Background:The relationship between 1/f¯uctuation of the heart rate variability and Helicobacter pylori infection was evaluated, in order to clarify whether autonomic nervous dysfunction is a necessary condition for chronic peptic ulcer formation. Methods: The subjects were 11 patients with recurrent chronic peptic ulcer and 20 age-matched normal subjects. Holter ECGs were recorded over 24 h, and the 1/f ±x¯u ctuation of the heart rate was computed. The 1/f ±x¯u ctuation of the heart rate is a novel index of autonomic function that has been shown to re¯ect a patient's pleasant mood. For 1/f ±x¯u ctuation, the slope of the regression line (±x) was determined and cosine ®tting of the absolute slope of the regression line over a 24-h period was performed.
Chemical modifications by photooxidation in the presence of rose bengal (RB) and with tetranitromethane (TNM) were carried out to elucidate the amino acid residues involved in the active site of inorganic pyrophosphatase (pyrophosphate phosphohydrolase) [EC 3.6.1.1] from Escherichia coli Q13. The photooxidation caused almost complete inactivation, which followed pseudo-first-order kinetics depending on pH and concentration of RB. The presence of Mg2+ or complex between Mg2+ and substrate or substrate analogues, imidodiphosphate and sodium methylenediphosphate, gave partial protection against the photoinactivation, whereas the substrate alone showed no protective effect. The enzyme was almost completely inactivated by chemical modification with TNM, depending upon the concentration of TNM. The amino acid analyses and enzyme activity measurements revealed that 2 histidyl residues among 5 photooxidized residues and 2 tyrosyl residues per subunit were essential for the enzyme activity. The circular dichroism (CD) spectra in the far ultraviolet region showed no significant alteration during these two modifications, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modifications altered considerably the CD bands in the near ultraviolet region and the fluorescence spectra, indicating that subtle change in conformation had occurred in the vicinity of the active site in the enzyme molecule. These results strongly suggest that histidyl and tyrosyl residues may be involved in the active site or be located in the vicinity of the active site and seem to participate in the mechanism of stability against heat inactivation.
Bilirubin oxidase [EC 1.3.3.5], purified from the culture medium of Myrothecium verrucaria, was found to contain two blue copper atoms per protein molecule with a molecular weight of ca. 52 kDa. The two copper atoms were estimated to be in the all cupric state by the cuproine colorimetric method and also atomic absorption analysis. We could remove the reduce cuprous ions from the holo enzyme by adding ascorbate, followed by a KCN solution, yielding an apo-enzyme with no activity. The apo-enzyme can be reconstituted with Cu or other divalent cations such as Co, Fe, and Cd, with accompanying recovery of the enzyme activity. The activity recovery depended upon the species of cation employed; Cu being most effective, an almost 100% recovery, and Cd the least, only a 25% recovery. We could obtain information on the copper ions and their coordination structure by spectroscopic analyses of the apo- and reconstituted enzymes, obtaining such as absorption, CD, MCD, and XPS spectra. The bilirubin oxidase catalyzed-reaction was a second order reaction with respect to copper bound with protein. The donor set was of the CuSS*N2 (S = Cys, S* = Met, N = His) type, i.e., the same as in the case of blue copper proteins. On studying the Co-substituted enzyme, it was revealed that the copper site of the enzyme had a 4-coordinated structure.
Autofluorescence observations enable scientists to sensitively identify various lesions. Non-steroidal anti-inflammatory drugs such as aspirin and indomethacin are well known to induce gastric mucosal injuries. Our purpose was to clarify whether the observation of mucosal autofluorescence could help us to recognize indomethacin-induced gastric lesion formation. Gastric mucosal fluorescence intensity and gastric lesion scores were time-sequentially measured after indomethacin treatment in rats. To identify the localization of autofluorescent substances, stomach cryosections were observed with an epifluorescence microscope. Fluorescent substances from damaged tissue were also analyzed by high-performance liquid chromatography. In addition, to elucidate whether oxidative stress directly generates fluorescent substances from heme, we investigated the reaction between hydrogen peroxide and hemoglobin in a cell-free system. Treatment with indomethacin induced gastric lesions by tissue peroxidation, with mucosal fluorescence intensity increasing time-dependently. The fluorescence products were mesoporphyrin and protoporphyrin, and they were localized in disrupted mucosal tissue. In the cell-free system, porphyrins were directly generated by hydrogen peroxide from hemoglobin. These findings indicate that indomethacin treatment increased the intensity of porphyrin fluorescence. Gastric mucosal lesion formation can be sensitively detected with fluorescence observations.
We previously reported that the gastric mucosa emits fluorescence of porphyrins at the onset of gastric lesions induced by hemorrhagic shock. In this study, we investigated whether the fluorescent substance concerns with the gastric mucosal injuries induced by diflofenac, a nonsteroidal antiinflammatory drug (NSAID). In the gastric mucosa treated with diclofenac, lesions were generated and myeloperoxidase activity increased. Diclofenac administration also increased thiobarbituric acid-reactive substances, a index of tissue peroxidation. After diclofenac treatment, the gastric mucosal fluorescence intensities rose. HPLC analysis demonstrated that the fluorescent substances were mesoporphyrin and protoporphyrin, which were the same as found in hemorrhagic shock. Pretreatment of the tissue with radical scavenging substances, catalase and troxipide, restrained the increase of mucosal fluorescence intensity, tissue peroxidation, and lesion formation. These findings indicate that diclofenac treatment induced the generation of porphyrins as well as tissue peroxidation in gastric mucosal tissue. This study suggests that autofluorescence observation is a useful tool to identify diclofenac-induced gastric injury.
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