Toyoyasu Kuwae

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.

Pro-apoptotic protein glyceraldehyde-3-phosphate dehydrogenase promotes the formation of Lewy body-like inclusions.

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) has long been recognized as a classical glycolytic protein; however, previous studies by our group and others have demonstrated that GAPDH is a general mediator initiating one or more apoptotic cascades. Our most recent findings have elucidated that an expression of a pro‐apoptotic protein GAPDH is critically regulated at the promoter region of the gene. Apoptotic signals for its subsequent aggregate formation and nuclear translocation are controlled by the respective functional domains harboured within its cDNA component. In this study, coexpression of GAPDH with either wild‐type or mutant (A53T) α‐synuclein and less likely with β‐synuclein in transfected COS‐7 cells was found to induce Lewy body‐like cytoplasmic inclusions. Unlike its full‐length construct, the deleted mutant GAPDH construct (C66) abolished these apoptotic signals, disfavouring the formation of inclusions. The generated inclusions were ubiquitin‐ and thioflavin S‐positive appearing fibrils. Furthermore, GAPDH coimmunoprecipitated with wild‐type α‐synuclein in this paradigm. Importantly, immunohistochemical examinations of post mortem materials from patients with sporadic Parkinsons disease revealed the colocalized profiles immunoreactive against these two proteins in the peripheral zone of Lewy bodies from the affected brain regions (i.e. locus coeruleus). Moreover, a quantitative assessment showed that about 20% of Lewy bodies displayed both antigenicities. These results suggest that pro‐apoptotic protein GAPDH may be involved in the Lewy body formation in vivo, probably associated with the apoptotic death pathway.

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 18O from H2 18O‐containing media into the amide carbonyls of both NAE and N‐acyl PE demonstrated a rapid, constitutive turnover of both lipids.

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.

Release of 6-keto-prostaglandin F1α 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.

Immunochemical Comparisons among Lipopolysaccharides from Symbiotic Luminous Bacteria Isolated from Several Luminous Marine Animals

The luminous marine fish may be divided into those that emit visible light by their own ability and those whose light is produced with the aid of symbiotic luminous bacteria or luminous substances of ingested crustaceans in their light organs (5). The symbiotic luminous bacteria inhabiting the light organs of their hosts are of great interest in regard to whether the relationship between the bacteria and the host is specific or not. Fitzgerald (3) and Reichelt et al (10) reported that certain species of luminous fish possess specific species of luminous bacteria in their light organs. On the other hand, Imai (6) reported that the luminous bacteria isolated from different fishes within the same species are not identical immunochemically. However, more detailed relationships between the hosts and the symbionts have not been clarified. Lipopolysaccharides (LPS) located in the cell walls of gram-negative bacteria not only have diverse biological effects, but also possess an a-antigenic region specific for the bacterial strain. In the present study, we investigated the relationship between the hosts and the symbionts by immunochemically studying the LPS from symbiotic luminous bacteria obtained from several different hosts. In this study, we used symbiotic luminous bacteria, facultative anaerobic enterobacteria, isolated from the light organs of five species (five genera) of luminous fish (Physiculus japonicus [PJ-I], Coelorhynchus kishinouyei [CK-I], and Ventrifossa garmani [VG-I] in the order Gadida; Chlorophihalmus albatrossis [CA-I] in the order Clupeida: and Acropoma japonicum [AJ-I b] in the order Percida) and from the light organ ofa luminous squid tDoryteuthis kensaki [DK-I]). For the name of the luminous bacterium isolated from each host, the abbreviation given in brackets ([]) is used hereafter. The morphological aspects and the derivations of these symbionts have been reported by Fukasawa (4). We identified luminous bacteria PJ-I, CK-I, VG-I, and CA-I as Photobacterium phosphoreum from the results obtained by comparison with the criteria of Bergeys manual (8th edition) (I) and with standard

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.

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.

Immunological Properties of Lipopolysaccharide from a Marine Bacterium,Photobacterium phosphoreum PJ-1

It is well known that lipopolysaccharides (LPS) localized in the cell walls of gram-negative bacteria perform diverse biological activities in animals. A number of LPS have already been isolated from terrestrial gram-negative bacteria and extensively studied regarding their chemical and biological properties (9, 12). Although some marine bacteria that have been isolated from seawater and from the surfaces and intestinal contents of fish and other marine animals have also been studied (2), there are only a few reports about LPS from such marine bacteria (4, 7). The genus of photobacteria among marine bacteria has the ability to emit light and has been widely employed in studies on the mechanism of bioluminescence in luminous bacteria (6). More recently, we found that purified LPS isolated by the hot phenol-water method ofWestphal andjann (14) from the symbiotic luminous marine bacterium Photobacterium phosphoreum PJ-l has a low content of 2-keto-3deoxyoctonate (KDO: 0.2% wfw in terms of KDO) and diverse biological activities similar in extent to those of the LPS from both Escherichia coli 0111 :B4 and Salmonella typhimurium (manuscript in preparation). Therefore, P. phosphoreum PJ-1 LPS may appear to merit further study. In this paper, we report that P. phosphoreum PJ-1 LPS has potency as an adjuvant, immunosuppressant, B-cell mitogen, and phagocytic stimulant of macrophages, as do the LPS from both E. coli 0111 :B4 and S. typhimurium. The derivation of P. phosphoreum PJ-1, a facultative anaerobic enterobacterium, was described in a previous paper (8). The mouse strains used in the present experiment were C3H(HeN and C57BL/6J (obtained from CLEA Japan Inc., Tokyo); C3HfHeJ (generously provided by Dr. H. Nariuchi of The University of Tokyo and subsequently bred in our laboratory); congenitally athymic (nude) mice, BALBfcA (generously provided by Dr. Y. Tanimoto of the Central Institute for Experimental Animals, Kawasaki); and ddY (obtained from specific pathogenfree colonies at the Shizuoka Experimental Animal Cooperative, Hamamatsu). Male mice (6 to 9 weeks old) of the above strains were employed. LPS from both E. coli 0111 :B4 and S. typhimurium (Westphals phenol-water method: Difco Laboratories, Detroit, Mich.) were purified by repeated ultracentrifugation (105,000 x g for 2 hr) as was the case with P. phosphoreum PJ-1 LPS and used for reference in all experiments. P. phosphoreum PJ-1 LPS obtained consisted of 40.6% carbohy-

Relationship between Luminous Fish and Symbiosis

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.