L-Amino acid oxidase (LAO) was purified from mouse milk. LAO reacted with L-amino acids in an apparent order of Phe > Met, Tyr > Cys, Leu > His > > other 11 amino acids tested and produced H 2 O 2 in a dose-and time-dependent manner. LAO in milk had a molecular mass of about 113 kDa and was converted to a 60-kDa protein by SDS-PAGE. LAO consisted of two subunits. The N-and C-terminal amino acid sequence determination followed by cDNA cloning showed that the 60-kDa protein consisted of 497 amino acids. LAO mRNA spanned about 2.0 kb, and its expression was found only in the mammary epithelial cells. Glucocorticoid was essential for LAO gene expression. Thus, the LAO gene is expressed acutely upon the onset of milk synthesis. LAO mRNA increased 1 day before parturition, peaked during early to mid-lactation, and decreased at the end of lactation. This is the first demonstration showing that LAO is present in milk. Mastitis is caused by an intramammary bacterial infection. As mouse milk produced H 2 O 2 using endogenous free amino acids, we suggest that LAO, together with free amino acids, is responsible for killing bacteria in the mammary gland.
To understand hair-discoloration in relation to swimming, we examined sixty-seven elite swimmers of the Japan National Swimming Team and fifty-four, age-matched subjects as controls. The incidence of hair discoloration (61%) in the swimmers' group was significantly higher than that in controls (0%) (p<0.0001). Interestingly, surface damage of the nail plates coexisted in the swimmers with the scalp-hair discoloration. The hairs picked from the eight swimmers and two age-matched individuals as controls were examined by electron microscope (EM) and EM X-ray microanalyzer. The swimmers' discolored, golden hair revealed complete disappearance of hair cuticle both by scanning EM (SEM) and transmission EM (TEM). The quantity of melanosomes in the cortex decreased, and their diameter was smaller than that of controls. In addition, irregularly shaped melanosomes with variable electron density and less electron-dense melanosomes with white haloes were frequently observed in the swimmers' golden hair. The X-ray elemental spectrograph by SEM revealed that the content of sulfur in all the swimmers' discoloured hair was lower than that in the normal controls and that the content of chlorine in the male swimmers' discoloured hair was higher than that in the female swimmers and the normal controls. The X-ray elemental microanalysis by TEM focused on melanosomes in the cortex of the cross section and detected elemental chlorine in all swimmers' golden hairs. It did not detect any element in the control hairs. The 14C-tyrosine uptake test of hairbulbs found no significant difference between the swimmers and the normal controls. These findings suggest that hair discoloration was mainly due to cuticle damage by friction with water. Hypochlorous acid in the swimming pool water can penetrate to the hair cortex through the cuticle. It can oxidize and degenerate melanosomes there.
Multidrug-resistant gram-negative rods such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae have presented problems.1,2) In particular, P. aeruginosa is an important species causing nosocomial infection, 3) and its development of multidrug-resistance has become a major social issue. [4][5][6] In Japan, multidrug-resistant P. aeruginosa was defined as isolates resistant to all of the following drugs: imipenem, MIC (minimum inhibitory concentration) Ն16 mg/ml; amikacin, MIC Ն32 mg/ml; and ciprofloxacin, MIC Ն4 mg/ ml. However, some of these isolates resistant to imipenem, ceftazidime, and amikacin are also resistant to all other commonly used antipseudomonal drugs such as piperacillintazobactam, aztreonam, and ciprofloxacin. For such isolates, drugs for treatment are not available at present. Our previous studies have shown that three drug combinations such as the combination of ceftazidime, aztreonam, and amikacin are more effective against multidrug-resistant P. aeruginosa than the combinations of b-lactam and aminoglycoside antibiotics, such as ceftazidime and amikacin. 7,8) Therefore, in this study, we evaluated the effects of three drug combinations of ceftazidime, aztreonam, and amikacin and ceftazidime, aztreonam, amikacin, or colistin alone at a concentration three times the breakpoint concentration on four strains of P. aeruginosa that were isolated from patients in two hospitals in 2002-2006 and resistant to all commonly used antipseudomonal drugs. MATERIALS AND METHODS Bacterial StrainsOf a total of 1720 P. aeruginosa strains isolated from clinical materials in two hospitals 733 and 315 beds, respectively in Yamaguchi Prefecture between January 2002 and December 2006, four strains that were resistant to all of piperacillin, piperacillin-tazobactam, imipenem, meropenem, ceftazidime, aztreonam, amikacin, and ciprofloxacin were used. The sources of the four strains were blood (two strains), bile (one), and frank hip pus (one).Random Amplified Polymorphic DNA (Deoxyribonucleic Acid) Analysis [9][10][11] Genomic DNA was purified by the phenol-chloroform method. The primers 272 (5Ј-AGC-GGGCCAA-3Ј) and ERIC2 (enterobacterial repetitive intragenic consensus; 5Ј-ATGTAAGCTCCTGGGATTTCA-3Ј) were employed. The reaction took place in 1 ml 10ϫPCR (polymerase chain reaction) buffer (Ex Taq, Takara, Tokyo, Japan), 0.8 ml dNTP (deoxyribonucleoside triphosphates) mixture (Ex Taq, Takara, Tokyo, Japan), 0.06 ml Ex Taq (Ex Taq, Takara, Tokyo, Japan), 0.2 ml primer, 80 ng DNA template, 6.94 ml water, and 10 ml mineral oil. Amplification was performed in a DNA thermal cycler (Perkin ELMER 9600) under the following conditions: primer 272; 94°C for 7 min and 94°C for 1 min, 35°C for 1 min, 72°C for 1 min (40 cycles), and 72°C for 16 min. Primer ERIC2 was used under the same conditions except for the following: the temperature was changed from 35 to 52°C, and the number of cycles from 40 to 35. Amplification products were resolved by electrophoresis in a 3% agarose gel and were detected by staining ...
Morphological changes in intraperitoneal free cancer cells of gastric cancer patients treated with continuous hyperthermic peritoneal perfusion
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