Preparation of a fluorescent derivative of benzoylecgonine, and preliminary studies of its application to the analysis of urine

A sensitive high-performance liquid chromatographic method using 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (Br-DMEQ) as a fluorescent labeling reagent is described for the determination of benzoylecgonine (BE) and ecgonine (EC). The Br-DMEQ derivatives of BE and EC were separated on a C₁₈ column and detected at 455 nm with excitation at 370 nm. The detection limits of the proposed method were 18.7 fmol for BE and 12.5 pmol for EC at a signal-to-noise ratio of 3. Relative standard deviations of five replicate measurements were 1.94% (10 pmol) and 2.98% (50 pmol) for BE and 6.3% (250 pmol) and 5.62% (1.25 pmol) for EC. This method was applied to the determination of BE in human urine. BE was extracted from urine by solvent extraction with chloroform—isopropyl alcohol (9:1, v/v) solution. Levels of 2.5 · 10⁻⁸ M BE in urine (25 pmol/ml) could be determined.     

Analysis of Genetic Relationships and Antimicrobial Susceptibility of Verotoxin-Producing Escherichia coli Strains Isolated in Nagasaki Prefecture,Japan in 1996

A total of 19 Escherichia coli O157 isolates were obtained in Nagasaki Prefecture, in the southwestern part of Japan, between 1990 and 1996. Pulsed-field gel electrophoresis (PFGE) and computer-assisted analysis were applied to determine genetic relationships among these strains. Fragment patterns of the isolates in Nagasaki, as determined by PFGE, were compared with those of isolates in other areas where large outbreaks and sporadic cases of E. coli O157 infection occurred. Similarity values of all the strains isolated in Nagasaki Prefecture were over 0.65 except for E. coli O26. Some strains were identical to the strains isolated from the areas where large outbreaks occurred. All strains were susceptible to ampicillin, fosfomycin, minocycline, amikacin, ofloxacin and sulfamethoxazole-trimethoprim.     

Low-temperature-induced structural changes in human lysozyme elucidated by three-dimensional NMR spectroscopy

The three-dimensional solution structures of human lysozyme were determined at 35 and 4 degrees C using the heteronuclear multidimensional NMR spectroscopy, which were compared with each other to clarify the structural response of this enzyme to lowering of the temperature. Together with the data of the temperature dependence experiments of the lytic activity against Micrococcus luteus, we consider the implication of the observed structural change for the low-temperature-induced reduction of the activity of human lysozyme. The structures of human lysozyme determined at the two temperatures are found to be similar, both of which comprise four alpha-helices (A- to D-helices) and three antiparallel beta-strands (beta(1)-beta(3)), leading to the constructions of the alpha- and beta-domains as previously identified in the X-ray crystal structure. A significant structural change was observed for the "active site lobe" comprising the loop region connecting C- and D-helices and the following D-helix, which moves toward the active site cleft located between the alpha- and beta-domains so as to obstruct the cleft according to the temperature lowering. It further appeared that the total volume as well as the accessible surface area of human lysozyme decreases with lowering of the temperature, suggesting that the internal cavity of this enzyme shrinks under low temperature environment. Because in human lysozyme the region comprising the active site lobe is responsible for turnover of the enzymatic reaction against the substrate, the low-temperature-induced structural change of the active site lobe presumably controls the efficiency of the lytic activity under low temperatures.     

Normalization of phospholipids concentration of the gastric mucosa was observed in patients with peptic ulcer after eradication of Helicobacter pylori

Background. Phospholipids concentration in the gastric mucosa decreased in patients with Helicobacter pylori infection. The aim of this study is to examine the effects of eradication of H. pylori on decreasing the phospholipids concentration in the gastric mucosa in patients with gastric or duodenal ulcer. Materials and Methods. Phospholipids (phosphatidylcholine, phosphatidylethanolamine, and sphingonomyeline) were measured in biopsy specimens from the antrum and corpus using thin‐layer chromatography. In H. pylori positive patients with gastric ulcer (n = 26) and duodenal ulcer (n = 13), and H. pylori negative controls (n = 20), the biopsy specimens were obtained before and 3 months after eradication. Eradication was performed using lansoprazole, amoxycillin, and clarithromycin. Results. Compared with the H. pylori negative control group, the concentrations of phosphatidylcholine and phosphatidylethanolamine decreased significantly in the gastric ulcer group in both antrum and corpus mucosa, and in the duodenal ulcer group in antrum mucosa. This decrease returned to the control level after eradication. Conclusions. This study demonstrates that the eradication of H. pylori in patients with peptic ulcer normalized the decrease of phosphatidylcholine and phosphatidylethanolamine in the gastric mucosa.     

Esterification of ferulic acid with polyols using a ferulic acid esterase from Aspergillus niger

Commercially available enzyme preparations were screened for enzymes that have a high ability to catalyze direct ester-synthesis of ferulic acid with glycerol. Only a preparation, Pectinase PL “Amano” produced by Aspergillus niger, feruloylated glycerol under the experimental conditions. The enzyme responsible for the esterification was purified and characterized. This enzyme, called FAE-PL, was found to be quite similar to an A. niger ferulic acid esterase (FAE-III) in terms of molecular mass, pH and temperature optima, substrate specificity on synthetic substrates, and the N-terminal amino acid sequence. FAE-PL highly catalyzed direct esterification of ferulic acid and sinapinic acid with glycerol. FAE-PL could feruloylate monomeric sugars including arabinose, fructose, galactose, glucose, and xylose. We determined the suitable conditions for direct esterification of ferulic acid with glycerol to be as follows: 1% ferulic acid in the presence of 85% glycerol and 5% dimethyl sulfoxide at pH 4.0 and 50 °C. Under these conditions, 81% of ferulic acid could be converted to 1-glyceryl ferulate, which was identified by ¹H-NMR. The ability of 1-glyceryl ferulate to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was higher than that of the anti-oxidant butyl hydroxytoluene.     

The B cell inhibitory Fc receptor triggers apoptosis by a novel c-Abl family kinase-dependent pathway

The inhibitory Fc receptors function to regulate the antigen-driven activation and expansion of lymphocytes. In B cells, the Fc gammaRIIB1 is a potent inhibitor of B cell antigen receptor (BCR) signaling when coligated to the BCR by engagement of antigen-containing immune complexes. Inhibition is mediated by the recruitment of the inositol phosphatase, SHIP, to the Fc gammaRIIB1 phosphorylated tyrosine-based inhibitory motif (ITIM). Here we show that BCR-independent aggregation of the Fc gammaRIIB1 transduces an ITIM- and SHIP-independent proapoptotic signal that is dependent on members of the c-Abl tyrosine kinase family. These results define a novel Abl family kinase-dependent Fc gammaRIIB1 signaling pathway that functions independently of the BCR in controlling antigen-driven B cell responses.     

Human corneal epithelial cells respond to ocular-pathogenic, but not to nonpathogenic-flagellin

In this study, we investigated the expression of TLR5 in human corneal epithelial cells (CEC), and the functional outcome of TLR5 triggering by flagellins of pathogenic- and nonpathogenic bacteria. Flagellins derived from Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescense or Bacillus subtilis were used. The TLR5 protein and TLR5 specific mRNA expression was evident on human CEC. In human corneal epithelium tissues, TLR5 protein was detected at the basal and wing cells of the tissues. Ocular pathogenic bacteria, namely P. aeruginosa and S. marcescense, derived flagellin induced the significantly increased level of gene activation and IL-6 and IL-8 production. In contrast, ocular nonpathogenic S. typhimurium- and B. subtilis-derived flagellin induced neither the gene activation nor the increased production of IL-6 and IL-8 in human CEC. Human CEC would respond only to flagellin derived of ocular pathogenic bacteria, but not to those derived of ocular nonpathogenic bacteria, to generate pro-inflammatory cytokines.     

Acute effect of static stretching on power output during concentric dynamic constant external resistance leg extension

The purpose of the present study was to clarify the effect of static stretching on muscular performance during concentric isotonic (dynamic constant external resistance [DCER]) muscle actions under various loads. Concentric DCER leg extension power outputs were assessed in 12 healthy male subjects after 2 types of pretreatment. The pretreatments included (a) static stretching treatment performing 6 types of static stretching on leg extensors (4 sets of 30 seconds each with 20-second rest periods; total duration 20 minutes) and (b) nonstretching treatment by resting for 20 minutes in a sitting position. Loads during assessment of the power output were set to 5, 30, and 60% of the maximum voluntary contractile (MVC) torque with isometric leg extension in each subject. The peak power output following the static stretching treatment was significantly (p < 0.05) lower than that following the nonstretching treatment under each load (5% MVC, 418.0 +/- 82.2 W vs. 466.2 +/- 89.5 W; 30% MVC, 506.4 +/- 82.8 W vs. 536.4 +/- 97.0 W; 60% MVC, 478.6 +/- 77.5 W vs. 523.8 +/- 97.8 W). The present study demonstrated that relatively extensive static stretching significantly reduces power output with concentric DCER muscle actions under various loads. Common power activities are carried out by DCER muscle actions under various loads. Therefore, the result of the present study suggests that relatively extensive static stretching decreases power performance.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: evidence for polymorphism among preprotemporin mRNAs

A previous study led to the isolation of antimicrobial peptides belonging to the temporin and brevinin-2 families from a pooled extract of the skin of adult specimens of the Japanese mountain brown frog Rana ornativentris Werner 1903. In order to ascertain whether individual frogs expressed the full complement of temporin genes, we individually cloned cDNAs encoding the temporin precursors from total RNA extracted from the skins of 12 frogs by RT-PCR using a set of preprotemporin-specific primers. All the specimens examined contained mRNAs directing the synthesis of the novel, but inactive, temporin-1Oe (ILPLLGNLLNGLL x NH2). Nucleotide sequence analysis revealed marked polymorphism among individual frogs. Twenty-seven distinct preprotemporin-1Oe mRNAs were identified that contained synonymous substitutions in the antimicrobial peptide region and both synonymous and non-synonymous substitutions in the signal peptide and intervening sequence regions. Up to eight preprotemporin-1Oe mRNA variants were found within a single frog. In addition, several cDNAs encoding preprotemporin-1Oa and -1Ob and a single cDNA encoding preprotemporin-1Oc were characterized. Peptidomic analysis of norepinephrine-stimulated skin secretions revealed the presence of temporin-1Oe, temporin-1Of (SLILKGLASIAKLF x NH2), temporin-1Og (FLSSLLSKVVSLFT x NH2), four members of the ranatuerin-2 family and one member of the palustrin-2 family in addition to previously characterized temporin and brevinin-2 peptides.