Gas chromatographic-mass spectrometric determination of erythrocyte 3-deoxyglucosone in diabetic patients

To determine if the erythrocyte levels of 3-deoxyglucosone (3-DG) are increased in diabetic patients, and if they correlate with glycemic status, they were measured in diabetic patients without renal disease as well as in healthy subjects. The erythrocyte levels of 3-DG were measured by a selected ion monitoring method of gas chromatography-chemical ionization mass spectrometry using [(13)C(6)]-3-DG as an internal standard. The erythrocyte levels of 3-DG were significantly higher in diabetic patients than in healthy subjects. The erythrocyte concentration of 3-DG was significantly and positively correlated with HbA1c (r=0.84, P<0.001). However, no significant correlation could be found between erythrocyte 3-DG and age, onset age of diabetes, or duration of diabetes in our group of diabetic patients. In diabetes, the production of 3-DG in the erythrocytes is increased via the polyol pathway and/or the Maillard reaction due to hyperglycemia.     

IL-17 stimulates inflammatory responses via NF-kappaB and MAP kinase pathways in human colonic myofibroblasts

Colonic subepithelial myofibroblasts (SEMFs) may play a role in the modulation of mucosal inflammatory responses. We investigated the effects of interleukin (IL)-17 on IL-6 and chemokine [IL-8 and monocyte chemoattractant protein (MCP)-1] secretion in colonic SEMFs. Cytokine expression was determined by ELISA and Northern blotting. Nuclear factor kappa B (NF-kappaB) DNA-binding activity was evaluated by electrophortetic gel mobility shift assay (EMSA). The activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-6, IL-8, and MCP-1 secretions were rapidly induced by IL-17. IL-17 induced NF-kappaB activation within 45 min after stimulation. A blockade of NF-kappaB activation markedly reduced these responses. MAPK inhibitors (SB-203580, PD-98059, and U-0126) significantly reduced the IL-17-induced IL-6 and chemokine secretion. The combination of either IL-17 + IL-1beta or IL-17 + tumor necrosis factor (TNF)-alpha enhanced cytokine secretion; in particular, the effects of IL-17 + TNF-alpha on IL-6 secretion were much stronger than the other responses. This was dependent on the enhancement of IL-6 mRNA stability. In conclusion, human SEMFs secreted IL-6, IL-8, and MCP-1 in response to IL-17. These responses might play an important role in the pathogenesis of gut inflammation.     

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.     

Scanning transmission X-ray,laser scanning,and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.     

Histidine-114 at subsites E and F can explain the characteristic enzymatic activity of guinea hen egg-white lysozyme

The courses of the reaction catalyzed by guinea hen egg-white lysozyme (GHL), in which Asn113 and Arg114 at subsites E and F in hen egg-white lysozyme (HEL) are replaced by Lys and His, respectively, was studied with the substrate N-acetylglucosamine pentamer, (GlcNAc)5. Although GHL was found to retain the main-chain folding similar to HEL as judged from CD spectroscopy, the courses of GHL showed increased production of (GlcNAc)4 and reduced production of (GlcNAc)2 when compared with HEL. To identify critical residue(s) involved in the alteration in the courses of GHL, two mutant enzymes as to subsites E and F in HEL, N113K and R114H, were prepared by site-directed mutagenesis. Kinetic analysis of these mutants revealed that the mutation of Asn113 to Lys had little effect on the courses of HEL, while the Arg114 to His mutation completely reproduced the courses of GHL, demonstrating that His114 in GHL is the key residue responsible for the characteristic courses of GHL. Computer simulation of the reaction courses of the R114H mutant revealed that this substitution decreased not only the binding free energies for subsites E and F, but also the rate constant of transglycosylation. The Arg residue at position 114 may play an important role in the transglycosylation activity of HEL.     

Desulfurization of Benzothiophene by the Gram-Negative Bacterium, Sinorhizobium sp. KT55

Sinorhizobium sp. KT55 was the first Gram-negative isolate to be capable of utilizing benzothiophene as the sole source of sulfur. By GC-MS analysis of metabolites of benzothiophene by this strain, benzothiophene sulfone, benzo[e][1,2]oxathiin S-oxide and o-hydroxystyrene were detected, suggesting that the benzothiophene desulfurization pathway of this strain is benzothiophene → benzothiophene sulfoxide → benzothiophene sulfone → benzo[e][1,2]oxathiin S-oxide →o-hydroxystyrene. Desulfurization activity of this strain was significantly repressed by methionine, cysteine, sulfate, dimethyl sulfoxide, and Casamino acids. Received: 5 January 2001/Accepted: 6 February 2001     

Effects of pH and chloride concentration on resonance Raman spectra of human myeloperoxidase and Raman microspectroscopic analysis of enzyme state in azurophilic granules

Resonance Raman spectra of human myeloperoxidase were examined at pH 3.3-10.5 in the absence and presence of chloride ions. Among the porphyrin vibrational bands, the core-size marker bands showed particularly large wavenumber downshifts on going from pH 8.7 to 5.3 with a transition midpoint at pH 6.5 in the absence of chloride ions. The chloride ions did not affect the spectrum at a pH below 5.3 and above 8.7 whereas an increase of chloride concentration at neutral pH caused spectral changes similar to those observed upon pH lowering. Analogous effects were also observed on the Raman intensity. In addition, the stretching mode of the bond between the heme Fe and proximal histidine shifted by -2 cm(-1) on going from pH 8.7 to 5.3. Decomposition of the nu(3) band revealed the presence of two components, which was confirmed by an isosbestic point in the absorption spectra. The observed spectral changes indicated the existence of alkaline and acidic forms of the enzyme. The pK of interconversion was 6.5, and it was increased by binding of chloride ions. The porphyrin core was slightly expanded in the acidic form compared to that in the alkaline form. A molecular mechanism of the porphyrin core expansion was proposed on the basis of the X-ray crystal structure. The pH-spectrum relationships obtained for the isolated enzyme were applied to in situ analysis of the state of myeloperoxidase in azurophilic granules of living neutrophils. The enzyme was stored in the acidic form and kept inactive in catalyzing HOCl production.     

Postextrasystolic contractile decay always contains exponential and alternans components in canine heart

In isolated, blood-perfused canine hearts, postextrasystolic potentiation (PESP) decays monotonically after a noncompensatory pause following a spontaneous extrasystole (ES). The monotonic PESP decay yields myocardial internal Ca(2+) recirculation fraction (RF). We have found that after a compensatory pause (CP), PESP decays in alternans, consisting of an exponential and a sinusoidal decay component. We have proposed that this exponential component also yields RF. In the present study, we examined the reliability of this alternative method by widely changing the ES coupling interval (ESI), CP, and heart rate in the canine excised, cross-circulated left ventricle. We found that all PESP decays consisted of the sum of an exponential and a sinusoidal decay component of variable magnitudes whether a CP existed or not. Their decay constants as well as the calculated RF were independent of the ESI and CP. This confirmed the utility of our alternative RF determination method regardless of the ESI, CP, and heart rate. Direct experimental evidence of Ca(2+) dynamics supportive of this alternative method, however, remains to be obtained.