Munetsugu Kurata

A possible mechanism of induction and translocation into blood stream of rat alkaline phosphatase activity by bile duct ligation.

We investigated the effects of bile duct ligation on alkaline phosphatase (ALP) activities in liver, calvarium, duodenum, and ileum in rats and its possible mechanism of action. ALP isozyme activities in the ligated rats were significantly elevated in the liver and duodenum, while those in the ileum and calvarium were markedly decreased. The ALP isozyme activity elevated by the ligation was obviously suppressed by prior administration of indomethacin, an inhibitor of prostaglandin synthesis. Moreover, phorbol ester also elevated the ALP activity as well as the phosphatase level in the ligated rat. However, other drugs, such as an inhibitor of protein kinase C and calmodulin, showed different effects: calmodulin stimulated an 11.0-, 1.3-, or 1.5-fold increase in ALP activity in the ileum, duodenum, or calvarium, respectively; whereas the hepatic enzyme activity was not affected. The induction by calmodulin was markedly different from that by the ligation. Moreover, imipramine, an inhibitor of protein kinase C, had little effect. These results suggest that prostaglandin is a possible ALP inducer in ligated rats, probably working by elevating the cAMP level. On the other hand, the ligation induced simultaneously de novo synthesis of the membranous and soluble ALP isozymes; and the release rate of the soluble enzyme was greater than that of the membranous isozymes, indicating that the soluble enzyme might be a main source of the induced serum ALP. Lectin affinity chromatography indicated that the soluble enzyme or induced serum enzyme may contain more fucose than that of the membranous one, suggesting that the sugar moiety in the ALP molecule may relate to the clearance of ALP from or its release into the circulation.

A possible mechanism for the changes in hepatic and intestinal alkaline phosphatase activities in bile-duct-ligated rats or guinea pigs

The effects of bile-duct ligation on hepatic and intestinal (jejunum) alkaline phosphatase activities were studied using rats and guinea pigs. In ligated rats, the enzyme activity was increased 4.1-fold in the liver after 24 h and 2.8-fold in the intestine after 12 h. In guinea pigs, the hepatic and intestinal enzyme activities were increased 2.3-fold and 1.5-fold after 100 and 24 h, respectively. The intestinal activity was induced sooner after ligation than hepatic activity. The induction of alkaline phosphatase was inhibited by prior treatment of animals with amanitin, an inhibitor of RNA polymerase activity. This result indicates that the induction is associated with de novo enzyme synthesis. The content of cyclic AMP in liver and intestine increased immediately after ligation. The increase in alkaline phosphatase activities was also inhibited by pretreatment with chlorpromazine, an inhibitor of adenylate cyclase activity. Hence, cellular cyclic AMP may be implicated in playing a role in the induction of alkaline phosphatase by bile-duct ligation.

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.

The effect of human thrombomodulin on the inactivation of thrombin by human antithrombin III.

The effect of human thrombomodulin (TM) on the inactivation of thrombin by human antithrombin III (ATIII) was evaluated in comparison with that produced from rabbits. Human TM did not accelerate the thrombin inhibition by ATIII but rabbit TM enhanced the activity of ATIII. Also inclusion of human TM at increasing concentration suppressed the thrombin inhibitory activity of ATIII. The intensity of ATIII activity in the presence of heparin (0.01U/ml) was also diminished by the human TM. However, this ATIII- heparin cofactor activity recovered with the addition of a 10-fold amount of heparin (0.1U/ml). In SDS-polyacrylamide gel electrophoresis and immunoblotting analysis, we found a complex formation of ATIII with both human and rabbit TM (and further confirmed their presence with isoelectrofocusing electrophoresis- data not shown). These results indicate that human TM is substantially different from rabbit TM. Our results suggest that human TM show the crucial role on protein C activation system via thrombin.

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.

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.

Chemical Properties of Thiobarbituric Acid‐Positive Substances Released from Photobacterial Lipopolysaccharides during Acid Hydrolysis

Lipopolysaccharides (LPSs) are located in the outer membranes of gramnegative bacterial cell walls and exhibit various biological activities (7, 14). Many LPSs have been isolated from various gram-negative bacteria and studied extensively with respect to their structure and biological activities. Most of these LPSs consist of three regions, namely, 1) an O-side chain of repeated sugar units which are specific for the bacterial species or strain, 2) a core region, and 3) lipid A which plays a role in most of their biological activities. Polysaccharides (containing the O-side chain and the core region) and lipid A in their LPSs are linked through 2-keto-3-deoxyoctonate (KDO) (6, 16). The LPS core region contains two or three residues of KDO , a unique acidic sugar, as one of its essential components. Although most LPSs reported so far contain KDO it is known that Vibrio cholerae LPS lacks KDO (10, 11). Recently, it was reported that the LPSs of most of the family Vibrionaceae also lack KDO (9). We also reported that LPSs from several marine photobacteria belonging to the family Vibrionaceae had undetectable or only small amounts of KDO in their constituents (12, 13). More recently, Brade and coworkers reported that both V. cholerae Ogawa and Inaba LPSs released thiobarbituric acid (TBA)-positive substances under strong acid hydrolysis (3) and that these substances were identified as KDO-5-phosphate (2). In the present study, we reexamined marine photobacterial LPSs to clarify whether they contain TBA-positive substances, and our results indicate that TBA-

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.