinical outcomes in patients with acute decompensated HF. METHODS: We prospectively evaluated 201 patients with acute decompensated HF, randomly divided into 2 groups based on the timing of tolvaptan initiation. The early group was administered tolvaptan approximately 1 week after day 1 or 2 (n = 104), whereas the late group was administered the same drug 1 week after the early group (n = 97). RESULTS: All-cause mortality, cardiovascular death, and hospitalization during the follow-up period were comparable between both groups. The early group had shorter durations of oxygenation, carperitide infusion, and hospitalization than the late group (p = 0.013, 0.003, 0.006, respectively). The early group demonstrated a significantly faster decrease in pleural effusion than the late group (p = 0.001). The 2 groups had comparable maximum and minimum serum sodium and potassium levels and minimum estimated glomerular filtration rates during hospitalization. The early group spent significantly less money on all diuretics administered over the first 2 weeks and on tolvaptan and carperitide administered during the hospitalization period than the late group (p < 0.001). CONCLUSIONS: Early and short-term administration of tolvaptan was feasible, contributed to a more rapid improvement in patients with acute decompensated HF, and reduced health-care costs.

Identification, characterization, and structural analyses of a fungal endo-ß-1,2-glucanase reveal a new glycoside hydrolase family.

endo-ß-1,2-Glucanase (SGL) is an enzyme that hydrolyzes ß-1,2-glucans, which play important physiological roles in some bacteria as a cyclic form. To date, no eukaryotic SGL has been identified. We purified an SGL from Talaromyces funiculosus (TfSGL), a soil fungus, to homogeneity and then cloned the complementary DNA encoding the enzyme. TfSGL shows no significant sequence similarity to any known glycoside hydrolase (GH) families, but shows significant similarity to certain eukaryotic proteins with unknown functions. The recombinant TfSGL (TfSGLr) specifically hydrolyzed linear and cyclic ß-1,2-glucans to sophorose (Glc-ß-1,2-Glc) as a main product. TfSGLr hydrolyzed reducing-end-modified ß-1,2-gluco-oligosaccharides to release a sophoroside with the modified moiety. These results indicate that TfSGL is an endo-type enzyme that preferably releases sophorose from the reducing end of substrates. Stereochemical analysis demonstrated that TfSGL is an inverting enzyme. The overall structure of TfSGLr includes an (α/α)6 toroid fold. The substrate-binding mode was revealed by the structure of a Michaelis complex of an inactive TfSGLr mutant with a ß-1,2-glucoheptasaccharide. Mutational analysis and action pattern analysis of ß-1,2-gluco-oligosaccharide derivatives revealed an unprecedented catalytic mechanism for substrate hydrolysis. Glu-262 (general acid) indirectly protonates the anomeric oxygen at subsite -1 via the 3-hydroxy group of the Glc moiety at subsite +2, and Asp-446 (general base) activates the nucleophilic water via another water. TfSGLr is apparently different from a GH144 SGL in the reaction and substrate recognition mechanism based on structural comparison. Overall, we propose that TfSGL and closely-related enzymes can be classified into a new family, GH162.

Characterization and Structural Analysis of a Novel exo-Type Enzyme Acting on ß-1,2-Glucooligosaccharides from Parabacteroides distasonis.

ß-1,2-Glucan is a polysaccharide produced mainly by some Gram-negative bacteria as a symbiosis and infectious factor. We recently identified endo-ß-1,2-glucanase from Chitinophaga pinensis ( CpSGL) as an enzyme comprising a new family. Here, we report the characteristics and crystal structure of a CpSGL homologue from Parabacteroides distasonis, an intestinal bacterium (BDI_3064 protein), which exhibits distinctive properties of known ß-1,2-glucan-degrading enzymes. BDI_3064 hydrolyzed linear ß-1,2-glucan and ß-1,2-glucooligosaccharides with degrees of polymerization (DPs) of ≥4 to produce sophorose specifically but did not hydrolyze cyclic ß-1,2-glucan. This result indicates that BDI_3064 is a new exo-type enzyme. BDI_3064 also produced sophorose from ß-1,2-glucooligosaccharide analogues that have a modified reducing end, indicating that BDI_3064 acts on its substrates from the nonreducing end. The crystal structure showed that BDI_3064 possesses additional N-terminal domains 1 and 2, unlike CpSGL. Superimposition of BDI_3064 and CpSGL complexed with ligands showed that R93 in domain 1 overlapped subsite -3 in CpSGL. Docking analysis involving a ß-1,2-glucooligosaccharide with DP4 showed that R93 completely blocks the nonreducing end of the docked ß-1,2-glucooligosaccharide. This indicates that BDI_3064 employs a distinct mechanism of recognition at the nonreducing end of substrates to act as an exo-type enzyme. Thus, we propose 2-ß-d-glucooligosaccharide sophorohydrolase (nonreducing end) as a systematic name for BDI_3064.

The role of soluble fibrin during anticoagulant therapy: a case report.

Warfarin, dabigatran, and apixaban are used for preventing ischemic stroke due to non-valvular atrial fibrillation (NVAF). However, it is often challenging to select the appropriate anticoagulant. We present the case of a 70-year-old male patient with persistent NVAF who developed pulmonary thromboembolism (PTE), deep vein thrombosis (DVT), and left atrial thrombus during anticoagulant therapy with warfarin. Intravenous recombinant tissue plasminogen activator was administered during his acute PTE. Heparin and apixaban were administered over 28 days; heparin was discontinued after the DVT resolved, while apixaban was administered to prevent ischemic stroke. Two days after heparin was discontinued, the patient experienced an ischemic stroke. Dabigatran was administered for secondary ischemic stroke prevention. Soluble fibrin (SF) levels remained elevated during treatment with heparin and apixaban and returned to normal after apixaban was replaced with dabigatran. Monitoring of SF may be useful as an index for selection of anticoagulants.

The relationship between paroxysmal atrial fibrillation and coronary artery spasm.

BACKGROUND: Coronary artery spasm has recently been reported as a complication of the ablation of atrial fibrillation (AF). It may be induced by cardiac autonomic nervous dysfunction during radiofrequency catheter ablation of AF. However, the underlying mechanism is not clear. We hypothesized that patients with paroxysmal AF coincidentally exhibit coronary artery spasm. METHODS: A total of 51 patients were enrolled in a case control study to clarify the relationship between paroxysmal AF and coronary artery spasm. We evaluated 17 patients with paroxysmal AF (AF group) and 34 patients without paroxysmal AF (control group). Drug-induced coronary artery spasm provocation tests were performed by intracoronary administration of acetylcholine or ergonovine. RESULTS: The AF group consisted of nine males and eight females, mean age 67 ± 10 years. The control group consisted of 16 males and 18 females, mean age 60 ± 14 years. In the AF group, coronary artery spasm was induced in 13 patients (76.5%) before AF ablation. In the control group, coronary artery spasm was induced in three patients (8.8%). Coronary artery spasm was more frequently induced in patients with AF (76.5% vs 8.8%; odds ratio, 33.583; 95% confidence interval, 6.5732-171.58; P < 0.0001). In the AF group, ventricular fibrillation and AF were recorded immediately after right coronary artery spasm induction in one patient. CONCLUSIONS: Paroxysmal AF patients have high positive rates of drug-provoked coronary artery spasm. Patients with paroxysmal AF may coincidentally exhibit coronary artery spasm.

Renal ultrasonography predicts worsening renal function in patients with heart failure under tolvaptan administration.

AIMS: Renal dysfunction in patients with chronic heart failure predicts a poor prognosis. Tolvaptan has a diuretic effect in patients with chronic kidney disease and heart failure without adverse effects on renal function. We aimed to determine the effects of tolvaptan and predictors of worsening renal function in patients with heart failure. METHODS AND RESULTS: This post hoc analysis was a sub-analysis of a single-centre prospectively randomized trial on the early and short-term tolvaptan administration. We enrolled 201 participants with decompensated heart failure between January 2014 and March 2019 (early group, n = 104; age: 79.0 ± 12.8 years; late group, n = 97; age: 80.3 ± 10.8 years). Renal ultrasonography was performed before and after the administration of tolvaptan. Urine output and oral water intake significantly increased during tolvaptan administration. The difference between water intake and urine volume increased during tolvaptan administration. Changes in body weight, blood pressure, heart rate, and estimated glomerular filtration rate (eGFR) in both groups were comparable. The changes in peak-systolic velocity (PSV), acceleration time (AT) of the renal arteries, and resistance index were comparable. The changes in PSV and end-diastolic velocity (EDV) of the interlobar arteries increased following tolvaptan administration (Δmax PSV: 0.0 ± 14.8 cm/s before tolvaptan vs. 5.6 ± 15.7 cm/s after tolvaptan, P = 0.002; Δmean PSV: 0.4 ± 12.3 vs. 4.9 ± 12.7 cm/s, P = 0.002; Δmax EDV: -0.2 ± 3.5 vs. 1.4 ± 4.0 cm/s, P = 0.001; Δmean EDV: -0.0 ± 3.1 vs. 1.1 ± 3.4 cm/s, P = 0.003). The renal artery AT was negatively correlated with the eGFR (Δmax AT: beta = -0.2354, P = 0.044; Δmean AT: beta = -0.2477, P = 0.035). CONCLUSIONS: Tolvaptan increased the PSV and EDV of the interlobar artery, which may mean tolvaptan increased renal blood flow. The renal artery AT may be a surrogate for worsening renal function.

Structural and biochemical analysis of family 92 carbohydrate-binding modules uncovers multivalent binding to ß-glucans.

Carbohydrate-binding modules (CBMs) are non-catalytic proteins found appended to carbohydrate-active enzymes. Soil and marine bacteria secrete such enzymes to scavenge nutrition, and they often use CBMs to improve reaction rates and retention of released sugars. Here we present a structural and functional analysis of the recently established CBM family 92. All proteins analysed bind preferentially to ß-1,6-glucans. This contrasts with the diversity of predicted substrates among the enzymes attached to CBM92 domains. We present crystal structures for two proteins, and confirm by mutagenesis that tryptophan residues permit ligand binding at three distinct functional binding sites on each protein. Multivalent CBM families are uncommon, so the establishment and structural characterisation of CBM92 enriches the classification database and will facilitate functional prediction in future projects. We propose that CBM92 proteins may cross-link polysaccharides in nature, and might have use in novel strategies for enzyme immobilisation.

Resolution of left atrial appendage thrombus with apixaban.

Left atrial appendage (LAA) thrombosis is an important cause of cardiogenic cerebral thromboembolism. Apixaban is a member of the class of novel oral anticoagulants (NOAC) and is superior to warfarin in preventing stroke or systemic embolism, causes less bleeding, and results in lower mortality in patients with atrial fibrillation. There are few reports of resolution of LAA thrombus with other NOAC. We present a 72-year-old male patient with persistent atrial fibrillation associated with left atrial thrombus. Sixteen days of apixaban treatment showed complete thrombus resolution. In this study, soluble fibrin and D-dimer levels decreased without prolongation of international normalized ratio (INR) and activated partial thromboplastin time (APTT).

The relationship between right ventricular lead position and paced QRS duration.

BACKGROUND: The relationship between the anatomical location of right ventricular pacing site and paced QRS duration is unclear. In this study, we aimed to investigate the relationship between right ventricular pacing site and paced QRS duration using cardiac angiography. METHODS: Fifty patients were implanted with pacemakers. The right ventricular lead position was determined from the findings of cardiac angiography and the paced QRS duration was measured. Cardiac angiography was used to display the right ventriculogram (RVG) and the left ventriculogram (LVG). The RVG view was divided into three areas and the LVG view was divided into four areas. RESULTS: The paced QRS duration value was significantly longer in the right ventricular apex area compared with the outflow and inflow areas (160 ± 15 ms vs 140 ± 15 ms, P = 0.02, and vs 133 ± 17 ms, P < 0.001, respectively), but those values were not statistically significantly different between the right ventricular outflow and the right ventricular inflow areas (140 ± 15 ms vs 133 ± 17 ms, P = 0.187). When assessed with LVG views, there were the statistically significant differences in the paced QRS duration values in all areas except the apex area. (LV mid-anterior: 147 ± 11 ms vs LV base: 127 ± 13 ms, P < 0.001, and vs LV mid-septum: 129 ± 12 ms, P = 0.001, respectively.) CONCLUSIONS: Cardiac angiography showed that there was a relationship between the anatomical right ventricular pacing site and paced QRS duration. Cardiac angiography can help determine the areas that produce shorter paced QRS duration.

Calmodulin interacts with ATP binding cassette transporter A1 to protect from calpain-mediated degradation and upregulates high-density lipoprotein generation.

OBJECTIVE: To investigate the interaction of ATP-binding cassette transporter A1 (ABCA1) with calmodulin in relation to its calpain-mediated degradation because many calpain substrates bind calmodulin to regulate cellular functions. METHODS AND RESULTS: The activity of ABCA1 is regulated through proteolysis by calpain. An immunoprecipitation and glutathione S-transferase pull-down assay revealed that ABCA1 directly binds calmodulin in a Ca(2+)-dependent manner. The cytoplasmic loop of ABCA1 contains a typical calmodulin binding sequence of 1-5-8-14 motifs (1245 to 1257 amino acids). The peptide of this region showed binding to calmodulin, and deletion of the 1-5-8-14 motif abolished this interaction. This motif is located near the ABCA1 Pro-Glu-Ser-Thr sequence, and the presence of calmodulin/Ca(2+) protected the peptides from proteolysis by calpain. The knockdown of calmodulin by a specific small and interfering RNA increased the degradation of ABCA1 and decreased ABCA1 protein and apolipoprotein A-I-mediated lipid release. Surprisingly, calmodulin inhibitor W7 increased calmodulin binding to ABCA1 and protected it from calpain-mediated degradation, consistent with our previous finding that this compound increased apolipoprotein A-I-mediated cell cholesterol release. CONCLUSIONS: Calmodulin directly binds and stabilizes ABCA1 in the presence of Ca(2+) and increases the generation of high-density lipoprotein.

Helical apolipoproteins of high-density lipoprotein enhance phagocytosis by stabilizing ATP-binding cassette transporter A7.

We previously reported that the endogenous ATP-binding cassette transporter (ABC)A7 strongly associates with phagocytic function rather than biogenesis of high-density lipoprotein (HDL), being regulated by sterol-regulatory element binding protein (SREBP)2. Phagocytic activity was found enhanced by apolipoprotein (apo)A-I and apoA-II more than twice the maximum in J774 and mouse peritoneal macrophages. Therefore we investigated the molecular basis of this reaction in association with the function of ABCA7. Similar to ABCA1, ABCA7 was degraded, likely by calpain, and apoA-I and apoA-II stabilize ABCA7 against degradation. Cell surface biotinylation experiments demonstrated that endogenous ABCA7 predominantly resides on the cell surface and that the apolipoproteins increase the surface ABCA7. The increase of phagocytosis by apolipoproteins was retained in the J774 cells treated with ABCA1 siRNA and in the peritoneal macrophages from ABCA1-knockout mice, but it was abolished in the J774 cells treated with ABCA7 siRNA and in the peritoneal macrophages from ABCA7-knockout mice. Phagocytosis was decreased in the cells in the peritoneal cavity of the ABCA7-knockout mouse compared with the wild-type control. We thus concluded that extracellular helical apolipoproteins augment ABCA7-associated phagocytosis by stabilizing ABCA7. The results demonstrated direct enhancement of the host defense system by HDL components.

ATP-binding cassette transporter A1 gene transcription is downregulated by activator protein 2alpha. Doxazosin inhibits activator protein 2alpha and increases high-density lipoprotein biogenesis independent of alpha1-adrenoceptor blockade.

ATP-binding cassette transporter A1 (ABCA1) is a rate-limiting factor for high-density lipoprotein (HDL) biogenesis. The ABCA1 gene expression is known to be upregulated by various transcriptional factors. However, negative regulation factors would be better targets for pharmacological modulation of HDL biogenesis. Doxazosin, an alpha(1)-adrenoceptor blocker, increased ABCA1 mRNA, its protein, and apolipoprotein A-I-mediated HDL biogenesis in THP-1 macrophages and CHO-K1 cells, independent of alpha(1)-adrenoceptor blockade. Analysis of the human ABCA1 promoter indicated that the region between the positions -368 and -147 that contains an activator protein (AP)2-binding site responsible for the effects of doxazosin. Overexpression of AP2alpha inhibited ABCA1 transcription in a dose-dependent fashion. Mutation in the AP2-binding site caused increase of the basal promoter activity and cancelling both the transactivation by doxazosin and the trans-repression by AP2alpha. Doxazosin had no effect on ABCA1 mRNA level in HepG2 cells, which lack endogenous AP2alpha, and it reversed the inhibitory effect of AP2alpha expression in this type of cells. Chromatin immunoprecipitation and gel shift assays revealed that doxazosin reduced specific binding of AP2alpha to the ABCA1 promoter, as it suppressed phosphorylation of AP2alpha. Finally, doxazosin increased ABCA1 expression and plasma HDL in mice. We thus concluded that AP2alpha negatively regulates the ABCA1 gene transcription. Doxazosin inhibits AP2alpha activity independent of alpha(1)-adrenoceptor blockade and increases the ABCA1 expression and HDL biogenesis. AP2alpha is a potent pharmacological target for the increase of HDL.

Effect of lipid-lowering therapy with atorvastatin on atherosclerotic aortic plaques: a 2-year follow-up by noninvasive MRI.

BACKGROUND: Using MRI, we reported plaque regression in thoracic aorta and retardation of plaque progression in abdominal aorta by 1-year atorvastatin. However, association between serial plaque changes and LDL-cholesterol levels was not fully elucidated. DESIGN: A prospective, randomized, open-label trial. METHODS: We investigated the long-term effect of 20 versus 5-mg atorvastatin on thoracic and abdominal plaques and the association between plaque progression and on-treatment LDL-cholesterol levels in 36 hypercholesterolemic patients. MRI was performed at baseline and 1 and 2 years of treatment. Vessel wall area change was evaluated. RESULTS: The 20-mg dose markedly reduced LDL-cholesterol levels (-47%) versus 5-mg (-35%) dose. After 2 years of treatment, regression of thoracic plaques was found in the 20-mg group (-15% vessel wall area reduction), but not in the 5-mg group (+7%). Although the 20-mg dose induced plaque regression (-14%) from baseline to 1 year, no further regression was seen from 1 to 2 years of treatment (-1%). Regarding abdominal plaques, progression was found in the 5-mg group (+10%), but not in the 20-mg group (+2%). Plaque progression in the 5-mg group was found from baseline to 1 year (+8%), but not from 1 to 2 years (+2%). The degree of thoracic plaque regression correlated with LDL-cholesterol reduction (r = 0.61), whereas thoracic plaque change from 1 to 2 years correlated with on-treatment LDL-cholesterol levels (r = 0.64). CONCLUSION: Twenty milligrams of atorvastatin regressed thoracic plaques. However, maintaining low LDL-cholesterol levels was needed to prevent plaque progression. In abdominal aorta, only retardation of plaque progression was found after 2 years of 20-mg treatment.

Synthesis of three deoxy-sophorose derivatives for evaluating the requirement of hydroxy groups at position 3 and/or 3' of sophorose by 1,2-ß-oligoglucan phosphorylases.

Sophorose (Sop2) is known as a powerful inducer of cellulases in Trichoderma reesei, and in recent years 1,2-ß-D-oligoglucan phosphorylase (SOGP) has been found to use Sop2 in synthetic reactions. From the structure of the complex of SOGP with Sop2, it was predicted that both the 3-hydroxy group at the reducing end glucose moiety of Sop2 and the 3'-hydroxy group at the non-reducing end glucose moiety of Sop2 were important for substrate recognition. In this study, three kinds of 3- and/or 3'-deoxy-Sop2 derivatives were synthesized to evaluate this mechanism. The deoxygenation of the 3-hydroxy group of D-glucopyranose derivative was performed by radical reduction using a toluoyl group as a leaving group. The utilization of a toluoyl group that plays two roles (a leaving group for the deoxygenation and a protecting group for a hydroxy group) resulted in efficient syntheses of the three target compounds. The NMR spectra of the two final compounds (3-deoxy- and 3,3'-dideoxy-Sop2) suggested that the glucose moiety of the reducing end of Sop2 can easily take on a furanose structure (five-membered ring structure) by deoxygenation of the 3-hydroxy group of Sop2. In addition, the ratio of the five- and six-membered ring structures changed depending on the temperature. The SOGPs exhibited remarkably lower specific activity for 3'-deoxy- and 3,3'-dideoxy-Sop2, indicating that the 3'-hydroxy group of Sop2 is important for substrate recognition by SOGPs.

Telmisartan enhances cholesterol efflux from THP-1 macrophages by activating PPARgamma.

AIM: The ATP binding cassette transporters A1 and G1 (ABCA1/G1) and scavenger receptor class B type I (SR-BI) are key molecules in cholesterol efflux and atherogenesis. These genes are regulated by peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor (LXR). Telmisartan is an angiotensin type 1 receptor blocker which has been reported to act as a ligand for PPARgamma. We investigated whether PPARgamma-activating ARBs affect the expression of these genes and cholesterol efflux from macrophages. METHODS AND RESULTS: Telmisartan increased ABCA1, ABCG1 and SR-BI mRNA levels in THP-1 macrophages in a dose- and time-dependent fashion. It also increased their protein levels and enhanced apoA-I- and HDL-mediated cholesterol efflux from macrophages. The knockdown of PPARgamma by siRNA abolished the telmisartan-induced expression of these genes. The knockdown of LXRalpha resulted in the complete and partial abolishment of telmisartan-induced ABCA1 and ABCG1 expression, respectively. We also demonstrated that telmisartan-induced SR-BI expression was dependent on the PPARgamma pathway but not on the LXRalpha pathway. A luciferase assay using an ABCA1 promoter construct showed that telmisartan activated ABCA1 transcription, which was abolished if the LXR binding element was mutated, indicating that increased ABCA1 transcription by telmisartan is LXR-dependent. CONCLUSION: Our results showed that telmisartan enhanced both apoA-I- and HDL-mediated cholesterol efflux from macrophages by increasing ABCA1, ABCG1 and SR-BI expression via PPARgamma-dependent and LXR-dependent/independent pathways.