Biotransport of metallic trace elements from marine to terrestrial ecosystems by seabirds.

Physical systems, such as currents and winds, have traditionally been considered responsible for transporting contaminants. Although evidence is mounting that animals play a role in this process through their movements, we still know little about how such contaminant biotransport occurs and the extent of effects at deposition sites. In the present study, we address this question by studying how rhinoceros auklets (Cerorhinca monocerata), a seabird that occurs in immense colonies (∼300 000 pairs at our study site, Teuri Island), affect contaminant levels at their colony and at nearby sites. More specifically, we hypothesize that contaminants are transported and deposited by seabirds at their colony and that these contaminants are passed on locally to the terrestrial ecosystem. To test this hypothesis, we analyzed the concentration of 9 heavy metal and metalloids, as well as δ13 C and δ15 N stable isotopes, in bird tissues, plants, and soil, both within and outside of the colony. The results show that rhinoceros auklets transport marine-derived mercury (Hg), possibly from their wintering location, and deposit Hg via their feces at their breeding site, thereby contaminating plants and soils within the breeding colony. The present study confirms not only that animals can transport contaminants from marine to terrestrial ecosystems, potentially over unexpectedly long distances, but also that bird tissues contribute locally to plant contamination. Environ Toxicol Chem 2019;38:106-114. © 2018 SETAC.

Role of carbonate burial in Blue Carbon budgets.

Calcium carbonates (CaCO3) often accumulate in mangrove and seagrass sediments. As CaCO3 production emits CO2, there is concern that this may partially offset the role of Blue Carbon ecosystems as CO2 sinks through the burial of organic carbon (Corg). A global collection of data on inorganic carbon burial rates (Cinorg, 12% of CaCO3 mass) revealed global rates of 0.8 TgCinorg yr-1 and 15-62 TgCinorg yr-1 in mangrove and seagrass ecosystems, respectively. In seagrass, CaCO3 burial may correspond to an offset of 30% of the net CO2 sequestration. However, a mass balance assessment highlights that the Cinorg burial is mainly supported by inputs from adjacent ecosystems rather than by local calcification, and that Blue Carbon ecosystems are sites of net CaCO3 dissolution. Hence, CaCO3 burial in Blue Carbon ecosystems contribute to seabed elevation and therefore buffers sea-level rise, without undermining their role as CO2 sinks.

Alterations of fatty acyl turnover in macrophage glycerolipids induced by stimulation. Evidence for enhanced recycling of arachidonic acid.

Glycerophospholipid biosynthesis by the de novo pathway was assessed in mouse peritoneal macrophages by pulse-labeling with [U-14C]glycerol. Phosphatidylcholine (PC), which amounts to about 35% of total cellular phospholipids, exhibited the highest rate of glycerol uptake, followed by phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Remodeling of PC molecular species by deacylation/reacylation was established by determining the redistribution of glycerol label over 2 h after a 1 h pulse of [U-14C]glycerol and by determining incorporation of 18O from H2(18)O-containing media. These data suggest that stearic and arachidonic acid enter PC primarily by the remodeling pathway but that small amounts of highly unsaturated molecular species, including 1,2-diarachidonoyl PC, are rapidly synthesized de novo, and subsequently remodeled or degraded. Treatment of the cells with the ionophore A23187 resulted in the selective enhancement of arachidonate turnover in PC, PI and neutral lipid, as well as enhanced de novo PI synthesis. [U-14C]Glycerol labeling experiments suggest that arachidonic acid liberated by Ca(2+)-dependent phospholipase A2 activity is also reacylated in part through de novo glycerolipid biosynthesis, leading to the formation and remodeling of 1,2-diarachidonoyl PC and other highly polyunsaturated molecular species.

Biosynthesis and turnover of anandamide and other N-acylethanolamines in peritoneal macrophages.

Polyunsaturated N-acylethanolamines (NAEs), including anandamide (20:4n-6 NAE), elicit a variety of biological effects through cannabinoid receptors, whereas saturated and monounsaturated NAEs are inactive. Arachidonic acid mobilization induced by treatment of intact mouse peritoneal macrophages with Ca2+ ionophore A23187 had no effect on the production of NAE or its precursor N-acylphosphatidylethanolamine (N-acyl PE). Addition of exogenous ethanolamine resulted in enhanced NAE synthesis by its N-acylation with endogenous fatty acids, but this pathway was not selective for arachidonic acid. Incorporation of (18)O from H2 (18)O-containing media into the amide carbonyls of both NAE and N-acyl PE demonstrated a rapid, constitutive turnover of both lipids.

Anandamide and other N-acylethanolamines in mouse peritoneal macrophages.

N-acyl phosphatidylethanolamine (N-acyl PE) and free N-acylethanolamine (NAE) in mouse peritoneal macrophages were identified and quantified by gas chromatography-mass spectrometry (GC-MS) of tertbutyldimethylsilyl derivatives in the presence of internal standards synthesized from [1,1,2,2-2H4]ethanolamine. N-acyl PE was present at a level of 123-187 pmol/mumol lipid P (521-768 pmol/10(8) cells), with arachidonic acid making up about 3-4% of the N-acyl moieties. NAE, on the other hand, was present at a level of only 17-30 pmol/mumol lipid P (70-121 pmol/10(8) cells), with N-arachidonoylethanolamine (anandamide) making up less than 1% of total NAE. Use of deuterium labeled internal standards and optimization of GC-MS conditions makes it possible to detect as little as 0.1 ng of saturated and 1 ng (3 pmol) of polyunsaturated NAEs in a lipid extract. The present method can be used to determine agonist-induced changes in the levels and compositions of N-acyl PE and NAE.

Rapid purification of extracted bacterial lipopolysaccharides by continuous free-flow electrophoresis.

The use of continuous free-flow electrophoresis for the purification of extracted lipopolysaccharides ( LPSs ) was investigated. Commercial (nucleic acid contaminated) LPS preparations, isolated by the hot phenol-water method of Westphal from Salmonella typhimurium and Escherichia coli 0111: B4, were analyzed. Continuous free-flow electrophoresis for purification of crude LPSs proved to be a rapid and useful means for the continuous purification of large amounts of LPS (more than 45 mg crude LPS per hr) and it showed good reproducibility and pure LPS. The electrophoretic profile of both crude LPSs obtained by continuous free-flow electrophoresis showed two distinct, sharp peaks; one representing the nucleic acid fraction and the other the LPS fraction. Under the continuous free-flow electrophoresis conditions employed, nucleic acid in the crude LPSs possessed low electrophoretic mobility, whereas LPS migration was negligible. Thus for both preparations pure LPS (no detectable nucleic acid) was obtained. Electrophoretic profiles of these purified LPSs on sodium dodecylsulfate-polyacrylamide gel electrophoresis were similar in both cases to those of crude LPS and of LPS purified by repeated ultracentrifugation. By immunological analysis using double immunodiffusion and immunoelectrophoresis, it was found that two components of crude E. coli 0111: B4 LPS were eliminated by continuous free-flow electrophoresis, but each component of purified E. coli 0111: B4 LPS was immunologically identical to the corresponding component in its crude LPS. In S. typhimurium LPS, none of its components were influenced by continuous free-flow electrophoresis but not by ultracentrifugation. In spite of these results, both purified LPSs possessed stronger mitogenic activity than each crude LPS. These results indicated that continuous free-flow electrophoresis is a useful means of purifying extracted crude LPS.

Chemical and biological properties of Lipopolysaccharides from symbiotic luminous bacteria from several luminous marine animals.

The chemical and biological properties of lipopolysaccharides (LPS) in five strains of symbiotic luminous bacteria isolated from four species of luminous marine fishes, Coelorhynchus kishinouyei (CK-1), Chlorophthalmus albatrossis (CA-1), Ventrifossa garmani (VG-1), and Acropoma japonicum (AJ-1b), as well as from a luminous squid, Doryteuthis kensaki (DK-1) were examined. The LPS isolated from these symbiotic luminous bacteria were characterized by the absence of 2-keto-3-deoxyoctonate, known to be a basic component of the usual gram-negative bacterial LPS. All LPS from these symbiotic luminous bacteria upon electrophoresis in sodium dodecylsulfate polyacrylamide gel exhibited one or two clear main bands with high mobility, and one or two obscure minor bands with low mobility when stained with periodate-Schiff reagent. LPS from CA-1 and VG-1 exhibited similar electrophoretic patterns, whereas the electrophoretic patterns of the LPS from CK-1, AJ-1b, and DK-1 were easily distinguishable from each other. All these LPS also had similarly potent and diverse biological activities in regard to their adjuvanticity, immunosuppression, polyclonal effect, B-cell mitogenicity, and activation of the phagocytic function of macrophages.

Determination of abundance and biovolume of bacteria in sediments by dual staining with 4',6-diamidino-2-phenylindole and acridine orange: relationship to dispersion treatment and sediment characteristics.

We measured the abundance and biovolume of bacteria in intertidal sediments from Tokyo Bay, Japan, by using a dual-staining technique (4',6-diamidino-2-phenylindole and acridine orange) and several dispersion techniques (ultrasonic cleaner, ultrasonic sonicator, and tissue homogenizer). Dual staining reduced serious background fluorescence, particularly when used for silt-, clay-, and detritus-rich sediments, and allowed us to distinguish bacteria from other objects during both counting and sizing. Within the studied samples, the number of bacterial cells ranged from 0.20 x 10(9) to 3. 54 x 10(9) g of wet sediment(-1). With the cleaner and sonicator treatments, the bacterial numbers for all of the sites initially increased with dispersion time and then became constant. For the homogenizer treatments, the highest bacterial numbers were observed with the shortest (0.5- to 2-min) treatments, and the counts then declined steeply as the homogenization time increased, indicating that cell destruction occurred. The cleaner treatment had the possibility of insufficient dispersion of bacteria for fine-grain sediments. Within the studied samples, the bacterial biovolume ranged from 0.07 to 0.22 microm(3). With the cleaner and sonicator treatments, the biovolume peaked during the shorter dispersion time. This pattern was caused not by cell destruction but by the incremental portion of dispersed small cells. We concluded that with the cleaner and sonicator treatments, the longer dispersion time reflected the real size spectrum and was preferable for accurate estimation of mean bacterial biovolumes.

Chemical and biological properties of lipopolysaccharide from a marine bacterium, Photobacterium phosphoreum PJ-1.

The chemical and biological properties of the lipopolysaccharide (LPS) isolated from a marine bacterium, Photobacterium phosphoreum PJ-1, were studied. This LPS consists of 40.6% carbohydrate, 27.3% fatty acid, 0.2% 2-keto-3-deoxyoctonate (KDO) and other components. One characteristic of this LPS is its small amount of KDO, the basic component of the usual LPS. Electrophoresis in sodium dodecylsulfate polyacrylamide gel revealed at least two staining bands for carbohydrates. These bands were continuous and broad, and showed rapid electrophoretic mobility which corresponded closely to the fastest moving band of LPS from Salmonella typhimurium. This LPS preparation had adjuvant activity, lethality for ddY mice, and the ability to gel Limulus amebocyte lysate, and the strength of these activities corresponded closely to those of LPS preparations from Escherichia coli 0111:B4 and S. typhimurium. In the test for lethality of the LPS for ddY mice, the lethal action appeared in two phases depending on the dose used for intravenous (i.v.) injection: the early lethal action appeared within 30 min after injection of 250 micrograms or less, and the late lethal action occurred gradually after 16 hr at doses of 500 microgrms or more. The total (both phases) LD50 of this LPS (i.v.) for ddY mice was 265 micrograms per mouse and in only the late phase it was 500 micrograms. These results show that in spite of structural differences in regard to KDO content, LPS from P. phosphoreum PJ-1 has some biological properties similar to those of LPS from E. coli 0111:B4 and S. typhimurium but it shows no immunological cross-reaction with other LPS.

Assessment of phospholipid deacylation-reacylation cycles by a stable isotope technique.

Incorporation of 18O into glycerophospholipids was determined after incubating mouse peritoneal exudate cells for 1 or 2 h in media containing 40% H(2)18O. Gas chromatography-mass spectrometry of hydrogenated fatty acid methyl esters showed highest amounts of 18O in choline phospholipids and phosphatidylinositol. Acyl groups generally present at the sn-1 position contained at least as much carbonyl 18O as those at the sn-2 position. Considering the route of 18O incorporation via free fatty acid derived through ester hydrolysis in H(2)18O, acyl turnover in certain peritoneal exudate cell phospholipids may equal or exceed 20% per h.

Effects of the release of arachidonic acid from 1-ether-linked phospholipids on opsonized-zymosan stimulated rabbit alveolar macrophages.

On rabbit alveolar macrophages prelabeled with double tracers of [3H]arachidonic acid (20:4) for 30 min (short-term labeling) and of [14C]20:4 for 12 h (long-term labeling), the relationship between phospholipid subclasses and the release of 20:4 on stimulation with opsonized-zymosan (OZ) was investigated. Stimulation with 500 micrograms of OZ/ml for 1 h caused a significant decrease in the radioactivity of incorporated [3H]20:4 in all phosphatidylcholine (PC) subclasses of the cells: by 48% in 1-O-alkyl-2-acyl-sn-glycerol-3-phosphocholine (alkylacyl-GPC), and by 24% in 1,2-diacyl-GPC (diacyl-GPC), and slightly in 1-O-alk-1'-enyl-2-acyl-GPC (alkylacyl-GPC). In phosphatidylethanolamine (PE) a significant decrease in the incorporated [3H]20:4 was also observed in the 1,2-diacyl-sn-glycerol-3-phosphoethanolamine (diacyl-GPE, 45%). On the other hand, the radio activity of [14C]20:4 incorporated by long-term labeling into PC decreased significantly, by approximately 40%, in the alkylacyl-GPC and slightly in the alkenylacyl-GPC. In PE, a significant decrease (approximately 32%) in radioactivity of [14C]20:4 was observed only in the alkenylacyl-GPE which incorporated the largest amount of [14C]20:4 among the cellular phospholipids in its labeling and which showed the biggest decrease in [14C]20:4 in the PC and PE subclasses on stimulation with OZ. These results suggest that on OZ stimulation of rabbit alveolar macrophages, PC and PE subclasses involved in the mobilization of 20:4 depend on each 20:4 pool which is extracted in the cells.

Release of 6-keto-prostaglandin F1 alpha and thromboxane B2 from mouse peritoneal macrophages during their adhesion and spreading on a glass surface.

The release of 6-keto-prostaglandin F1 alpha (6KF1 alpha) and of thromboxane B2 (TXB2) from cells were investigated using mouse peritoneal exudate cells (PECs) and non-cultured peritoneal macrophages. They were prepared by adhesion to glass dishes and incubated for 1 hr at 37 degrees C in 5% CO2 in air. Both the percentage of spreading macrophages and the release of 6KF1 alpha and TXB2 increased in proportion to the incubation time. 6KF1 alpha and TXB2 were released from the macrophages, not from the non-coated glass dishes, the spreading of macrophages was hardly detected and lower amounts of 6KF1 alpha and TXB2 were released from these cells compared with cells incubated in non-treated glass dishes. These findings suggest that adhesion with the correlated spreading of macrophages on glass dishes serve as a considerable physical factor for the release of 6KF1 alpha and TXB2.

Relationship between luminous fish and symbiosis. I. Comparative studies of lipopolysaccharides isolated from symbiotic luminous bacteria of the luminous marine fish, Physiculus japonicus.

In order to investigate the relationship between host and symbiosis in the luminous marine fish, Physiculus japonicus, the bacterial lipopolysaccharides (LPS) of symbiotic luminous bacteria were compared serologically and electrophoretically. Five symbiotic luminous bacteria (PJ strains) were separately isolated from five individuals of this fish species caught at three points, off the coasts of Chiba, Nakaminato, and Oharai. LPS preparations were made from these bacteria by Westphal's phenol-water method and highly purified by repeated ultracentrifugation. These LPSs contained little or no 2-keto-3-deoxyoctonate and had powerful mitogenic activity. In sodium dodecylsulfate polyacrylamide gel electrophoresis, these PJ-1 to -5 LPSs were separated by their electrophoretic patterns into three groups; the first group included PJ-1 and PJ-4, the second group PJ-2 and PJ-3, and the third group PJ-5 alone. The results agreed with those of the double immunodiffusion test; precipitin lines completely coalesced within each group but not with other groups. In immunoelectrophoresis, one precipitin line was observed between anti PJ-2 LPS serum and PJ-5 LPS but the electrophoretic mobility of PJ-5 LPS was clearly different from that of the PJ-2 LPS group. Furthermore, in a 50% inhibition test with PJ-2 LPS by the passive hemolysis system, the doses of PJ-2 LPS, PJ-3 LPS, and PJ-5 LPS required for 50% inhibition (ID50) in this system were 0.25, 0.25, and 21.6 micrograms/ml for each alkali-treated LPS, respectively, and the ID50's of both PJ-1 LPS and PJ-4 LPS were above 1,000 micrograms/ml. These results indicate that PJ-5 LPS has an antigenic determinant partially in common with LPS from the PJ-2 group but not with LPS from the PJ-1 group and that the symbiotic luminous bacterium PJ-5 is more closely related to the PJ-2 group than to the PJ-1 group. These results show that the species Physiculus japonicus is symbiotically associated with at least three immunologically different strains of luminous marine bacteria in its specialized light organ.

Partial purification and immunological aspects of carboxylesterase from rat liver microsomes.

Carboxylesterase (CEase) was solubilized from rat liver microsomes by autolysis followed by cholate treatment and then purified by the combination of ammonium sulfate fractionation, gel filtration, chromatography on DEAE Sephadex A-50 and hydroxyapatite and preparative Disc electrophoresis. The overall purification was 25-fold with a yield of 6% of the original enzyme activity. Analytical Disc electrophoresis of the final enzyme preparation showed a single band. However, SDS polyacrylamide gel electrophoresis revealed one main band of 93% and three other minor bands. To investigate the interaction between CEases of rat, monkey, pig and rabbit liver microsomes, rabbit antibody to the above enzyme preparation was prepared and immunological analyses, i.e., Ouchterlony's test and immunoelectrophoresis, were performed. In the comparative double diffusion test, the partial fusion of precipitation line between anti-rat CEase and the enzymes of other species was observed. In the second analysis, sharp arc precipitation lines also could be seen in all specimens and, furthermore, mobilities of each enzyme were different. These observations suggest that rat liver CEase seems to be immunologically related in part but not completely identical with the CEases of other species and the charge difference may exist in these specimens.