Utility of glycated albumin for the diagnosis of diabetes mellitus in a Japanese population study: results from the Kyushu and Okinawa Population Study (KOPS).

AIMS/HYPOTHESIS: Glycated albumin is a measure of the mean plasma glucose concentration over approximately 2-3 weeks. We determined reference values for glycated albumin, and assessed its utility for the diagnosis of type 2 diabetes mellitus in the general population. METHODS: We studied 1,575 men and women (mean age, 49.9 years; range, 26-78 years) who participated in a periodic health examination in a suburban Japanese town. HbA(1c) and fasting plasma concentrations of glucose (FPG) and glycated albumin were measured. Participants with FPG ≥ 7.0 mmol/l or HbA(1c) ≥ 6.5% (48 mmol/mol) were diagnosed as having diabetes. In our laboratory, the glycated albumin assay had intra-assay and inter-assay CVs of 1.1% and 1.6%, respectively. RESULTS: Glycated albumin levels were significantly correlated with HbA(1c) levels (r = 0.766, p < 0.001) and FPG (r = 0.706, p < 0.001). The presence of diabetes was significantly higher in participants with glycated albumin levels between 15.0% and 15.9% (five of 276, 1.81%) than in those with glycated albumin <14% (three of 672, 0.45%) (p = 0.037), and was markedly increased in those with a glycated albumin level >16% (58 of 207, 28.0%). Receiver operating characteristic curve analysis indicated that a glycated albumin level of ≥15.5% was optimal for predicting diabetes, with a sensitivity of 83.3% and a specificity of 83.3%. CONCLUSIONS/INTERPRETATION: There is merit to further investigating the potential for glycated albumin to be used as an alternative measure of dysglycaemia for future research and clinical practice.

Spectroscopic and electrochemical studies on structural change of plastocyanin and its tyrosine 83 mutants induced by interaction with lysine peptides.

Interactions of wild-type and Tyr83 mutant (Y83F, Y83S, Y83L, and Y83H) plastocyanins (PCs) with lysine peptides as models for the PC interacting site of cytochrome f have been studied by absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopies and electrochemical measurements. The spectral and electrochemical properties of PCs corresponded well with each other; species having a longer wavelength maximum for the S(Cys) pi --> Cu 3d(x)()()2(-)(y)()()2 charge transfer (CT) band observed around 600 nm and a stronger intensity for the 460-nm absorption band exhibited stronger intensities for the positive Met --> Cu 3d(x)()()2(-)(y)()()2 and negative His pi(1) --> Cu 3d(x)()()2(-)(y)()()2 circular dichroism (CD) bands at about 420 and 470 nm, respectively, a lower average nu(Cu)(-)(S) frequency, a smaller |A( parallel)| EPR parameter, and a higher redox potential, properties all related to a weaker Cu-S(Cys) bond and a more tetrahedral planar geometry for the Cu site. Similarly, on oligolysine binding to wild-type and several Tyr83 mutant PCs, a longer absorption maximum for the 600-nm CT band, a stronger intensity for the 460-nm absorption band, stronger 420-nm positive and 470-nm negative CD bands, and a lower average nu(Cu)(-)(S) frequency were observed, suggesting that PC assumes a slight more tetrahedral geometry on binding of oligolysine. Since changes were observed for both wild-type and Tyr83 mutant PCs, the structural change due to binding of oligolysine to PC may not be transmitted through the path of Tyr83-Cys84-copper by a cation-pi interaction which is proposed for electron transfer.

Structure comparison between oxidized and reduced plastocyanin from a fern, Dryopteris crassirhizoma.

The X-ray crystal structures of oxidized and reduced plastocyanin obtained from the fern Dryopteris crassirhizoma have been determined at 1.7 and 1.8 A resolution, respectively. The fern plastocyanin is unique in the longer main chain composed of 102 amino acid residues and in the unusual pH dependence due to the pi-pi stacking interaction around the copper site [Kohzuma, T., et al. (1999) J. Biol. Chem. 274, 11817-11823]. Here we report the structural comparison between the fern plastocyanin and other plastocyanins from cyanobacteria, green algae, and other higher plants, together with the structural changes of fern plastocyanin upon reduction. Glu59 hydrogen bonds to the OH of Tyr83, which is thought to be a possible conduit for electrons, in the oxidized state. However, it moves away from Tyr83 upon reduction like poplar plastocyanin.

Crystal structure determinations of oxidized and reduced pseudoazurins from Achromobacter cycloclastes. Concerted movement of copper site in redox forms with the rearrangement of hydrogen bond at a remote histidine.

The crystal structures of oxidized and reduced pseudoazurins from a denitrifying bacterium, Achromobacter cycloclastes IAM1013, have been determined at 1.35- and 1.6-A resolutions, respectively. The copper site in the oxidized state exhibits a distorted tetrahedral structure like those of other pseudoazurins. However, not only a small change of the copper geometry, but concerted peptide bond flips are identified. The imidazole ring of remote His6 has a hydrogen bonding distance of 2.73 A between N-delta1(His6) and O-gamma1(Thr36) in the oxidized protein. When the protein is reduced at pH 6.0, the imidazole ring rotates by 30.3 degrees and moves 1.00 A away from the position of the oxidized state. A new hydrogen bond between N-epsilon2(His6) and O-epsilon1(Glu4) is formed with a distance of 3.03 A, while the hydrogen bond between N-delta1(His6)-O-gamma1(Thr36) is maintained with an interatomic distance of 2.81 A. A concomitant peptide bond flip of main chain between Ile34 and Thr36 occurs.

Crystal structure determinations of oxidized and reduced plastocyanin from the cyanobacterium Synechococcus sp. PCC 7942.

The crystal structures of oxidized and reduced plastocyanins from Synechococcus sp. PCC 7942 have been determined at 1.9 and 1.8 A resolution, respectively, at pH 5.0. The protein consists of only 91 amino acid residues, the smallest number known for a plastocyanin, and apparently lacks the mostly conserved acidic patch that is believed to be important for recognition with electron-transfer partners. The protein has two acidic residues, Glu42 and Glu85, around Tyr83, which is thought to be a possible conduit for electrons, but these are neutralized by Arg88 and Lys58. Residue Arg88 interacts with Tyr83 through a pi-pi interaction in which the guanidinium group of the former completely overlaps the aromatic ring of the tyrosine. Reduction of the protein at pH 5.0 causes a lengthening of one Cu-N(His) bond by 0.36 A, despite the small rms deviation of 0.08 A calculated for the backbone atoms. Moreover, significant conformational changes of Arg88 and Lys58, along with the movement of a water molecule adjacent to the OH group of Tyr83, were observed on reduction; the guanidinium group of Arg88 rotates by more than 11 degrees, and the water molecule moves by 0.42 A. The changes around the copper site and the alterations around Tyr83 may be linked to the reduction of the copper.

The structure and unusual pH dependence of plastocyanin from the fern Dryopteris crassirhizoma. The protonation of an active site histidine is hindered by pi-pi interactions.

Spectroscopic properties, amino acid sequence, electron transfer kinetics, and crystal structures of the oxidized (at 1.7 A resolution) and reduced form (at 1.8 A resolution) of a novel plastocyanin from the fern Dryopteris crassirhizoma are presented. Kinetic studies show that the reduced form of Dryopteris plastocyanin remains redox-active at low pH, under conditions where the oxidation of the reduced form of other plastocyanins is inhibited by the protonation of a solvent-exposed active site residue, His87 (equivalent to His90 in Dryopteris plastocyanin). The x-ray crystal structure analysis of Dryopteris plastocyanin reveals pi-pi stacking between Phe12 and His90, suggesting that the active site is uniquely protected against inactivation. Like higher plant plastocyanins, Dryopteris plastocyanin has an acidic patch, but this patch is located closer to the solvent-exposed active site His residue, and the total number of acidic residues is smaller. In the reactions of Dryopteris plastocyanin with inorganic redox reagents, the acidic patch (the "remote" site) and the hydrophobic patch surrounding His90 (the "adjacent" site) are equally efficient for electron transfer. These results indicate the significance of the lack of protonation at the active site of Dryopteris plastocyanin, the equivalence of the two electron transfer sites in this protein, and a possibility of obtaining a novel insight into the photosynthetic electron transfer system of the first vascular plant fern, including its molecular evolutionary aspects. This is the first report on the characterization of plastocyanin and the first three-dimensional protein structure from fern plant.

Crystallization and preliminary X-ray analysis of plastocyanin from cyanobacterium Synechococcus sp. PCC 7942.

A plastocyanin from the cyanobacterium Synechococcus sp. PCC 7942 has been crystallized in two different forms by hanging-drop vapour diffusion with ammonium sulfate as precipitant. Form I is hexagonal, space group P61 or P65, with unit-cell dimensions a = b = 34.62 and c = 107.22 A. Form II is tetragonal, space group P41 or P43, with unit-cell dimensions a = b = 43.05 and c = 56.94 A. Form I crystals diffract to 2.5 A using graphite-monochromated Cu Kalpha radiation from a Rigaku RU-300 rotating-anode generator operated at 40 kV and 100 mA. Form II crystals diffract to 1.9 A using synchrotron radiation at beamline BL6A of the Photon Factory (KEK). Molecular-replacement calculations using the structure of plastocyanin from Ulva pertusa have been performed.

Cloning, sequencing, and transcriptional studies of the gene encoding copper-containing nitrite reductase from Alcaligenes xylosoxidans NCIMB 11015.

Gene encoding of the blue copper-containing nitrite reductase (nir) from Alcaligenes xylosoxidans NCIMB 11015 has been cloned and characterized. The nir is translated into a polypeptide of 360 amino acid residues as a precursor, and the N-terminal 24 residues are subsequently removed upon transport into the periplasm as a mature protein. A specific transcription product of nir was detected only in the presence of nitrate. The aeration level of the culture medium did not show a significant effect on the transcriptional level. A varsigma54 binding sequence is identified upstream of the transcriptional initiation at 53 to 26 nucleotides. A putative fnr box has also been identified in the sequence of the upstream region. The mature polypeptide showed 70% sequence identity with those of the Achromobacter cycloclastes enzyme. The transcriptional start point has been determined at 92 nucleotides upstream of the initiation codon and is preceded by the binding sites for varsigma54 and the fnr box. These results suggest that gene expression depends on the presence of nitrate and is stimulated under an anaerobic environment.

Novel insight into the copper-ligand geometry in the crystal structure of Ulva pertusa plastocyanin at 1.6-A resolution. Structural basis for regulation of the copper site by residue 88.

The crystal structure of plastocyanin from a green alga, Ulva pertusa, has been determined at 1.6-A resolution. At its copper site, U. pertusa plastocyanin has a distorted tetrahedral coordination geometry similar to other plastocyanins. In comparison with structures of plastocyanins reported formerly, a Cu(II)-Sdelta(Met92) bond distance (2.69 A) is shorter by about 0.2 A and a Cu(II)-Sgamma(Cys84) distance is longer by less than 0.1 A in U. pertusa plastocyanin. These subtle but significant differences are caused by the structural change at a His-Met loop (His87-Met92) due to an absence of a O(Asp85)-Ogamma(Ser88) hydrogen bond which is found in Enteromorpha prolifera plastocyanin. In addition, poplar and Chlamydomonas reinhardtii plastocyanins with a glutamine at residue 88 have a weak cation-pi interaction with Tyr83. This interaction lengthens the Cu(II)-Sdelta(Met92) bond of poplar and C. reinhardtii plastocyanins by 0.14 and 0.20 A, respectively. As a result of structural differences, U. pertusa plastocyanin has a less distorted geometry than the other plastocyanins. Thus, the cupric geometry is finely tuned by the interactions between residues 85 and 88 and between residues 83 and 88. This result implies that the copper site is more flexible than reported formerly and that the rack mechanism would be preferable to the entatic theory. The His-Met loop may regulate the electron transfer rate within the complex between plastocyanin and cytochrome f.

The influence of conserved aromatic residues on the electron transfer reactivity of 2[4Fe-4S] ferredoxins.

The detailed mechanism used by [4Fe-4S] ferredoxins to exchange electrons is not known. The importance of two highly conserved aromatic residues, each located close to one cluster of 2[4Fe-4S] ferredoxins has been probed by site-directed mutagenesis of Clostridium pasteurianum ferredoxin. All generated variants are less stable than the native protein and only hydrophobic residues can replace one of the two conserved aromatic residues. With leucine substituting both aromatics, Clostridium pasteurianum ferredoxin cannot even be completely purified because of its deleterious instability. The reduction potentials of Clostridium pasteurianum ferredoxin variants do not depend on the presence of aromatic residues near the clusters. However, the ferredoxin from Entamoeba histolytica which is naturally devoid of aromatic residues displays a reduction potential nearly 60 mV less negative than that of Clostridium pasteurianum ferredoxin. The rate constants for the oxidation of the reduced ferredoxins by the inorganic complexes hexaamine-cobalt(III) chloride and sodium ethylenediaminetetra-acetatecobaltate(III) are similar. This implies that electron transfer from the clusters of these molecules is not mediated by the conserved aromatic residues. These residues rather appear to be involved in maintaining the overall stability of ferredoxins.

Redox reactivity of the type 1 copper protein amicyanin from Thiobacillus versutus with its physiological partner cytochrome C550 and inter-protein cross-reaction studies.

Reduction potentials Eo' for the T. versutus amicyanin couple, AmCuII/I, were determined at pH values in the range 4.4-9.0 by direct measurement using cyclic voltammetry, and from rate constants for the reactions AmCu1 + [Co(terpy)2]3+ and [Co(terpy)2]2+ + AmCuII, using an Eo' for the [Co(terpy)2]2+/3+ couple of 260 mV. At pH > 7.5 the value obtained is 236 mV, which increases with decreasing pH in keeping with proton inactivation of AmCuI. Together with previously determined Eo' values for the T. versutus cytochrome C550 FeIII/FeII couple, it is concluded that the physiologically relevant reaction AmCuI + cyt C550FeIII (kf) is thermodynamically favourable at pH > 6.25, but that the back reaction cyt C550FeII + AmCuII (kb) is favourable at pH < 6.25. Values of kf (25 degrees C) at pH > 6.25 were determined directly by the stopped-flow method, I = 0.100 M (NaCl). At pH < 6.25 kf values were obtained indirectly from the measured kb and equilibrium constants from delta Eo'. The combined kf variations with pH give an acid dissociation pKa for AmCuIH+ of 6.6. In further studies (25 degrees C) rate constants/M-1 S-1 (pH 6.0-8.6) were determined for the cross-reactions of AmCuI with P. aeruginosa azurin AzCuII, and AmCuI with P. aeruginosa cyt C550FeIII, and are 11.0 x 10(5) and 6.4 x 10(5) M-1 S-1 respectively at pH 8.6. Using the Marcus equations corresponding electron self-exchange rate constants (kese/M-1 S-1) of 1.3 x 10(5) and 0.6 x 10(5) M-1 S-1 were calculated for the exchange of AmCuII with unprotonated AmCuI, in good agreement with the value 1.2 x 10(5) M-1 S-1 determined by NMR at pH 8.6. Information was also obtained as to the effect of pH on these kese values.

Spectroscopic and electrochemical studies on active-site transitions of the type 1 copper protein pseudoazurin from Achromobacter cycloclastes.

The single type 1 copper protein pseudoazurin from Achromobacter cycloclastes gives reversible electrochemical behavior at a (4-pyridyl)disulfide-modified gold electrode. Measurements carried out at 25.0 degrees C indicate a midpoint reduction potential of E 1/2 = 260 mV versus normal hydrogen electrode at pH 7.0 and a peak-to-peak separation of delta Ep = 59 mV. The diffusion coefficient and heterogeneous electron transfer rate constant are estimated to be 2.23 x 10(-6) cm2 s-1 and 3.7 x 10(-2) cm s-1, respectively. Also, controlled potential electrolysis indicates a 1-electron transfer process and a formal reduction potential of 259 mV versus normal hydrogen electrode for the Cu(II)/Cu(I) couple. The heterogeneous electron transfer rate constant determined at the (4-pyridyl)disulfide-modified gold electrode at pH 4.6 is 6.7 x 10(-3) cm s-1, consistent with a slower process at the positively charged electrode surface. At pH 11.3, UV-visible, EPR, and resonance Raman spectra indicate a conversion of the distorted tetrahedral copper geometry to a trigonal structure. The trigonal form has elongated axial bonding and an axial EPR spectrum. At pH 11.3, the reduction potential is further decreased, and Cu-S bands in resonance Raman spectra at 330-460 cm-1 are shifted to higher energy (approximately 10 cm-1), consistent with a stronger Cu-S bond.

Refined crystal structure of pseudoazurin from Methylobacterium extorquens AM1 at 1.5 A resolution.

The crystal structure of pseudoazurin from Methylobacterium extorquens AM1 (PAZAM1) has been solved by the molecular replacement method using copper-copper distances as translation parameters, which were obtained from difference Patterson maps calculated with the synchrotron radiation data containing the multiwavelength anomalous-dispersion effect. The structure refinement was carried out by the use of molecular dynamics optimization and the restrained least-squares method. The final crystallographic R factor was 19.9% for the 14 365 reflections greater than 3sigma between 1.5 and 8.0 A resolution. This report describes the characteristic features of the structure of PAZAM 1 as well as the effectiveness of synchrotron radiation for structure analysis of metalloproteins. The environment of the metal active site and the structural differences among blue-copper proteins are discussed.

Anastomotic recurrence at hepaticojejunostomy in a long-term survivor of bile duct carcinoma: report of a case.

A rare case of autopsy-proven recurrence 10 years after a radical resection for lower bile duct carcinoma is herein reported. The subject is a 53-year-old man who underwent a curative resection of distal bile duct carcinoma with pancreatoduodenectomy in 1981. The lesion was a 1.0 x 1.5 x 1.0 cm well-differentiated papillotubular adenocarcinoma invading the fibromuscular layer of the bile duct with a slight infiltration to the lymphatics but without any extension to the vessels, nerves, connective tissues, or nodes. The patient demonstrated a recurrence 10 years after the initial operation and died 4 months later. An autopsy revealed a 2.0 x 2.5 x 1.6 cm mass at the anastomotic site of hepaticojejunostomy without any distant metastases. Although a late anastomotic recurrence after more than 10 years is unique, this case highlights the difficulty of the operative eradication of bile duct carcinoma. As a result, all possible maneuvers either during or after operation to promote the prophylaxis of recurrence are warranted.

Crystallization and preliminary X-ray studies on pseudoazurin from Achromobacter cycloclastes IAM1013.

New crystals of a blue copper protein, pseudoazurin from denitrifier Achromobacter cycloclastes IAM1013, have been obtained by means of vapor diffusion with ammonium sulfate as a precipitant at pH 6.0 and 4 degrees C. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 56.69(2), b = 61.53(2), and c = 30.20(1) A. The asymmetric unit includes one molecule of pseudoazurin with a Vm value of 2.04 A3/Da. The crystals are so stable against X-ray irradiation that a complete data set up to 1.54 A has been collected using a single native crystal. Solution of the structure was performed by means of the Patterson search techniques, and the current crystallographic R-factor is 17.5% at 3.0 A resolution. Refinement at higher resolution is in progress.

Validity of putative calcium binding loops of photoprotein aequorin.

Three peptides containing the putative Ca2+ binding loops, I, II and III, respectively, of a photoprotein, aequorin, from jellyfish Aequorea victoria were synthesized by a solid-phase procedure. The peptides bound Ca2+ with dissociation constants of 10(-3) to 10(-4) M, providing evidence for the assumption that Ca2+ binding loops are actually responsible for the binding of Ca2+. When the highly conserved 6th glycine residue in the 12-residue loops was replaced by arginine, no large effect was observed on Ca2+ binding. Exposure to a hydrophobic environment and the binding of Ca2+ brought about conformational changes to the peptides.

Preliminary crystallographic study of a pseudoazurin from methylotrophic bacterium, Methylobacterium extorquens AM1.

Single crystals of pseudoazurin, one of the blue copper proteins produced by methylotrophic bacterium Methylobacterium extorquens AM1, have been obtained by the method of vapor diffusion with ammonium sulfate as a precipitant at pH 8.0. Crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 52.619(4) A, b = 63.280(6) A, c = 35.133(4) A. The asymmetric unit includes one molecule of pseudoazurin (Vm = 2.18 A3/dalton). The crystals are so stable against X-ray irradiation that diffraction intensities of the native crystal up to 1.68 A resolution could be collected from only one crystal. Among the many heavy-metal reagents examined, uranyl acetate gave an effective isomorphous derivative.

EPR spectra of ferric cytochromes c' from five strains of Achromobacter xylosoxidans at low temperature and their temperature dependence.

The EPR spectra at low temperature (6 K) and their temperature dependence (10-93 K) for five ferric cytochromes c' isolated from chemoheterotrophic bacteria, Achromobacter xylosoxidans NCIB 11015 (formerly Alcaligenes sp. NCIB 11015), GIFU 543, GIFU 1048, GIFU 1051, and GIFU 1764 are reported. The EPR spectral results indicate that the ground state of the heme iron(III) of cytochromes c' from these chemoheterotrophic bacteria can appear to be in an admixed spin state which consists of predominant S = 5/2 with a slight S = 3/2 character. The EPR spectra were compared with those for ferric cytochromes c' from photosynthetic bacteria and the other ferric hemoproteins.

Investigation of an active site histidine-aspartate couple in Trimeresurus flavoviridis phospholipase A2.

When Trimeresurus flavoviridis phospholipase A2 was reacted with methyl p-nitrobenzenesulfonate, its activity decreased following first-order kinetics. The pH dependence of the rate constants of inactivation showed that His-48 with an apparent pKa of 6.5 controls the reaction. In the pH region below 6.5, N1-methylhistidine was predominantly formed. On the other hand, N1,N3-dimethylhistidine was almost exclusively produced in the pH region above 6.5. No N3-methylhistidine was detected at any pH tested. Such observations suggested that the first methylation occurred at the N1-position of the imidazole ring followed by a second methylation at the N3-position, and that His-48 couples the carboxylate of Asp-99 at the N3-position of the imidazole ring, in accord with the interaction observed in the crystal structure of homologous Crotalus atrox phospholipase A2. As it has been reported that, in the reaction of chymotrypsin with methyl p-nitrobenzenesulfonate at pH 7.8, only monomethylation occurred at the N1-position of the His-57 imidazole group (Nakagawa, Y. & Bender, M.L. (1970) Biochemistry 9, 259-267), the nature of the active site histidine-aspartate couple of T. flavoviridis phospholipase A2 seems not to be identical with that of chymotrypsin.

Spectroscopic evidence for a copper-nitrosyl intermediate in nitrite reduction by blue copper-containing nitrite reductase.

The reactions of nitrogen monoxide (NO) with the blue copper-containing nitrite reductases from Alcaligenes sp. NCIB 11015 and Achromobacter cycloclastes IAM 1013 were investigated spectroscopically. The electron paramagnetic resonance (EPR) signals of the blue coppers vanished in the presence of NO at 77 K, being fully restored by the removal of NO. The additions of NO to the enzyme solutions resulted in the substantial bleaching of the visible absorption bands at room temperature. The reactions were also completely reversible. These results suggest the formation of a cuprous nitrosyl complex (Cu+-NO+), which is likely the intermediate in the enzymatic nitrite reduction.