Fatty acid composition of choline and ethanolamine glycerophospholipid subclasses in heart tissue of mammals and migratory and demersal fish.

The distribution and fatty acid composition of cardiac choline and ethanolamine glycerophospholipids in both migratory and demersal fish and bovine and pig were determined. Phospholipid contents (mg/g heart) were 4.7-9.4 in demersal fish, 14.0-16.5 in migratory fish, and 16.8-20.6 in mammals. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the major components in the phospholipid fraction. Diacyl forms represented 50.2-88.1% of PC in all animals, while plasmalogens comprised 47.0% in bovine, 8.2% in pig and 6.2-7.2% in four species of fish. In PE, plasmalogens varied from 45.0% in bovine and 57.9% in pig to 26.1-29.7% in fish. This glycerophospholipid subclass was identified as containing higher proportions of polyunsaturated fatty acids (PUFAs; 20:4, 20:5, and 22:6) than found in alkylacyl- and diacyl-glycerophospholipids. Qualitative and quantitative differences were found in PE-plasmalogen between land mammals and fish, especially with regard to n-3 fatty acid composition, but no significant difference was noted between migratory and demersal fish.

Mechanism of oxidative damage to fish red blood cells by ozone.

The present study was conducted to elucidate the adverse effects of ozone exposure on rainbow trout (Oncorhynchus mykiss) red blood cells (RBCs). We evaluated whether hemoglobin (Hb) or Hb-derived free iron could participate in the RBC damage using an in vitro ozone exposure system. Ozone exposure induced hemolysis, formation of methemoglobin, and RBC membrane lipid peroxidation. This RBC damage was not suppressed by the addition of a specific iron chelator (deferoxamine mesilate) to the medium but was suppressed by carbon monoxide (CO) treatment before ozone exposure. Generation of hydrogen peroxide (H2O2) in RBC was observed upon ozone exposure but was significantly suppressed by CO treatment before ozone exposure. Thus the Hb status (i.e., Hb redox condition) and H2O2 generation in RBC should play important roles in mediating RBC damage by ozone exposure. In other words, neither ozone nor its derivative directly attacked from the outside of the cell, but ozone that penetrated through the membrane derived the reactive oxygen species from Hb inside of the cell.

Effect of Several Solutions Including Mineral-encaging Zeolites on the Restoration of Cell Motility of Tributyltin-intoxicated Euglena gracilis Z.

To examine the detoxification effect of mineral-encaged zeolites on cells impaired by pollutant-intoxication, we used a bioassay system involving Euglena gracilis Z as the model organism and TBTCl as a pollutant. TBTCl exposure causes Euglena cells to quickly change shape from a spherical to spindle form, with the process being reversible by detoxification. Taking advantage of this morphological characteristic, we examined the restoration of motility by water containing zeolites encaging different minerals. TBTCl-intoxicated Euglena cells were incubated in processed water with different types of mineral-encaging zeolites for up to 3 hours. The restoration of motility was evaluated by observing the number of motile cells with a video microscope. Remarkable recovery was observed in the incubation systems with water containing Fe-, Zn-, and Mn-encaging zeolites. However, the effect was suppressed when the water species were treated with the chelator, Chelex-100(®). An equivalent concentration of FeCl3 to that in the Fe-encaging zeolite processed water did not show significant restoration effect.

Susceptibilities of plasma antioxidants and erythrocyte constituents to low levels of ozone.

To evaluate the susceptibilities of human blood constituents to the low levels of ozone used in ozonated autohemotherapy (40 microgO3/ml), we quantified plasma antioxidants and erythrocyte constituents after rapid mixing of human whole blood with ozone at 20, 40, 60, and 100 microg/ml blood. Ascorbic acid, uric acid, and alpha-tocopherol in plasma decreased as ozone increased, but bilirubin was unaffected. The content of thiobarbituric acid-reactive substances in plasma was increased by ozone. However, the content of thiobarbituric acid-reactive substances and alpha-tocopherol in the erythrocyte membrane was not significantly affected. No significant changes occurred in the content of methemoglobin, cytoskeleton proteins or erythrocyte enzymes such as Na+/K+-ATPase, acetylcholinesterase, catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase at all the ozone levels tested. A decrease in reduced glutathione in erythrocytes was the only significant change caused by the ozone level used for autohemotherapy. It may be one of the chemical events responsible for the beneficial effects of ozonated autohemotherapy.

Effect of haemin on the enterotoxin production of Campylobacter jejuni.

When Campylobacter jejuni was exposed to iron compounds, alterations in enterotoxin production were monitored by enzyme linked immunosorbent assay (ELISA) and Western blotting. By ELISA, treatment with 10 microM haemin increased the ganglioside binding activity of the toxin in the extracellular fraction but not in the intracellular fraction. By Western blotting, a 68 kD subunit of the enterotoxin was observed to increase in concentration. The results suggest that haemin may have a positive regulation effect on enterotoxin maturation when released from bacterial cells.

Reactivity of mammalian 15-lipoxygenase with phospholipids in large unilamellar liposomes.

The reactivity of rabbit reticulocyte 15-lipoxygenase (15-LOX) with phosphatidylcholine (PC) possessing linoleic acid (LA-PC), arachidonic acid (AA-PC), or docosahexaenoic acid (DHA-PC) was investigated by measuring oxygen uptake in the suspension of large unilamellar liposomes (LUV). The 15-LOX reaction with PC LUV was found to follow Michaelis-Menten kinetics. The apparent values of the Michaelis constants (K(m)) of LUV of LA-PC, AA-PC, and DHA-PC were nearly the same (1.0-1.2 mM). However, the maximum velocity (Vmax) of DHA-PC was obviously two to three times lower than those of LA-PC and AA-PC. On the other hand, the oxidizability of PC per bis-allylic H of unsaturated fatty acids in the free radical chain reaction of LUV decreased with increasing degree of unsaturation, that is, LA-PC > AA-PC > DHA-PC. These results suggested that the oxidizability of the DHA moiety incorporated into biomembranes is not necessarily high in lipoxygenase reaction as in free radical chain reaction.

Coulometric detection in high-performance liquid chromatographic analysis of cholesteryl ester hydroperoxides.

A highly sensitive and simple method for the determination of cholesteryl ester hydroperoxides (ChE-OOH) was developed using high-performance liquid chromatography (HPLC) with coulometric electrochemical detection. The lowest detectable level by this technique was 2 pmol for cholesteryl linoleate hydroperoxides at the signal-to-noise ratio of 3. This method was applied to the determination of ChE-OOH presumably present in human plasma. Although ChE-OOH could not be detected, the ChE-OOH level in the fluid was estimated to be less than 27 nM. It was found that the extraction efficiency of an internal standard, cholesteryl nervonate, was decreased by lowering its amount spiked to the plasma. The concentration of ChE-OOH in human plasma and plasma lipoprotein, which were peroxidized with a radical initiator in vitro, could be determined by use of this standard. HPLC-coulometric technique is, therefore, useful to measure the peroxidation of plasma lipids in vitro.

High-performance liquid chromatographic determination of plasma malondialdehyde level without a solvent extraction procedure.

A highly sensitive and simple HPLC method for measuring malondialdehyde (MA) in plasma was developed. For sample preparation, an aliquot of plasma (10 microliters) was mixed with 2.0% thiobarbituric acid (TBA) in 2 M sodium acetate buffer (containing 1 mM DTPA and 0.05% BHT), pH 3.5. After incubating at 95 degrees C for 45 min, the reaction solution was passed through a 0.2-micron membrane filter. An aliquot of 10 microliters filtrate was injected into HPLC system without any extraction procedures. The thiobarbituric acid-malondialdehyde (TBA-MA) complex was separated on reversed-phase HPLC and quantitated with a fluorescence detector. The mobile phase was mixture of acetonitrile:water (7:3, v/v) under isocratic conditions at ambient temperature, and a single analysis was completed in ca. 2.5 min. The minimum detection level for MA was 0.01 pmol. A very close correlation was observed between this method and the Yagi method, which is widely used for clinical diagnosis. The simple and inert mobile phase along with a very simple sample preparation makes this method applicable to numerous clinical samples without any difficulties.

Effect of d-alpha-tocopherol analogues on lipoxygenase-dependent peroxidation of phospholipid-bile salt micelles.

In order to know whether or not vitamin E acts as an effective antioxidant in lipoxygenase-dependent peroxidation of phospholipids, the effect of vitamin E and vitamin E analogues, 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMC) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox C), was investigated in enzymatic lipid peroxidation of bile salt micelles of pig liver phosphatidylcholine (PC) using soybean lipoxygenase. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid was exclusively produced by the reaction with the PC molecular species containing arachidonic acid moiety, indicating that the hydroperoxidation of pig liver PC entirely progresses through the enzymatic reaction. PMC suppressed the accumulation of PC-hydroperoxides (PC-OOH) more efficiently than either d-alpha-tocopherol (alpha-Toc) or Trolox C, and 50% inhibition concentration by PMC was close to that of quercetin, a known lipoxygenase inhibitor from natural origin. The antioxidant activity of PMC was also superior to that of either alpha-Toc or Trolox C in ferrous ion-induced nonenzymatic oxidation of PC micelles in the presence of a trace amount of PC-OOH, although the radical-scavenging activities of these compounds in solution were similar or comparable to one another. In conclusion, PMC is more effective than alpha-Toc as an inhibitor of lipoxygenase reaction with phospholipids and of autoxidation in phospholipids. The phytyl chain of alpha-Toc seems to be unfavorable for exerting an inhibitory effect on lipoxygenase reaction with phospholipid-bile salt micelles.

Immunological properties and ganglioside recognitions by Campylobacter jejuni-enterotoxin and cholera toxin.

The immunological properties of Campylobacter jejuni enterotoxin (CJT) and cholera toxin (CT) were compared by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis with antiserum against each toxin. Antibody against CJT recognized the 68, 54 and 43 kDa polypeptides of CJT and the 11 kDa subunit of CT, whereas antibody against CT recognized the 68 and 54 kDa polypeptides of CJT and 11 kDa subunit of CT. The immunological reactions between the heterogeneous combinations of toxins and the antibodies were weaker than those between the homogenous systems. Thus, different antigenicity was found in CJT and CT at the subunit level, although they possessed cross-reactive epitope(s). The binding of CJT and CT to gangliosides was also examined. CJT and CT bound to GM1 ganglioside preferentially than to other ganglioside species. However, CJT did not bind to GD1b in spite of the fact that CT preferred GD1b. This suggests that both toxins recognize different receptors on the surface of the target cell. This study is the first demonstration of the different properties between CJT and CT in immunological character and ganglioside recognition.

Comparative effects of gamma rays and electron beams on spores of Bacillus pumilus.

The effects of gamma rays and electron beams on the germination, outgrowth and the synthesis of protein and RNA of Bacillus pumilus spores were investigated to clarify the difference in the effects of the two types of radiations on bacterial spores. Gamma irradiation facilitated the germination to a slightly larger degree than electron irradiation. The outgrowth, growth and the synthesis of protein and RNA were inhibited by gamma irradiation to a greater extent than electron irradiation, when the spores were irradiated at the same dose. However, the effects of the two types of radiations were the same when the spores were irradiated with electron beams at a dose 30% higher than gamma rays. The results indicate that the effects of electron beams on bacterial spores and those of gamma rays are qualitatively the same but quantitatively different.

Characteristics of cytotoxin produced by Campylobacter jejuni strains.

The cytotoxic activity against cultured cells of nineteen clinically isolated strains of Campylobacter jejuni was tested. These strains were found to have different profiles in cytotoxin and enterotoxin production, and the characteristics of cytotoxin were further investigated. The cytotoxin showed cell killing toxicity against CHO and HeLa cells. Vacuole formation was observed in the case of rat hepatocyte primary culture. Treatment with trypsin at 80 degrees C for 30 min inactivated the cytotoxin activity, suggesting that the toxin was protein. The toxin was produced in the culture supernatant with high specific activity per protein, followed by polymyxin and CHAPS treatment fractions in this order. This suggests that the cytotoxin was a cell-releasing toxin and that the active toxin was present as a membrane-associated form. The cytotoxin activity was separated from the enterotoxin activity by DEAE-cellulose column chromatography. The washed fraction contained enterotoxin and cytotoxin, whereas the KCl eluted fraction showed mainly cytotoxic activity.

Changes in cell morphology and polyamine composition during growth of Campylobacter jejuni.

Resting cells of Campylobacter jejuni were spherical whereas growing cells were mainly spiral. Content of cadaverine increased with the decrease in spherical forms prior to growth commencing but production of spermidine increased in early log phase. Cadaverine and spermidine are possibly involved in changes in cell morphology and growth, respectively.

Highly sensitive high-performance liquid chromatography for the measurement of malondialdehyde in biological samples.

A highly sensitive method for the measurement of malondialdehyde as thiobarbituric acid-malondialdehyde complex by reversed-phase high-performance liquid chromatography with fluorescence detection in biological samples is described. As samples, 20 microliters of rat plasma or 10% (w/v) liver homogenate mixed with 2.0% thiobarbituric acid in 2 M sodium acetate buffer containing 0.05% butyl hydroxytoluene (pH 3.5) were heated at 95 degrees C for 45 min to give the complex. The complex, extracted with n-butanol, was chromatographed on a system equipped with a reversed-phase column, and the eluted peak was monitored with a fluorescence detector (excitation wavelength 515 nm, emission wavelength 553 nm). The mobile phase was a acetonitrile-water (2:8, v/v) under isocratic conditions at ambient temperature, and a single analysis was done in ca. 4 min. The minimum detection level for malondialdehyde was as low as 0.05 pmol. The n-butanol extract was stable at least for 3 days. The simple mobile phase, the extremely sensitive detection limit, and the stability of the complex make this system applicable to routine clinical analysis with a small amount of tissue or biopsy sample.

Occurrence of tributyltin-tolerant bacteria in tributyltin- or cadmium-containing seawater.

Tributyltin chloride (TBTCl)-tolerant bacteria accounted for 90% of the flora in natural seawater to which TBTCl was added. These tolerant bacteria were insensitive to 250 nmol of TBTCl per disc, and all were Vibrio species. Total counts of viable bacteria did not decrease upon storage of the TBTCl-treated seawater, indicating that enrichment of tolerant strains took place. Addition of CdSO4 to seawater resulted in the occurrence of TBTCl-tolerant bacteria as well as Cd-tolerant bacteria, suggesting some correlation of Cd tolerance and TBTCl tolerance.

Antioxidant activity of xanthophylls on peroxyl radical-mediated phospholipid peroxidation.

The ability of xanthophylls (canthaxanthin, zeaxanthin, and astaxanthin) as chain-breaking antioxidants was investigated in peroxyl radical-mediated peroxidation of phosphatidylcholine (PC) liposomes under atmospheric conditions using lipid-soluble and water-soluble radical generators. These xanthophylls retarded the chain propagation reaction of phosphatidylcholine hydroperoxides (PC-OOH) formation, although their activities to trap chain-carrying peroxyl radical were much less than that of alpha-tocopherol. In chick plasma studies, it was observed that endogenious xanthophylls participated in the antioxidant defenses against the attack of aqueous peroxyl radical. It was concluded that xanthophylls possess the ability to act as chain-breaking antioxidants in the peroxidation of membraneous phospholipids. Dietary xanthophylls may, therefore, be helpful in resisting membraneous phospholipids against oxidative damage in vivo.

Isolation and characterization of tributyltin chloride-resistant marine Vibrio.

Tributyltin chloride (TBTCl)-resistant marine bacteria were isolated from coastal sea water. One of these bacteria (Vibrio M-1) was highly resistant when grown in medium containing 125 microM of TBTCl. This strain was sensitive to other metals. Two polypeptides, 30 kDa and 12 kDa, increased when the strain was cultured in the medium supplemented with TBTCl. Initially TBTCl was taken up by the cell; however, the amount of TBTCl determined in the cellular fraction was low after the exponential growth phase of Vibrio M-1, suggesting the existence of a TBTCl-efflux system.

Acute toxicity of ozone against morphology of gill and erythrocytes of Japanese charr (Salvelinus leucomaenis).

1. Acute toxicity of ozone exposure to Japanese charr (Salvelinus leucomaenis) was studied histopathologically, and hematologically on gill tissue and red blood cells (RBC) under different ozone concentrations (0-0.7 ppm). 2. Exposure of ozone above 0.7 ppm led to characteristic symptoms and all died of choking in 30 min. 3. Many swollen RBC were seen under the scanning electron microscope. 4. RBC congestion was serious in the gill where degeneration of lamellar epithelium was observed. However, injury to chloride cells was not clear.

Inhibition of the electron transport system in Staphylococcus aureus by trimethylamine-N-oxide.

Trimethylamine-N-oxide (TMAO) inhibited the growth of Staphylococcus aureus but not of S. epidermidis, which is the main flora in ripening squid, the Japanese traditional sea food Ika-shiokara. This selective inhibition of S. aureus was based on the inhibition of the electron transport system. The cytochrome fraction isolated from S. aureus was converted from the reduced to the oxidized form by the addition of TMAO. It is suggested that the inhibition occurred between cytochrome B and cytochrome o.