24-Hour Glycemic Variations in Drug-Naïve Patients with Type 2 Diabetes: A Continuous Glucose Monitoring (CGM)-Based Study

OBJECTIVE: To investigate a 24-hour glycemic variation in drug-naïve, type 2 diabetic patients by using CGM. METHODS: A total of 30 inpatients with type 2 diabetes were included in the study to analyze the 24-hour CGM data. RESULTS: The patients' median age was 58 years old (interquartile range: 42-66 years), and their median HbA1c value was 7.6 (6.7-8.8)%. The median time to postprandial peak glucose levels(Peak Time) for each meal was 70-85 minutes, with the range of postprandial glucose increases(Increase Range) for each meal being 83-109 mg/dL. There was a significant positive correlation between the HbA1c values and Increases Range, Peak Time observed after breakfast and dinner, respectively. When the patients were stratified by a median HbA1c value of 7.6% into 2 groups, Increases Range and Peak Time, after breakfast, were shown to be significantly higher in the high-HbA1c group (H) than in the low-HbA1c (L) group. When the subjects were divided into four groups according to HbA1c levels:1 (<7.0%, n = 8), 2 (7.0-7.9%, n = 8), 3 (8.0-8.9%, n = 8), and 4 (≥9%, n = 6), the average glucose level, pre-meal glucose level and postprandial peak glucose level increased steadily from group 1 to 4 in a stepwise manner. CONCLUSIONS: In drug-naïve, Japanese type 2 diabetic patients, the Peak Time and the Increase Range were maximal after dinner. It was shown that the greater the HbA1c values, the longer Peak time and the higher Increase Range after breakfast and dinner. The average glucose level, pre meal glucose level and postprandial peak glucose level increased steadily as HbA1c level increased

Photoreduction and Reoxidation of the Three Iron-Sulfur Clusters of Reaction Centers of Green Sulfur Bacteria

AbstractIron-sulfur clusters are the terminal electron acceptors of the photosynthetic reaction centers of green sulfur bacteria and photosystem I. We have studied electron-transfer reactions involving these clusters in the green sulfur bacterium Chlorobium tepidum, using flash-absorption spectroscopic measurements. We show for the first time that three different clusters, named FX, F1, and F2, can be photoreduced at room temperature during a series of consecutive flashes. The rates of electron escape to exogenous acceptors depend strongly upon the number of reduced clusters. When two or three clusters are reduced, the escape is biphasic, with the fastest phase being 12–14-fold faster than the slowest phase, which is similar to that observed after single reduction. This is explained by assuming that escape involves mostly the second reducible cluster. Evidence is thus provided for a functional asymmetry between the two terminal acceptors F1 and F2. From multiple-flash experiments, it was possible to derive the intrinsic recombination rates between P840+ and reduced iron-sulfur clusters: values of 7, 14, and 59s−1 were found after one, two and three electron reduction of the clusters, respectively. The implications of our results for the relative redox potentials of the three clusters are discussed

Purification and characterization of ferredoxin–NAD(P)+ reductase from the green sulfur bacterium Chlorobium tepidum

Ferredoxin–NAD(P)+ reductase [EC 1.18.1.3, 1.18.1.2] was isolated from the green sulfur bacterium Chlorobium tepidum and purified to homogeneity. The molecular mass of the subunit is 42 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the native enzyme is approximately 90 kDa, estimated by gel-permeation chromatography, and is thus a homodimer. The enzyme contains one FAD per subunit and has absorption maxima at about 272, 385, and 466 nm. In the presence of ferredoxin (Fd) and reaction center (RC) complex from C. tepidum, it efficiently catalyzes photoreduction of both NADP+ and NAD+. When concentrations of NADP+ exceeded 10 μM, NADP+ photoreduction rates decreased with increased concentration. The inhibition by high concentrations of substrate was not observed with NAD+. It also reduces 2,6-dichlorophenol-indophenol (DPIP) and molecular oxygen with either NADPH or NADH as efficient electron donors. It showed NADPH diaphorase activity about two times higher than NADH diaphorase activity in DPIP reduction assays at NAD(P)H concentrations less than 0.1 mM. At 0.5 mM NAD(P)H, the two activities were about the same, and at 1 mM, the former activity was slightly lower than the latter

Crystallization and preliminary X-ray studies of ferredoxin-NADP + oxidoreductase encoded by Bacillus subtilis yumC

金沢大学理工研究域物質化学系Ferredoxin-NADP+ oxidoreductase encoded by Bacillus subtilis yumC has been purified and successfully crystallized in complex with NADP + in two forms. Diffraction data from crystals of these two forms were collected at resolutions of 1.8 and 1.9 Å. The former belonged to space group P21212, with unit-cell parameters a = 63.90, b = 135.72, c = 39.19 Å, and the latter to space group C2, with unit-cell parameters a = 207.47, b = 64.85, c = 61.12 Å, Β = 105.82°. The initial structure was determined by the molecular-replacement method using a thioredoxin reductase-like protein as a search model. © 2010 International Union of Crystallography All rights reserved

Kinetics of NADP+/NADPH reduction–oxidation catalyzed by the ferredoxin-NAD(P)+ reductase from the green sulfur bacterium Chlorobaculum tepidum

Ferredoxin-NAD(P)+ oxidoreductase (FNR, [EC 1.18.1.2], [EC 1.18.1.3]) from the green sulfur bacterium Chlorobaculum tepidum (CtFNR) is a homodimeric flavoprotein with significant structural homology to bacterial NADPH-thioredoxin reductases. CtFNR homologs have been found in many bacteria, but only in green sulfur bacteria among photoautotrophs. In this work, we examined the reactions of CtFNR with NADP+, NADPH, and (4S-2H)-NADPD by stopped-flow spectrophotometry. Mixing CtFNRox with NADPH yielded a rapid decrease of the absorbance in flavin band I centered at 460 nm within 1 ms, and then the absorbance further decreased gradually. The magnitude of the decrease increased with increasing NADPH concentration, but even with ~50-fold molar excess NADPH, the absorbance change was only ~45 % of that expected for fully reduced protein. The absorbance in the charge transfer (CT) band centered around 600 nm increased rapidly within 1 ms, then slowly decreased to about 70 % of the maximum. When CtFNRred was mixed with excess NADP+, the absorbance in the flavin band I increased to about 70 % of that of CtFNRox with an apparent rate of ~4 s−1, whereas almost no absorption changes were observed in the CT band. Obtained data suggest that the reaction between CtFNR and NADP+/NADPH is reversible, in accordance with its physiological function. © 2016 Springer Science+Business Media DordrechtEmbargo period 12 month

Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP+ oxidoreductase activity toward NADPH

Ferredoxin-NAD(P)+ oxidoreductases ([EC 1.18.1.2], [EC 1.18.1.3], FNRs) from green sulfur bacteria, purple non-sulfur bacteria and most of Firmicutes, such as Bacillus subtilis (BsFNR) are homo-dimeric flavoproteins homologous to bacterial NADPH-thioredoxin reductase. These FNRs contain two unique aromatic residues stacked on the si- and re-face of the isoalloxazine ring moiety of the FAD prosthetic group whose configurations are often found among other types of flavoproteins including plant-type FNR and flavodoxin, but not in bacterial NADPH-thioredoxin reductase. To investigate the role of the si-face Tyr50 residue in BsFNR, we replaced Tyr50 with Gly, Ser, and Trp and examined its spectroscopic properties and enzymatic activities in the presence of NADPH and ferredoxin (Fd) from B. subtilis (BsFd). The replacement of Tyr50 to Gly (Y50G), Ser (Y50S), and Trp (Y50W) in BsFNR resulted in a blue shift of the FAD transition bands. The Y50G and Y50S mutations enhanced the FAD fluorescence emission, whereas those of the wild type and Y50W mutant were quenched. All three mutants decreased thermal stabilities compared to wild type. Using a diaphorase assay, the kcat values for the Y50G and Y50S mutants in the presence of NADPH and ferricyanide were decreased to less than 5 % of the wild type activity. The Y50W mutant retained approximately 20 % reactivity in the diaphorase assay and BsFd-dependent cytochrome c reduction assay relative to wild type. The present results suggest that Tyr50 modulates the electronic properties and positioning of the prosthetic group

Role of the C-terminal extension stacked on the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group in ferredoxin-NADP+ oxidoreductase from Bacillus subtilis

Ferredoxin-NADP+ oxidoreductase [EC 1.18.1.2] from Bacillus subtilis (BsFNR) is homologous to the bacterial NADPH-thioredoxin reductase, but possesses a unique C-terminal extension that covers the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide (FAD) prosthetic group. In this report, we utilize BsFNR mutants depleted of their C-terminal residues to examine the importance of the C-terminal extension in reactions with NADPH and ferredoxin (Fd) from B. subtilis by spectroscopic and steady-state reaction analyses. The depletions of residues Y313 to K332 (whole C-terminal extension region) and S325 to K332 (His324 intact) resulted in significant increases in the catalytic efficiency with NADPH in diaphorase assay with ferricyanide, whereas Km values for ferricyanide were increased. In the cytochrome c reduction assay in the presence of B. subtilis ferredoxin, the S325-K332 depleted mutant displayed a significant decrease in the turnover rate with an Fd concentration range of 1-10 μM. The Y313-K332 depleted mutant demonstrated an increase in the rate of the direct reduction of horse heart cytochrome c in the absence of Fd. These data indicated that depletion of the C-terminal extension plays an important role in the reaction of BsFNR with ferredoxin. © 2014 Elsevier Masson SAS. All rights reserved

Studies of interaction of homo-dimeric ferredoxin-NAD(P)+ oxidoreductases of Bacillus subtilis and Rhodopseudomonas palustris, that are closely related to thioredoxin reductases in amino acid sequence, with ferredoxins and pyridine nucleotide coenzymes

金沢大学理工研究域物質化学系Ferredoxin-NADP+ oxidoreductases (FNRs) of Bacillus subtilis (YumC) and Rhodopseudomonas palustris CGA009 (RPA3954) belong to a novel homo-dimeric type of FNR with high amino acid sequence homology to NADPH-thioredoxin reductases. These FNRs were purified from expression constructs in Escherichia coli cells, and their steady-state reactions with [2Fe-2S] type ferredoxins (Fds) from spinach and R. palustris, [4Fe-4S] type Fd from B. subtilis, NAD(P)+/NAD(P)H and ferricyanide were studied. From the Km and kcat values for the diaphorase activity with ferricyanide, it is demonstrated that both FNRs are far more specific for NADPH than for NADH. The UV-visible spectral changes induced by NADP+ and B. subtilis Fd indicated that both FNRs form a ternary complex with NADP+ and Fd, and that each of the two ligands decreases the affinities of the others. The steady-state kinetics of NADPH-cytochrome c reduction activity of YumC is consistent with formation of a ternary complex of NADPH and Fd during catalysis. These results indicate that despite their low sequence homology to other FNRs, these enzymes possess high FNR activity but with measurable differences in affinity for different types of Fds as compared to other more conventional FNRs. © 2008 Elsevier B.V. All rights reserved