ABSTRACT
We attempted to develop an efficient method for producing isomaltose, a disaccharide consisting of an α-(1â6)-linkage, from starch by combining enzymes of known activity. We found that the combination of 1,4-α-glucan 6-α-glucosyltransferase from Bacillus globisporus N75 and isopullulanase from Aspergillus brasiliensis ATCC 9642 led to the efficient synthesis of isomaltose. Inclusion of isoamylase and cyclomaltodextrin glucanotransferase resulted in increased efficiency, with production yields exceeding 70%. Furthermore, we considered that isomaltooligosaccharides could be synthesized from starch by combining 1,4-α-glucan 6-α-glucosyltransferase from Paenibacillus sp. PP710 and isopullulanase. In reactions that additionally utilized isoamylase and α-amylase, the total concentration of product, which included a series of isomaltooligosaccharides from isomaltose to isomaltodecaose, was 131 m m, and the ratio of 6-linked glucopyranosyl bonds to all bonds was 91.7% at a substrate concentration of 10%. The development of these manufacturing methods will accelerate the industrial production of isomaltose and isomaltooligosaccharides.
Subject(s)
IsomaltoseABSTRACT
We performed whole genome sequence analyses of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006 that secrete enzymes to produce cyclo-{â6)-α-d-Glcp-(1â6)-α-d-Glcp-(1â6)-α-d-Glcp-(1â6)-α-d-Glcp-(1â} (CI4) from dextran. Full-length amino acid sequences of CI4-forming enzymes were identified by matching known N-terminal amino acid sequences with products of the draft genome. Domain searches revealed that the CI4-forming enzymes are composed of Glycoside Hydrolase family 66 (GH66) domain, Carbohydrate Binding Module family 35 (CBM35) domain, and CBM13 domain, categorizing the CI4-forming enzymes in the GH66. Furthermore, the amino acid sequences of the two CI4-forming enzymes were 71% similar to each other and up to 51% similar to cycloisomaltooligosaccharide glucanotransferases (CITases) categorized in GH66. Differences in sequence between the CI4-forming enzymes and the CITases suggest mechanisms to produce specific cycloisomaltooligosaccharides, and whole genome sequence analyses identified a gene cluster whose gene products likely work in concert with the CI4-forming enzymes.
Subject(s)
MicrobacteriumABSTRACT
Glucanotransferases that can synthesize cyclo-{â6)-α-d-Glcp-(1â6)-α-d-Glcp-(1â6)-α-d-Glcp-(1â6)-α-d-Glcp-(1â} (CI4) from dextran were purified to homogeneity from the culture supernatant of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006. The molecular mass of both enzymes was estimated to be 86 kDa by SDS-PAGE. The glucanotransferase, named CI4-forming enzyme, from Agreia sp. exhibited the highest activity at pH 6.0 and 40 °C. The enzyme was stable on the pH range of 4.6-9.9 and up to 40 °C. On the other hand, the enzyme from M. trichothecenolyticum exhibited the highest activity at pH 5.7 and 40 °C. The enzyme was stable on the pH range of 5.0-6.9 and up to 35 °C. Both enzymes catalyzed 4 reactions, namely, intramolecular α-1,6-transglycosylation (cyclization), intermolecular α-1,6-transglycosylation, hydrolysis of CI4, and coupling reaction. Furthermore, the CI4-forming enzyme produced CI4 from α-1,6-linked glucan synthesized from starch by 6-α-glucosyltransferase. These findings will enable the production of CI4 from starch.
Subject(s)
Actinobacteria/enzymology , Glycogen Debranching Enzyme System/isolation & purification , Oligosaccharides/chemistry , Culture Media , Cyclization , Electrophoresis, Polyacrylamide Gel , Glycogen Debranching Enzyme System/chemistry , Glycosylation , Hot Temperature , Hydrogen-Ion Concentration , Microbacterium/enzymology , Molecular WeightABSTRACT
Cyclic α-maltosyl-(1â6)-maltose (CMM, cyclo-{â6)-α-d-Glcp-(1â4)-α-d-Glcp-(1â6)-α-d-Glcp-(1â4)-α-d-Glcp-(1â})is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. CMM is composed of two maltose units and is one of the smallest cyclic glucooligosaccharides. Although CMM is resistant to usual amylases, it is efficiently hydrolyzed by CMM hydrolase (CMMase), belonging to subfamily 20 of glycoside hydrolase family 13 (GH13_20). Here, we determined the ligand-free crystal structure of CMMase from the soil-associated bacterium Arthrobacter globiformis and its structures in complex with maltose, panose, and CMM to elucidate the structural basis of substrate recognition by CMMase. The structures disclosed that although the monomer structure consists of three domains commonly adopted by GH13 and other α-amylase-related enzymes, CMMase forms a unique wing-like dimer structure. The complex structure with CMM revealed four specific subsites, namely -3', -2, -1, and +1'. We also observed that the bound CMM molecule adopts a low-energy conformer compared with the X-ray structure of a single CMM crystal, also determined here. Comparison of the CMMase active site with those in other enzymes of the GH13_20 family revealed that three regions forming the wall of the cleft, denoted PYF (Pro-203/Tyr-204/Phe-205), CS (Cys-163/Ser-164), and Y (Tyr-168), are present only in CMMase and are involved in CMM recognition. Combinations of multiple substitutions in these regions markedly decreased the activity toward CMM, indicating that the specificity for this cyclic tetrasaccharide is supported by the entire shape of the pocket. In summary, our work uncovers the mechanistic basis for the highly specific interactions of CMMase with its substrate CMM.
Subject(s)
Arthrobacter/enzymology , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/metabolism , Macrocyclic Compounds/metabolism , Oligosaccharides/metabolism , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Hydrolysis , Macrocyclic Compounds/chemistry , Models, Molecular , Oligosaccharides/chemistry , Protein Conformation , Sequence HomologyABSTRACT
ß1-adrenergic receptor (Adrb1) belongs to the superfamily of G-protein-coupled receptors (GPCRs) and plays a critical role in the regulation of heart rate and myocardial contraction force. GPCRs are phosphorylated at multiple sites to regulate distinct signal transduction pathways in different tissues. However, little is known about the location and function of distinct phosphorylation sites of Adrb1 in vivo. To clarify the mechanisms underlying functional regulation associated with Adrb1 phosphorylation in vivo, we aimed to identify Adrb1 phosphorylation sites in the mouse heart using phosphoproteomics techniques with nano-flow liquid chromatography/tandem mass spectrometry (LC-MS/MS). We revealed the phosphorylation residues of Adrb1 to be Ser274 and Ser280 in the third intracellular loop and Ser412, Ser417, Ser450, Ser451, and Ser462 at the C-terminus. We also found that phosphorylation at Ser274, Ser280, and Ser462 was enhanced in response to stimulation with an Adrb1 agonist. This is the first study to identify Adrb1 phosphorylation sites in vivo. These findings will provide novel insights into the regulatory mechanisms mediated by Adrb1 phosphorylation.
Subject(s)
Myocardium/chemistry , Myocardium/metabolism , Receptors, Adrenergic, beta-1/chemistry , Receptors, Adrenergic, beta-1/metabolism , Animals , Chromatography, Liquid , Heart , Male , Mice , Mice, Inbred C57BL , Phosphorylation , Proteomics , Signal Transduction , Tandem Mass SpectrometryABSTRACT
Our study measured circulating microRNA (miRNA) levels in the plasma of calsequestrin (CSQ)-tg mouse, a severe heart failure model, and evaluated whether treatment with angiotensin II type 1 receptor blocker, azilsartan medoxomil (AZL-M) influenced their levels using miRNA array analysis. MiR-146a, miR-149, miR-150, and miR-342-3p were reproducibly reduced in the plasma of CSQ-tg mice. Among them, miR-146a and miR-342-3p were significantly restored by AZL-M, which were associated with improvement of survival rate and reduction of congestion. These results suggest that miRNA, especially miR-146a and miR-342-3p, could be used as potential biomarkers for evaluating the efficacy of anti-heart failure drugs.
Subject(s)
Benzimidazoles/pharmacology , Heart Failure/drug therapy , MicroRNAs/blood , Oxadiazoles/pharmacology , Angiotensin II Type 1 Receptor Blockers/pharmacology , Angiotensin II Type 1 Receptor Blockers/therapeutic use , Animals , Benzimidazoles/therapeutic use , Calsequestrin , Cardiomyopathy, Dilated/drug therapy , Disease Models, Animal , Heart Failure/genetics , Mice , Oxadiazoles/therapeutic use , Survival RateABSTRACT
Cyclic nigerosylnigerose (CNN), a saucer-shaped cyclic tetrasaccharide with a shallow concave surface, was reacted with pyromellitic dianhydride in 1:2 and 1:4 ratios to give two CNN-based polymers of different degrees of crosslinking, both of which swelled upon soaking in water, acting as a 'nanosponge' (NS). These NSs evolved several phases from isotropic solution to flowing and rigid gels via suspension by gradually increasing the concentration in water. The CNN-NSs thus prepared effectively mediated the enantiodifferentiating photoisomerization of (Z)-cyclooctene (1Z) to chiral (E)-isomer (1E). The enantiomeric excess (ee) of 1E obtained was a critical function of the solvent composition and the phase evolved at different CNN-NS concentrations in water. In isotropic solution, the enantioselectivity was generally low (−4% to +6% ee) but the chiral sense of 1E was inverted by increasing the methanol content. Interestingly, the product's ee was controlled more dramatically by the phase evolved, as was the case with the cyclodextrin-based nanosponge (CD-NS) reported previously. Thus, the ee of 1E was low in solution and suspension, but suddenly leaped at the phase border of flowing gel and rigid gel to give the highest ee of 2224%, which are much higher than those obtained with CD-NSs (612% ee), revealing the positive roles of the chiral void space formed upon gelation of the crosslinked saccharide polymer.
Subject(s)
Benzoates/chemistry , Cyclodextrins/chemistry , Glucans/chemistry , Nanostructures/chemistry , Photochemistry/methods , Solvents/chemistry , Circular Dichroism , Cross-Linking Reagents/chemistry , Gels , Methanol/chemistry , Molecular Conformation , Nanotechnology/methods , Photosensitizing Agents/chemistry , Polymers/chemistry , Polysaccharides/chemistry , Spectrophotometry, Ultraviolet , Stereoisomerism , Water/chemistryABSTRACT
Current differentiation protocols for human induced pluripotent stem cells (hiPSCs) produce heterogeneous cardiomyocytes (CMs). Although chamber-specific CM selection using cell surface antigens enhances biomedical applications, a cell surface marker that accurately distinguishes between hiPSC-derived atrial CMs (ACMs) and ventricular CMs (VCMs) has not yet been identified. We have developed an approach for obtaining functional hiPSC-ACMs and -VCMs based on CD151 expression. For ACM differentiation, we found that ACMs are enriched in the CD151low population and that CD151 expression is correlated with the expression of Notch4 and its ligands. Furthermore, Notch signaling inhibition followed by selecting the CD151low population during atrial differentiation leads to the highly efficient generation of ACMs as evidenced by gene expression and electrophysiology. In contrast, for VCM differentiation, VCMs exhibiting a ventricular-related gene signature and uniform action potentials are enriched in the CD151high population. Our findings enable the production of high-quality ACMs and VCMs appropriate for hiPSC-derived chamber-specific disease models and other applications.
Subject(s)
Induced Pluripotent Stem Cells , Humans , Cell Differentiation/physiology , Heart Ventricles , Myocytes, Cardiac/metabolism , Tetraspanin 24/genetics , Tetraspanin 24/metabolismABSTRACT
From a complex mixture of mono- and di-2-anthracenecarboxylic acid (AC) esters of cyclic nigerosylnigerose (CNN), two monoesters (2(B) and 6(A)) and four diesters in which AC was introduced on the transannular B/D (2(B)2(D)), adjacent A/B and A/D (6(A)2(B) and 6(A)2(D)), and same B/B (2(B)3(B)) nigerose rings were isolated. Possessing two ACs at distant positions, 2(B)2(D) and 6(A)2(D) showed negative Cotton effects for the (1)Bb band, the intensities of which were stronger than that of 6(A). 2(B)2(D) and 6(A)2(D) slowly photocyclodimerized to give HH dimers 3* and 4 with 57% and 81% HH selectivity, respectively, which were appreciably higher than that for 6(A) (34%), while the enantiomeric excesses (ee's) of anti-HH dimer 3* were 2% and -18%, respectively. In contrast, 6(A)2(B) and 2(B)3(B) carrying two ACs on adjacent A and B rings or at vicinal positions on the B ring, respectively, exhibited strong positive CD couplets, the amplitudes of which amounted to 97 and 409 M(-1) cm(-1), respectively. Upon irradiation, 6(A)2(B) afforded 3* with -62% ee and 4 in 96% combined yield, whereas 2(B)3(B) gave almost exclusively 3* with -99% ee in 96% yield, likely as a result of the introduction of two ACs at the vicinal positions of the rigid CNN scaffold.
ABSTRACT
Engineered cardiac tissue (ECT) using human induced pluripotent stem cell-derived cardiomyocytes is a promising tool for modeling heart disease. However, tissue immaturity makes robust disease modeling difficult. Here, we established a method for modeling hypertrophic cardiomyopathy (HCM) malignant (MYH7 R719Q) and nonmalignant (MYBPC3 G115∗) pathogenic sarcomere gene mutations by accelerating ECT maturation using an ERRγ agonist, T112, and mechanical stretching. ECTs treated with T112 under 10% elongation stimulation exhibited more organized and mature characteristics. Whereas matured ECTs with the MYH7 R719Q mutation showed broad HCM phenotypes, including hypertrophy, hypercontraction, diastolic dysfunction, myofibril misalignment, fibrotic change, and glycolytic activation, matured MYBPC3 G115∗ ECTs displayed limited phenotypes, which were primarily observed only under our new maturation protocol (i.e., hypertrophy). Altogether, ERRγ activation combined with mechanical stimulation enhanced ECT maturation, leading to a more accurate manifestation of HCM phenotypes, including non-cardiomyocyte activation, consistent with clinical observations.
Subject(s)
Cardiomyopathy, Hypertrophic , Induced Pluripotent Stem Cells , Humans , Tissue Engineering , Carrier Proteins/genetics , Induced Pluripotent Stem Cells/pathology , Cardiomyopathy, Hypertrophic/pathology , Phenotype , Myocytes, Cardiac/physiology , Mutation , Hypertrophy/pathologyABSTRACT
Highly branched α-glucan molecules exhibit low digestibility for α-amylase and glucoamylase, and abundant in α-(1â3)-, α-(1â6)-glucosidic linkages and α-(1â6)-linked branch points where another glucosyl chain is initiated through an α-(1â3)-linkage. From a culture supernatant of Paenibacillus sp. PP710, we purified α-glucosidase (AGL) and α-amylase (AMY), which were involved in the production of highly branched α-glucan from maltodextrin. AGL catalyzed the transglucosylation reaction of a glucosyl residue to a nonreducing-end glucosyl residue by α-1,6-, α-1,4-, and α-1,3-linkages. AMY catalyzed the hydrolysis of the α-1,4-linkage and the intermolecular or intramolecular transfer of maltooligosaccharide like cyclodextrin glucanotransferase (CGTase). It also catalyzed the transfer of an α-1,4-glucosyl chain to a C3- or C4-hydroxyl group in the α-1,4- or α-1,6-linked nonreducing-end residue or the α-1,6-linked residue located in the other chains. Hence AMY was regarded as a novel enzyme. We think that the mechanism of formation of highly branched α-glucan from maltodextrin is as follows: α-1,6- and α-1,3-linked residues are generated by the transglucosylation of AGL at the nonreducing ends of glucosyl chains. Then AMY catalyzes the transfer of α-1,4-chains to C3- or C4-hydroxyl groups in the α-1,4- or α-1,6-linked residues generated by AGL. Thus the concerted reactions of both AGL and AMY are necessary to produce the highly branched α-glucan from maltodextrin.
Subject(s)
Glucans/biosynthesis , Oligosaccharides, Branched-Chain/biosynthesis , Paenibacillus/enzymology , Polysaccharides/metabolism , alpha-Amylases/isolation & purification , alpha-Glucosidases/isolation & purification , Biocatalysis , Carbohydrate Sequence , Chromatography, Gel , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Glycosylation , Oligosaccharides/metabolism , Oxidation-Reduction , Paenibacillus/chemistry , alpha-Amylases/metabolism , alpha-Glucosidases/metabolismABSTRACT
One kojibiose phoshorylase (KP) homolog gene was cloned from Caldicellulosiruptor saccharolyticus ATCC43494. Recombinant KP from C. saccharolyticus (Cs-KP) expressed in Escherichia coli showed highest activity at pH 6.0 at 85 °C, and was stable from pH 3.5 to 10.0 and up to 85 °C for phosphorolysis. Cs-KP showed higher productivity of kojioligosaccharides of DP ⧠4 than KP from Thermoanaerobacter brockii ATCC35047.
Subject(s)
Bacterial Proteins/metabolism , Disaccharides/metabolism , Phosphorylases/metabolism , Recombinant Proteins/metabolism , Thermoanaerobacterium/enzymology , Bacterial Proteins/genetics , Cloning, Molecular , Escherichia coli , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Phosphorylases/genetics , Plasmids , Recombinant Proteins/genetics , Substrate Specificity , Thermoanaerobacterium/chemistry , Transformation, BacterialABSTRACT
Engineered cardiac tissue (ECT) derived from human induced pluripotent stem cells (iPSCs) can replicate human heart in vitro and be applied to drug discovery and heart disease models. The contraction force of ECT is an important indicator of its function and of the disease phenotype. Here we describe a construction method of ECT using the Flexcell® Tissue Train® culture system and a contraction force measurement method based on the Frank-Starling law.
Subject(s)
Induced Pluripotent Stem Cells/cytology , Myocardial Contraction/physiology , Myocytes, Cardiac/cytology , Tissue Engineering/methods , Cells, Cultured , HumansABSTRACT
One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1EmGFP and TNNI3mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.
Subject(s)
Induced Pluripotent Stem Cells/cytology , Myocytes, Cardiac/cytology , Receptors, Estrogen/physiology , Cell Differentiation/drug effects , Cell Differentiation/genetics , Gene Expression Regulation/drug effects , Genes, Reporter , Humans , Induced Pluripotent Stem Cells/metabolism , Models, Biological , Myocytes, Cardiac/metabolism , Receptors, Estrogen/chemistry , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Sarcolemma/drug effects , Sarcolemma/metabolism , Sarcomeres/drug effects , Sarcomeres/metabolism , Transcriptome/drug effects , Troponin I/genetics , Troponin I/metabolismABSTRACT
OBJECTIVE: Fish oil (FO), and specifically omega 3 fatty acids, has favorable effects on cardiovascular outcomes. The aim of this study was to investigate the effects of FO on the process of macrophage reverse cholesterol transport (RCT) in an in vivo mouse model. METHODS AND RESULTS: C57BL/6J mice were fed a FO diet, whereas control mice were fed diets containing alternative sources of fats, soybean oil (SO), and coconut oil (CO) for 4 weeks. Macrophage RCT was assessed by injecting [(3)H]cholesterol-labeled J774 macrophages intraperitoneally into mice. After 48 hours, tissues were harvested and feces were collected. An increase in the excretion of macrophage-derived [(3)H]-tracer recovered in fecal neutral sterols for FO-fed mice was observed (273% versus SO and 182% versus CO). FO also decreased [(3)H]-tracer in hepatic cholesteryl ester compared to SO and CO by 76% and 56%, respectively. To specifically determine the effect of FO on the fate of HDL-derived cholesterol, mice fed FO or SO diets were injected with HDL labeled with [(3)H]cholesteryl oleate, and the disappearance of [(3)H]-tracer from blood and its excretion in feces was measured. There was no significant difference in the fractional catabolic rate of [(3)H]cholesteryl oleate-HDL between the 2 groups. However, there was a 242% increase in the excretion of HDL-derived [(3)H]-tracer recovered in fecal neutral sterols in FO-fed mice, concordant with significantly increased expression of hepatic Abcg5 and Abcg8 mRNA. CONCLUSIONS: As measured by this tracer-based assay, FO promoted reverse cholesterol transport, primarily by enhancement of the hepatic excretion of macrophage-derived and HDL-derived cholesterol.
Subject(s)
Cholesterol/metabolism , Fish Oils/pharmacology , Macrophages/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 5 , ATP Binding Cassette Transporter, Subfamily G, Member 8 , ATP-Binding Cassette Transporters/physiology , Animals , Biological Transport , Cells, Cultured , Cholesterol Esters/metabolism , Female , Lipids/blood , Lipoproteins/physiology , Lipoproteins, HDL/metabolism , Membrane Transport Proteins/physiology , Mice , Mice, Inbred C57BLABSTRACT
Cyclic α-maltosyl-(1â6)-maltose (CMM) is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. Here, we report functional and structural analyses on CMM-binding protein (CMMBP), which is a substrate-binding protein (SBP) of an ABC importer system of the bacteria Arthrobacter globiformis. Isothermal titration calorimetry analysis revealed that CMMBP specifically bound to CMM with a Kd value of 9.6 nM. The crystal structure of CMMBP was determined at a resolution of 1.47 Å, and a panose molecule was bound in a cleft between two domains. To delineate its structural features, the crystal structure of CMMBP was compared with other SBPs specific for carbohydrates, such as cyclic α-nigerosyl-(1â6)-nigerose and cyclodextrins. These results indicate that A. globiformis has a unique metabolic pathway specialized for CMM.
Subject(s)
Arthrobacter/metabolism , Maltose-Binding Proteins/chemistry , Maltose-Binding Proteins/metabolism , Calorimetry , Crystallography, X-Ray , Cyclodextrins/metabolism , Disaccharides/metabolism , Metabolic Networks and Pathways , Models, Molecular , Protein Conformation , Protein DomainsABSTRACT
Renin is the rate-limiting enzyme of the renin-angiotensin system cascade, which drives the pathophysiological progression of heart failure. Species differences in the amino acid sequence of the catalytic domain of renin limit evaluations of the potency and efficacy of human renin inhibitors in animal models, and a high dose of inhibitors is usually needed to show its organ-protective effects in rodents. In the present study, we developed a novel murine heart failure model (triple-tg) to enable us to evaluate the cardioprotective effect of renin inhibitors at more relevant doses for humans, by cross-breeding calsequestrin transgenic (CSQ-tg) mice with human renin and human angiotensinogen double-transgenic mice. The triple-tg mice exhibited increased plasma renin activity, worsened cardiac hypertrophy, and higher mortality compared to CSQ-tg mice. Triple-tg mice treated with 10 mg·kg-1 of TAK-272 (imarikiren/SCO-272), an orally active direct renin inhibitor, exhibited improvements in heart failure phenotypes, such as cardiac hypertrophy and survival rate; however, a dose of 300 mg·kg-1 was required to improve symptoms in CSQ-tg mice. Our results suggest that this newly generated triple-tg heart failure model is useful to evaluate the cardioprotective effects of human renin inhibitors at clinically relevant doses, thereby minimizing the concerns of off-target effects related to much higher drug exposure than that achieved in clinical study.
Subject(s)
Angiotensinogen/metabolism , Heart Failure/physiopathology , Renin/metabolism , Angiotensinogen/genetics , Angiotensinogen/pharmacology , Animals , Antihypertensive Agents/pharmacology , Benzimidazoles/pharmacology , Blood Pressure/drug effects , Blood Pressure/genetics , Calsequestrin/pharmacology , Disease Models, Animal , Female , Gene Expression/genetics , Gene Expression Regulation/genetics , Heart Failure/drug therapy , Heart Failure/metabolism , Humans , Male , Mice , Mice, Inbred DBA , Mice, Transgenic , Morpholines/pharmacology , Piperidines/pharmacology , Renin/genetics , Renin-Angiotensin System/drug effects , Renin-Angiotensin System/geneticsABSTRACT
Two bacterial strains isolated from soil, namely Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006, were found to produce a novel oligosaccharide. The oligosaccharide was enzymatically produced from dextran using the culture supernatant of Agreia sp. D1110 or M. trichothecenolyticum D2006. LC-MS and NMR analysis identified the novel oligosaccharide as cyclo-{â6)-α-d-Glcp-(1â6)-α-d-Glcp-(1â6)-α-d-Glcp-(1â6)-α-d-Glcp-(1â}, which was named cycloisomaltotetraose, and abbreviated as CI4. CI4 was subsequently crystalized and its X-ray crystallographic structure was determined. CI4 crystals were shown to be pentahydrate, with the CI4 molecules in the crystal structure displaying a unique 3D structure, in which two glucosyl residues in the molecule were facing each other. This unique 3D structure was quite different from the 3D structure of known cyclic tetrasaccharides. This is the first report of CI4 molecules and their unique crystal structure.
Subject(s)
Dextrans/metabolism , Oligosaccharides/biosynthesis , Oligosaccharides/chemistry , Actinobacteria/enzymology , Actinobacteria/metabolism , Carbohydrate Conformation , Carbohydrate Sequence , Crystallography, X-Ray , Microbacterium/enzymology , Microbacterium/metabolism , Models, MolecularABSTRACT
Cyclic maltosyl-maltose [CMM, cyclo-[-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]], a novel cyclic tetrasaccharide, has a unique structure. Its four glucose residues are joined by alternate alpha-1,4 and alpha-1,6 linkages. CMM is synthesized from starch by the action of 6-alpha-maltosyltransferase from Arthrobacter globiformis M6. Recently, we determined the mechanism of extracellular synthesis of CMM, but the degrading pathway of the saccharide remains unknown. Hence we tried to identify the enzymes involved in the degradation of CMM to glucose from the cell-free extract of the strain, and identified CMM hydrolase (CMMase) and alpha-glucosidase as the responsible enzymes. The molecular mass of CMMase was determined to be 48.6 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and 136 kDa by gel filtration column chromatography. The optimal pH and temperature for CMMase activity were 6.5 and 30 degrees C. The enzyme remained stable from pH 5.5 to 8.0 and up to 25 degrees C. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates, but it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the alpha-1,6-linkage of CMM. The molecular mass of alpha-glucosidase was determined to be 60.1 kDa by SDS-PAGE and 69.5 kDa by gel filtration column chromatography. The optimal pH and temperature for alpha-glucosidase activity were 7.0 and 35 degrees C. The enzyme remained stable from pH 7.0 to 9.5 and up to 35 degrees C. alpha-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates, but it did not degrade CMM. Furthermore, when CMMase and alpha-glucosidase existed simultaneously in a reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by the synergistic action of CMMase and alpha-glucosidase.
Subject(s)
Arthrobacter/enzymology , Glycoside Hydrolases , Macrocyclic Compounds/metabolism , Oligosaccharides/metabolism , alpha-Glucosidases , Glucose , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/isolation & purification , Glycoside Hydrolases/metabolism , Hydrogen-Ion Concentration , Molecular Weight , Temperature , alpha-Glucosidases/chemistry , alpha-Glucosidases/isolation & purification , alpha-Glucosidases/metabolismABSTRACT
The renin-angiotensin system (RAS), which plays an important role in the progression of heart failure, is efficiently blocked by the inhibition of renin, the rate-limiting enzyme in the RAS cascade. In the present study, we investigated the cardioprotective effects of TAK-272 (SCO-272, imarikiren), a novel, orally effective direct renin inhibitor (DRI), and compared its efficacy with that of aliskiren, a DRI that is already available in the market. TAK-272 was administered to calsequestrin transgenic (CSQ-tg) heart failure mouse model that show severe symptoms and high mortality. The CSQ-tg mice treated with 300 mg/kg, the highest dose tested, of TAK-272 showed significantly reduced plasma renin activity (PRA), cardiac hypertrophy, and lung congestion. Further, TAK-272 reduced cardiomyocyte injury accompanied by an attenuation of the increase in NADPH oxidase 4 and nitric oxide synthase 3 expressions. TAK-272 also prolonged the survival of CSQ-tg mice in a dose-dependent manner (30 mg/kg: P = 0.42, 100 mg/kg: P = 0.12, 300 mg/kg: P < 0.01). Additionally, when compared at the same dose level (300 mg/kg), TAK-272 showed strong and sustained PRA inhibition and reduced the heart weight and plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) concentration, a heart failure biomarker, while aliskiren showed a significant weaker PRA inhibition and failed to demonstrate any cardioprotective effects. Our results showed that TAK-272 is an orally active and persistent renin inhibitor, which reduced the mortality of CSQ-tg mice and conferred protection against cardiac hypertrophy and injury. Thus, TAK-272 treatment could provide a new therapeutic approach for heart failure.