Fat and fish intake and asthma in Japanese women: baseline data from the Osaka Maternal and Child Health Study.

OBJECTIVE: It remains controversial whether the intake of n-3 polyunsaturated fatty acids and fish is preventive against asthma. This cross-sectional study investigated the relationship between fat and fish intake and the prevalence of asthma using baseline data from a prospective study. DESIGN: The subjects were 1002 pregnant Japanese females. A diet history questionnaire was used to assess dietary habits. Current asthma and asthma after age 18 were defined as present if subjects had been treated with medications at some time in the previous 12 months and after reaching the age of 18, respectively. RESULTS: Fish consumption was independently associated with a decreased prevalence of asthma after age 18 and current asthma. A significant inverse relationship was observed between the ratio of n-3 to n-6 polyunsaturated fatty acid intake and the prevalence of current asthma, but not asthma after age 18. Intake of total fat, saturated, monounsaturated, n-3 polyunsaturated and n-6 polyunsaturated fatty acids, cholesterol, meat, eggs or dairy products was not evidently related to either outcome for asthma. CONCLUSION: Our results suggest that fish consumption and the high ratio of n-3 to n-6 polyunsaturated fatty acid intake may be associated with a reduced prevalence of asthma in young female Japanese adults.

Magnesium intake is inversely associated with the prevalence of tooth loss in Japanese pregnant women: the Osaka Maternal and Child Health Study.

There have only been a few studies on the role of mineral intake in tooth loss. We investigated the association between mineral intake and the prevalence of tooth loss in Japan. We used the baseline data on 1002 pregnant women who were enrolled in the Osaka Maternal and Child Health Study between November 2001 and March 2003. Tooth loss was defined as the previous extraction of one or more teeth. Nutrient intake was assessed by a validated diet history questionnaire. Prevalence odds ratios and confidence intervals were estimated by applying a multiple logistic regression model. The adjusted odds ratio upon comparison of the highest quartile with the lowest quartile of magnesium intake was 0.64 (95% confidence interval, 0.42-0.99), showing a tendency for an inverse dose-response relationship (p for linear trend = 0.05). There were no associations between the level of consumption of calcium, phosphate, iron, zinc, or copper and tooth loss. The present findings suggest that intake of magnesium is related to reduced prevalence of tooth loss among young Japanese women.

Comparative therapeutic effects of vitamin D3 and its derivatives on experimental renal osteodystrophy.

The comparative effectiveness of vitamin D3 and its derivatives in curing hyperparathyroidism and osteodystrophic bone lesions was examined in a laboratory model of renal osteodystrophy associated with marked secondary hyperparathyroidism in rats. The experimental model was prepared by a single injection of homologous glycopeptide. Plasma levels of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] appeared to decrease in the rats receiving glycopeptide. Various doses of vitamin D3 derivatives [2 or 10 microgram/kg D3, 2 microgram/kg 25-hydroxyvitamin D3 (25OHD3), 0.1 microgram/kg 1 alpha,25(OH)2D3, and 0.1 or 0.2 microgram/kg 1 alpha-hydroxyvitamin D3 (1 alpha OHD3)] were daily administered orally to the nephritic rats for 23 days before sacrifice. 1 alpha,25(OH)2D3 and 1 alpha OHD3 were much more potent than 25OHD3 and D3 in reducing the hyperplasia of parathyroir glands. The potency of 1 alpha OHD3 in curing the histological changes of osteodystrophy appeared to be greater than that of 1 alpha,25(OH)2D3. The same dose level of 1 alpha OHD3 was more effective than 1 alpha,25(OH)2D3 in enhancing plasma 1 alpha,25(OH)2D3 levels.

Direct involvement of hydrogen peroxide in bacterial alpha-hydroxylation of fatty acid.

We have reported that fatty-acid alpha-hydroxylase partially purified from Sphingomonas paucimobilis required NADH and molecular oxygen. In this study, we found that the reaction was greatly inhibited by catalase. Glutathione and glutathione peroxidase also inhibited alpha-hydroxylation, but superoxide dismutase and mannitol did not. Replacement of NADH and molecular oxygen by hydrogen peroxide increased the alpha-hydroxylation activity. In the presence of hydrogen peroxide, molecular oxygen was not required for the activity. These findings suggest that hydrogen peroxide was essential for bacterial alpha-hydroxylase.

Mechanism of the differentiating action of 25-hydroxyvitamin D3 endoperoxides in human myeloid leukemia cells (HL-60).

The action of 25-hydroxy-6,19-dihydro-6,19-epidioxyvitamin D3 [25-(OH)D3 endoperoxides, 2a and 3a] in inducing differentiation of human myeloid leukemia cells (HL-60) was studied by using their radioactive derivatives (2a' and 3a'). When HL-60 cells were incubated with the labeled endoperoxides (2a' and 3a') in serum-free RPMI 1640 medium, no radioactivity was incorporated into either the cytosol or the chromatin fraction of the cells. When the radioactive endoperoxide (2a') was incubated in the culture medium for 3 days, with or without HL-60 cells, about 45% of the compound was similarly converted to 19,25-dihydroxy-6,19-dihydro-6,19-epoxyvitamin D3 (4a) and about 10% to 25-hydroxy-6,19-epoxyvitamin D3 (6a). These two new vitamin D derivatives were synthesized chemically and tested for their biological activities. Both compounds (4a and 6a) were about 2 times as active as 25-(OH)D3 endoperoxides (2a and 3a) and about 7 times as active as 25-hydroxyvitamin D3 (1a) in inducing differentiation of HL-60 cells. The differentiating activity of these compounds was well correlated with their activity in binding to the cytosol receptor for 1 alpha, 25-dihydroxyvitamin D3 in HL-60 cells. The in vitro bone-resorbing activity of 25-hydroxy-6,19-epoxyvitamin D3 (6a) and 25-(OH)D3 endoperoxide (2a) was higher than that of 25-hydroxyvitamin D3 (1a), indicating that the differentiating activity also paralleled the bone-resorbing activity in these vitamin D derivatives. These results suggest that 25-(OH)D3 endoperoxides (2a and 3a) induce differentiation of HL-60 cells and bone resorption after being converted to these two compounds.

Binding of fluorescein monoglucuronide to human serum albumin.

The binding to human albumin of fluorescein monoglucuronide, a fluorescent metabolite of fluorescein, was studied using two methods: pressure dialysis and fluorescence polarization. Both methods indicated that fluorescein monoglucuronide binds to human albumin more loosely than fluorescein. The free fraction in human plasma estimated from the dissociation constant and the number of binding sites was in a range from 31 to 37%. Fluorescence of fluorescein was significantly quenched by the albumin binding, but fluorescence of fluorescein monoglucuronide was not affected by albumin. The relative molar intensity of fluorescence between these fluorophores varied from 3.2 to 37.3, depending on the excitation wavelength.

Cell surface expression of immature H glycoprotein in measles virus-infected cells.

Two forms of hemagglutinin (H) protein, one with an apparent molecular mass of 78 kDa (78K H protein) and the other with that of 74 kDa (74K H protein), are present in cells infected with measles virus (MV). We previously observed that only the mature 78K H protein, a completely glycosylated form of the 74K H protein, was expressed on the cell surface of the infected cells. In the present study, we detected transient expression of the 74K H protein on the cell surface of infected cells by pulse-chase studies, although the level of this expression was much lower than that of the 78K H protein. On the cell surface the 74K H protein was present as dimers and sensitive to endo-beta-N-acetylglucosaminidase H digestion. Treatment with brefeldin A, which blocks the transport of membrane and secretory proteins from the endoplasmic reticulum to the Golgi apparatus, inhibited the cell surface expression of the 78K H protein, but not that of the 74K H protein. These data suggest that a part of the MV 74K H proteins could be transported directly to the cell surface - probably via an alternative pathway - without processing to the complex form in the Golgi apparatus.

Relationship between induction of macrophage chemotactic factors and formation of granulomas caused by mycoloyl glycolipids from Rhodococcus ruber (Nocardia rubra).

Mycoloyl glycolipids cause granulomas in the lungs, liver, and spleen of mice, but the mechanism is not fully understood. To understand the role of macrophage chemotactic factors (MCFs) in granuloma formation, we prepared various mycoloyl glycolipids with different carbohydrate moieties: trehalose dimycolate (TDM), glucose mycolate (GM), mannose mycolate (MM), and fructose mycolate (FM) from Rhodococcus ruber, and examined the relationship between their MCF induction in peritoneal macrophages and the extent of granuloma formation. The molecular mass of each glycolipid was confirmed by fast-atom-bombardment mass-spectrometry. TDM or GM caused granulomas in the lungs, spleen, and liver of ICR mice, but MM and FM did not. The culture supernatant of peritoneal macrophages stimulated with TDM or GM increased macrophage migration, whereas MM and FM had no chemotactic activity. The activity of interleukin-1 (IL-1) in the supernatant was increased equally by each glycolipid and was therefore not related to chemotaxis. Tumor necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were not detected in the four supernatants. The TDM-induced MCF was heat-stable, trypsin-labile, and undialyzable. Furthermore, we separated two MCF active fractions from the supernatant of TDM-stimulated macrophages by gel filtration. These factors acted on macrophages but not on neutrophils. Our results suggested that macrophages recognize the sugar moieties of mycoloyl glycolipids and may, in response, generate a MCF that may play an important role in the macrophage or monocyte recruitment which is essential prior to granuloma formation.

Unique heme environment at the putative distal region of hydrogen peroxide-dependent fatty acid alpha-hydroxylase from Sphingomonas paucimobilis (peroxygenase P450(SPalpha).

Fatty acid alpha-hydroxylase from Sphingomonas paucimobilis is a hydrogen peroxide-dependent cytochrome P450 (P450) enzyme (P450(SPalpha)). In this study, heme-ligand exchange reactions of P450(SPalpha) were investigated using the optical spectroscopic method and compared with those of various P450s. Alkylamines (C >/= 5) induced changes in the spectrum of ferric P450(SPalpha) to one typical of a nitrogenous ligand-bound low-spin form of ferric P450, although their affinities were lower than those for other P450s, and a substrate, laurate, did not interfere with the binding in contrast with in the cases of other P450s. Other compounds having a nitrogen donor atom to the heme iron of P450, including pyridine or 1-methylimidazole, induced no change in the spectrum of P450(SPalpha) in either the ferric or ferrous state. Practically no spectral change was observed on the addition of alkyl isocyanides to ferric P450s. On the other hand, cyanide induced a change in the spectrum of ferric P450(SPalpha) to one characteristic of cyanide-bound form of ferric P450. The affinity of cyanide increased when the substrate was added, in contrast with in the cases of other P450s. Ferrous P450(SPalpha) combined with CO and alkyl isocyanides, and the affinity for CO was of the same order of magnitude as in the cases of other P450s. These findings suggest a unique heme environment of P450(SPalpha), in which most compounds usually acting as external ligands of ferric P450s are prevented from gaining access to the heme iron of P450(SPalpha). The unique properties of the hydroxylase reaction catalyzed by P450(SPalpha), where an oxygen atom of hydrogen peroxide but not of molecular oxygen is utilized and incorporated into a fatty acid at its alpha position, is possibly related with such a specific heme environment of this P450. A possible mechanism for the peroxygenase reaction of P450(SPalpha) is proposed.

Further characterization of hydrogen peroxide-dependent fatty acid alpha-hydroxylase from Sphingomonas paucimobilis.

Although fatty acid alpha-hydroxylase (FAAH) activity has been detected in various species, FAAH has not been sufficiently characterized. In this report, we describe the properties of FAAH highly purified from Sphingomonas paucimobilis. The FAAH was purified by about 5,200-fold. Blotting analysis with a specific antibody against the FAAH showed that its apparent molecular mass was approximately 43 kDa. FAAH showed alpha-hydroxylation activity in the presence of H2O2, but little if any activity with cumene hydroperoxide, t-butyl hydroperoxide, or t-butyl peroxybenzonate. The Km value for H2O2 was 72 microM. Highly purified FAAH oxidized various non-esterified saturated and unsaturated fatty acids including myristic acid, but not myristoyl-CoA. Potassium cyanide and sodium azide inhibited the FAAH activity in a concentration-dependent manner. Other respiratory chain inhibitors such as rotenone and antimycin A did not inhibit the activity. Among cytochrome P450 inhibitors, SKF-525A markedly inhibited the activity at the concentration of 2 mM, but CO did not. Imidazole, an inhibitor of plant alpha-oxidation, showed no inhibitory effect at 1 mM.

Mycolyl glycolipids stimulate macrophages to release a chemotactic factor.

We have previously reported that mycolyl glycolipids from Nocardia rubra such as glucose or trehalose mycolates induced granuloma formation in mice. The structure of the carbohydrate moiety of the mycolyl glycolipids influenced the granuloma forming activity profoundly. Here, we have examined the macrophage-chemotactic activity in the culture supernatants stimulated with various glycolipids differing in carbohydrate moiety (trehalose 6,6'-dimycolate, or TDM; glucose monomycolate, or GM; mannose monomycolate, or MM; and fructose monomycolate, or FM). A distinctive chemotactic activity was detected with TDM or GM, but, little or none with MM or FM.

Uptake of 10 polar organic solvents during short-term respiration.

Respiratory uptake was investigated for 10 polar organic solvents with high blood/air partition coefficients (lambda(blood/air)): ethyl acetate (lambda(blood/air), 77), methyl iso-butyl ketone (90), methyl acetate (90), methyl propyl ketone (150), acetone (245), iso-pentyl alcohol (381), iso-propyl alcohol (848), methyl alcohol (2590), ethylene glycol monobutyl ether (EGBE, 7970), and propylene glycol monomethyl ether (PGME, 12380). Test-air concentrations (Cinh) were 25 to 200 ppm. Four healthy male volunteers inhaled the test air for 10 min at rest and then room air for 5 min. The percentage of solvent in the end-exhaled air and in the mixed-exhaled air increased after the start of the test-air respiration, and reached a quasi-steady-state level within a few min. The speeds of these increases at the start of the test-air respiration became lower as lambda(blood/air) increased. The mean uptakes (U) for the last five min of the test air respiration were 67.3, 52.9, 60.4, 53.0, 52.6, 63.0, 60.3, 60.8, 79.7, and 81.3%, respectively, for ethyl acetate, methyl iso-butyl ketone, methyl acetate, methyl propyl ketone, acetone, iso-pentyl alcohol, iso-propyl alcohol, methyl alcohol, EGBE and PGME. Thus, U values of the alcohols were higher than those of the ketones and lower than the glycol ethers. The overall view, except for esters, showed that U increased with lambda(water/air) increases. This tendency can be explained by a hypothesis that solvent absorbed in the mucus layer of the respiratory tract is removed by the bronchial blood circulation. U values of ethyl acetate and methyl acetate were higher than those of methyl iso-butyl ketone and methyl propyl ketone, though the lambda(blood/air) values of these esters were nearly equal to those of the ketones. For the respiration of the esters, their metabolites, ethyl alcohol and methyl alcohol, were detected in the exhaled air. The exhalation percentage of the metabolites increased after the start of test-air respiration and reached a quasi-steady-state level of 2 and 3%, respectively, by the 5th min. These data suggest that removal of the solvent via metabolism in the wall tissue of the respiratory tract plays an important role for the esters.

Synthesis of desmosterol and epidesmosterol from hyodeoxycholic acid.

A new synthesis of desmosterol was described using hyodeoxycholic acid (3alpha,6alpha-dihydroxy-5beta-cholanic acid) as a starting material. Epidesmosterol (3alpha-hydroxycholesta-5,24-diene) was also synthesized for the first time from the same starting material.

Semisynthetic beta-lactam antibiotics. I. Synthesis and antibacterial activity of new ureidopenicillin derivatives having catechol moieties.

The synthesis and antibacterial activity of new ureidopenicillin derivatives having catechol moieties in the 6-acyl side chain are described. These compounds showed remarkably strong activities against Pseudomonas aeruginosa. Especially, 6-[(R)-2-[3-(3,4-dihydroxybenzoyl)-3-methyl-1-ureido]-2- phenylacetamido]penicillanic acid (7a) had the most potent activity in vitro against Gram-negative bacteria, its activity being 30 approximately 60-fold greater than that of piperacillin against most strains of P. aeruginosa.

Semisynthetic beta-lactam antibiotics. II. Effect on antibacterial activity of ureido N-substituents in the 6-[(R)-2-[3-(3,4-dihydroxybenzoyl)-1- ureido]-2-phenylacetamido]penicillanic acids.

The synthesis and the relationship between in vitro and in vivo activities of 6-[(R)-2-[3-(3,4-dihydroxybenzoyl)-3-R1-1-ureido]-2- phenylacetamido]penicillanic acids having C2 approximately 8 alkyl or substituted alkyl groups as the substituents (R1) are described. In this series, 6-[(R)-2-[3-(3,4-dihydroxybenzoyl)-3-(3-hydroxypropyl)-1-ureido] -2-phenylacetamido]penicillanic acid (1b, AO-1100) showed the most potent protective effect on mice in experimental Pseudomonas aeruginosa infections, although it did not have the strongest in vitro activity among the penicillins we synthesized.

Phytanic acid alpha-hydroxylation by bacterial cytochrome P450.

Fatty acid alpha-hydroxylase, a cytochrome P450 enzyme, from Sphingomonas paucimobilis, utilizes various straight-chain fatty acids as substrates. We investigated whether a recombinant fatty acid alpha-hydroxylase is able to metabolize phytanic acid, a methyl-branched fatty acid. When phytanic acid was incubated with the recombinant enzyme in the presence of H2O2, a reaction product was detected by gas chromatography, whereas a reaction product was not detected in the absence of H2O2. When a heat-inactivated enzyme was used, a reaction product was not detected with any concentration of H2O2. Analysis of the methylated product by gas chromatography-mass spectrometry revealed a fragmentation pattern of 2-hydroxyphytanic acid methyl ester. By single-ion monitoring, the mass ion and the characteristic fragmentation ions of 2-hydroxyphytanic acid methyl ester were detected at the retention time corresponding to the time of the product observed on the gas chromatogram. The Km value for phytanic acid was approximately 50 microM, which was similar to that for myristic acid, although the calculated Vmax for phytanic acid was about 15-fold lower than that for myristic acid. These results indicate that a bacterial cytochrome P450 is able to oxidize phytanic acid to form 2-hydroxyphytanic acid.

Fatty acid-specific, regiospecific, and stereospecific hydroxylation by cytochrome P450 (CYP152B1) from Sphingomonas paucimobilis: substrate structure required for alpha-hydroxylation.

Fatty acid alpha-hydroxylase from Sphingomonas paucimobilis is an unusual cytochrome P450 enzyme that hydroxylates the alpha-carbon of fatty acids in the presence of H2O2. Herein, we describe our investigation concerning the utilization of various substrates and the optical configuration of the alpha-hydroxyl product using a recombinant form of this enzyme. This enzyme can metabolize saturated fatty acids with carbon chain lengths of more than 10. The Km value for pentadecanoic acid (C15) was the smallest among the saturated fatty acids tested (C10-C18) and that for myristic acid (C14) showed similar enzyme kinetics to those seen for C15. As shorter or longer carbon chain lengths were used, Km values increased. The turnover numbers for fatty acids with carbon chain lengths of more than 11 were of the same order of magnitude (10(3) min(-1)), but the turnover number for undecanoic acid (C11) was less. Dicarboxylic fatty acids and methyl myristate were not metabolized, but monomethyl hexadecanedioate and omega-hydroxypalmitic acid were metabolized, though with lower turnover values. Arachidonic acid was a good substrate, comparable to C14 or C15. The metabolite of arachidonic acid was only alpha-hydroxyarachidonic acid. Alkanes, fatty alcohols, and fatty aldehydes were not utilized as substrates. Analysis of the optical configurations of the alpha-hydroxylated products demonstrated that the products were S-enantiomers (more than 98% enantiomerically pure). These results suggested that this P450 enzyme is strictly responsible for fatty acids and catalyzes highly stereo- and regioselective hydroxylation, where structure of omega-carbon and carboxyl carbon as well as carbon chain length of fatty acids are important for substrate-enzyme interaction.

Characterization of the ybdT gene product of Bacillus subtilis: novel fatty acid beta-hydroxylating cytochrome P450.

We have characterized the gene encoding fatty acid alpha-hydroxylase, a cytochrome P450 (P450) enzyme, from Sphingomonas paucimobilis. A database homology search indicated that the deduced amino acid sequence of this gene product was 44% identical to that of the ybdT gene product that is a 48 kDa protein of unknown function from Bacillus subtilis. In this study, we cloned the ybdT gene and characterized this gene product using a recombinant enzyme to clarify function of the ybdT gene product. The carbon monoxide difference spectrum of the recombinant enzyme showed the characteristic one of P450. In the presence of H2O2, the recombinant ybdT gene product hydroxylated myristic acid to produce beta-hydroxymyristic acid and alpha-hydroxymyristic acid which were determined by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry. The amount of these products increased with increasing reaction period and amount of H2O2 in the reaction mixture. The amount of beta-hydroxyl product was slightly higher than that of alpha-hydroxyl product at all times during the reaction. However, no reaction products were detected at any time or at any concentration of H2O2 when heat-inactivated enzyme was used. HPLC analysis with a chiral column showed that the beta-hydroxyl product was nearly enantiomerically pure R-form. These results suggest that this P450 enzyme is involved in a novel biosynthesis of beta-hydroxy fatty acid.

Idiopathic colonic phlebitis with massive ascites.

We describe a 35-year-old woman with colonic phlebitis of unknown origin accompanied by effusion of serum protein into the peritoneal cavity. Abdominal ultrasonography and computed tomography showed massive ascites and edematous wall thickness of the colon. Laboratory examination of the peritoneal fluid showed a high concentration of protein, probably due to nonselective efflux of serum protein. The main histopathological finding was extensive edema of the submucosa with vasculitis in the colon. This kind of phlebitis with massive ascites, but without systemic involvement, and with the ascitic fluid almost identical to the serum protein level, has not been reported previously.

Precise day of ovulation determined by real-time ultrasound evidence of graafian follicular development.

Real-time ultrasonic scanning was performed in 21 infertile Japanese women during 37 menstrual cycles. The maximum diameter prior to ovulation was 23.3 +/- 2.9 mm in spontaneous ovulation cycles, 29.6 +/- 5.2 mm in case of clomiphene therapies, and 26.7 +/- 3.9 mm in HMG-HCG therapies, respectively. Size of the graafian follicles was maximum at almost the same time as the LH peak in the plasma and urine, respectively. The LH peak in the urine was determined by the hemagglutination inhibition assay, the results of which were obtainable within 2 h. Four patients became pregnant (19.0%). There was no statistical correlation between the diameter of the largest follicle and the plasma estradiols (r = 0.28, 0.2 less than P less than 0.3) or between the diameter of the largest follicle and the peak luteinising hormone level (r = 0.27, 0.3 less than P less than 0.4). Therefore, the combination of the real-time ultrasound and a hemagglutination inhibition assay for LH in urine can be clinically applied to detect the precise day of the ovulation.