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Biomedical subjects

K Tanamoto

Publications and source records attributed to K Tanamoto.

At least 37 records · Page 2Linked to original sources
PubMed1993

A quantitative microanalysis of bacterial endotoxin using [3H]-labeled L-glycero-D-mannoheptitol as a marker.

Quantitative microanalysis of bacterial endotoxin was performed using [3H]-labeled L-glycero-D-mannoheptitol (LD-Heptitol) as a marker. Several different amounts of authentic L-glycero-D-mannoheptose (LD-Heptose) were reduced with 20 micrograms of cold NaBH4 containing 2 micrograms of NaB3H4 (40 Ci/mmol) in 20 microliters of 1 mM NaOH at 4 C for 48 hr. The product, [1-3H]-labeled LD-Heptitol, has high specific activity, and was purified by HPLC and detected using a liquid-scintillation counter. As little as 50 pg of LD-Heptose was detectable, and the radioactivity increased dose-dependently in the 100 pg to 80 ng range tested. More than 2 ng of Salmonella abortus equi endotoxin could be accurately determined by this method. It is possible to detect 50 pg of endotoxin by this method, if 100% hot material (NaB3H4) is used for [3H]-labeling.

Chromatography, High Pressure Liquid↗
PubMed1992

Succinylated lipid A is a potent and specific inhibitor of endotoxin mitogenicity.

Chemically modified lipopolysaccharides of Salmonella abortus-equi were tested for mitogenicity on mouse spleen cells as well as antagonism of the mitogenicity of intact lipopolysaccharide (LPS). All the lipopolysaccharide preparations deacylated by different alkaline treatments suffered a drastic loss of mitogenicity. The mitogenic activity of lipid A was also lost when succinic residues were introduced on hydroxyl groups. Partially deacylated alkaline-treated preparations (but not completely deacylated preparations) inhibited the activation of splenic B-cells by LPS. They were found to be toxic to spleen cells, however, and to suppress not only the mitogenicity of LPS but that of concanavalin A as well. This inhibitory action was not exhibited when all of the fatty acid was eliminated. Succinylated lipid A, on the other hand, was not toxic to the cells and inhibited the B-cell mitogenicity of lipopolysaccharide (but not the T-cell mitogenicity of concanavalin A). Chemical analysis revealed that about 4.6 mol of succinic acid had been introduced into lipid A by succinylation, and that the fatty acid and phosphate composition was unchanged by this treatment. Macrophages do not seem to participate in this inhibition. Inhibition was observed when succinylated lipid A was added either at the same time or after lipid A mitogen, but optimal inhibition was expressed when it was added to the culture 3 h before LPS. Inhibition was not affected by washing the cells before adding LPS. Inhibition increased as the ratio of suppressor to mitogen increased, suggesting that the succinylated lipid A competes with intact LPS.

Animals↗
PubMed1990

Development of a new quantitative method for detection of endotoxin by fluorescence labeling of 3-hydroxy fatty acid.

New quantitative method for the detection of minute amounts of endotoxin has been developed using 3-hydroxytetradecanoic acid as a chemical marker. After converting 3-hydroxytetradecanoic acid to methyl ester, it was coupled with a fluorescent probe, anthracene-9-carboxyl chloride, obtained by chlorization of 9-anthroic acid with oxalyl chloride. The resulting ester was isolated by HPLC on silica column. The purified product, methyl-3-0-(9-carboxy-anthracenyl) tetradecanoate (M/Z 462), was highly responsive to a fluorescence spectrophotometer, showing maximum emission with excitation wavelength at 257 nm and emission wavelength at 458 nm in dichloromethane, the limit of detection being as little as 10 f mol. Using this method it is currently possible to detect Salmonella abortus equi endotoxin in aqueous solution at a level of 100 pg.

Anthracenes↗
PubMed1990

Endotoxic induction of prostaglandin release from macrophages by nontoxic lipid A analogs synthesized chemically.

The ability of synthetic lipid A analogs to induce prostaglandin synthesis in macrophages was compared with that of native lipopolysaccharide. The synthetic preparations comprised monomeric or dimeric derivatives of D-glucosamine with different patterns of substitution by phosphate and tetradecanoic, (R)-3-hydroxytetradecanoic, and (R)-3-tetradecanoyloxytetradecanoic acid. All of these preparations are structurally distinct from native lipid A (principally regarding the position of fatty acid substitution) and hence have been previously shown to be endotoxically inactive in many biological tests. It was found that many of these synthetic samples exhibit strong activity in inducing prostaglandin E2 and prostaglandin F2 alpha, with some of them having activity comparable to that of lipopolysaccharide. Experiments with macrophages of C3H/HeJ mice enabled us to differentiate between endotoxin-specific (in the case of dimeric preparations) and endotoxin-nonspecific (for monomeric preparations) mechanisms for the induction of prostaglandins. These results indicate that there is a difference in the mechanism of induction of prostaglandin synthesis between monomeric lipid A's and dimeric or native lipid A structures.

Animals↗
PubMed1989

Sensitization of alveolar macrophages to lipopolysaccharide-induced prostaglandin synthesis by exogenous prostaglandins.

Rabbit alveolar macrophages were found to produce extraordinary amounts of prostaglandin E2 and F2 alpha with the stimulation of lipopolysaccharide or lipid A. Exogenous prostaglandin E2 greatly enhanced the lipopolysaccharide action on rabbit alveolar macrophages for the induction of prostaglandin F2 alpha release (3-5 fold), while prostaglandin E2 alone did not cause any effect. The enhancement expressed was especially strong when prostaglandin E2 was administered to the cells simultaneously with lipopolysaccharide. The effect of prostaglandin E2 was observed neither with a nonstimulating dose of lipopolysaccharide nor with a stimulating dose of zymosan. This phenomenon was even more pronounced when prostaglandin I2 was used instead of prostaglandin E2, while no sensitization was demonstrated by prostaglandin F2 alpha. These observations suggest that prostaglandins can modulate the activation of the cyclooxygenase pathway of arachidonate metabolism in the activated macrophages by lipopolysaccharide.

Animals↗
PubMed1989

Deacylation of bacterial lipopolysaccharide in rat hepatocytes in vitro.

The possible role of liver parenchymal cells in the uptake and degradation of bacterial lipopolysaccharide (LPS) was investigated in vitro by employing radiolabelled LPS as substrate. Hepatocytes obtained from Wistar rats by collagenase treatment were found to take up LPS only when it was not linked to the polysaccharide of O-antigen. The amount of LPS taken up increased with time and after 48 h incubation it increased in a dose-dependent manner up to at least 30 micrograms. When incubated with LPS radiolabelled exclusively in the fatty-acid moiety, cultured hepatocytes released lipophilic materials into the culture medium. These were identified as beta-hydroxytetradecanoic acid and triglyceride, in the ratio of 7:I. These results indicate that the R-form of LPS which lacks the O-antigen polysaccharide is taken up and deacylated in hepatocytes, and the derived fatty acids are released into the culture medium either in the free form or after conversion to triglyceride.

Animals↗
PubMed1986

Structure-activity relationship of lipid A: comparison of biological activities of natural and synthetic lipid A's with different fatty acid compositions.

To investigate the structure-activity relationships, various biological activities, including pyrogenicity, lethal toxicity, elicitation of Shwartzman reaction, mitogenicity and tumor necrosis factor (TNF)-inducing activity, were compared among natural and synthetic lipid A's differing in fatty acid composition. In all these tests, natural lipid A's from Escherichia coli and Salmonella minnesota and synthetic LA-15-PP, which carries 3-hydroxy- and 3-acyloxy-tetradecanoyl groups at the 2, 3 and 2', 3' positions, respectively, showed the strongest activities among the tested lipid A's. In contrast, LA-16-PP, in which the amide-bound 3-hydroxytetradecanoic acid at position 2 of LA-15-PP is replaced by 3-hexadecanoyloxytetradecanoic acid, exhibited lower activity than LA-15-PP and natural lipid A's. Although LA-16-PP has been assumed to have a typical Salmonella lipid A structure (and, in fact, it has a structure corresponding to one of the components of Salmonella lipid A), the activity of this synthetic compound was not comparable to that of natural Salmonella lipid A. LA-17-PP, in which tetradecanoic acid is the sole fatty acid component, exhibited relatively strong mitogenicity and TNF-inducing activity, but very low pyrogenicity. The activities of LA-18-PP, which has ester-bound tetradecanoic acid and amide-bound 3-hydroxytetradecanoic acid, were lower than those of LA-17-PP. The results indicate that the differences in fatty acid composition of lipid A's have important influences on the biological activities studied.

Animals↗
PubMed1985

Structural requirements of lipid A responsible for the functions: a study with chemically synthesized lipid A and its analogues.

To confirm the revised lipid A structure of Escherichia coli and to establish the structure responsible for its functions, biological activities of the synthetic compounds based on the presented structure of E. coli lipid A were investigated. Compound 506, 2-deoxy-6-O-(2-deoxy-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O [(R)3-tetradecanoyloxytetradecanoyl]-beta-D-glucopyranosyl]-3-O-[(R) -3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-alpha -D-glucopyranose 1,4'-bis(phosphate), exhibited activities identical to those of natural E. coli lipid A in eliciting Shwartzman reaction and tests on lethality, pyrogenicity, interferon- and tumor necrosis factor-inducing activities as well as in B-cell activating activity and Limulus amebocyte lysate gelating activity. With the exception of the Shwartzman reaction the monophosphorylated synthetic compounds at either the 1 or 4' position showed slightly lower activities than the compound with the bisphosphorylated compound (Compound 506). The compound without the phosphate group showed no or only very weak activities. The structural requirements for each activity (i.e. binding position and composition of fatty acids and presence of phosphate groups) are discussed taking into account the results of previous investigations.

Animals↗
PubMed1984

Biological activities of analogues of lipid A based chemically on the revised structural model. Comparison of mediator-inducing, immunomodulating and endotoxic activities.

Lipid A analogues were chemically synthesized based on the model structure recently revised, and biological activities of the analogues were tested. The analogue, (beta-1,6)-linked glucosamine disaccharide carrying ester-bound 3-hydroxytetradecanoic acids at 3 and 3' position of reducing and nonreducing glucosamine in addition to amide-bound 3-hydroxytetradecanoic acids and glycosidic-linked and ester-linked phosphate groups, showed much stronger activities for mediator inducing and immunomodulating as well as endotoxic activities than those exhibited by the previously synthesized analogues based on the old model. Among the activities tested, induction of interferon and tumor necrosis factor as well as mitogenicity, adjuvanticity and pyrogenicity were, however, not expressed so strongly as natural lipid A used as controls. In contrast, the analogue exhibited comparable activities to those of control lipid A in the test of lethal toxicity to mice and gelating activity of Limulus amebocyte lysate. Other synthetic analogues carrying a phosphate group showed comparable, slightly stronger or weaker activities depending on the test, but nonphosphorylated analogue exhibited no apparent or only very weak activities.

Adjuvants, Immunologic↗
PubMed1984

Biological activities of synthetic lipid A analogs: pyrogenicity, lethal toxicity, anticomplement activity, and induction of gelation of Limulus amoebocyte lysate.

Chemically synthesized lipid A analogs were investigated for several endotoxic activities, including pyrogenicity, lethal toxicity, anticomplement activity, and the capacity to gelate Limulus amoebocyte lysate in comparison to natural lipid A. The synthetic preparations contained D-glucosamine or D-glucosamine-beta-1,6-D-glucosamine disaccharide substituted by ester- and amide-bound hydroxylated or non-hydroxylated fatty acids and by phosphate groups in different combinations. Some preparations which were insoluble in water were succinylated and thus rendered more soluble. Strong biphasic pyrogenic responses with a maximal increase in body temperature of 1 to 2 degrees C were obtained with 50 micrograms/kg doses of 3 disaccharide preparations of 15 tested. With two preparations (50 micrograms/kg) moderate pyrogenicity with monophasic fever curves and a maximal temperature increase of about 0.6 degrees C was obtained. Lethal toxicity tests were carried out in galactosamine-sensitized mice. Of 15 synthetic preparations, 4 exhibited lethal toxicity under these conditions. The effective doses of the lipid A analogs in both in vivo tests were, however, several hundred times higher than those of bacterial lipid A. For the activities in vivo, hydroxyacyl residues seemed to be important. Anticomplement activity was demonstrable in seven preparations, one of which expressed an activity comparable to that of lipid A. Preparations containing non-hydroxylated fatty acids seemed to be most active in this test. None of the synthetic preparations was found to exhibit gelation activity for Limulus amoebocyte lysate when tested in doses up to 0.4 micrograms, whereas bacterial free lipid A was active in doses of about 2 pg. None of the monosaccharide derivatives exhibited any of these activities.

Animals↗
PubMed1984

Mitogenic activities of synthetic lipid A analogs and suppression of mitogenicity of lipid A.

The effect of synthetic lipid A analogs on murine spleen cells was studied. The preparations represented D-glucosamine and D-glucosaminyl-beta 1,6-D-glucosamine disaccharide derivatives substituted in different combinations by ester- and amide-bound fatty acids and by phosphate groups. Significant mitogenic activity was demonstrated with a number of synthetic disaccharide preparations; however, their potency was lower than that of lipid A. The synthetic preparations were not mitogenic for spleen cells from C3H/HeJ mice. Furthermore, the mitogenicity of the synthetic preparations was abolished after binding with polymyxin B. The results indicate that for expression of mitogenicity, a phosphate group at position 1 of the reducing glucosamine and amide-bound acyloxyacyl residues are important factors. Some of the synthetic preparations containing the diglucosamine backbone and expressing relatively low mitogenicity suppressed B-cell mitogenicity of lipid A. Although these preparations were lytic for erythrocytes, they did not affect the viability of the splenic lymphocytes. Suppression was seen when the synthetic preparations were added simultaneously with or after the lipid A mitogen, but optimal suppression was expressed when the preparations were added to the system 3 h before lipid A. Washing of the cells before the addition of lipid A did not affect the results. The suppression was not due to the induction of suppressor cells by the synthetic preparations. The disaccharide preparations did not inhibit T-cell mitogenicity of concanavalin A. In contrast to the disaccharide preparations, the monosaccharide preparations suppressed mitogenicity of both lipid A and concanavalin A, probably because of their direct toxicity for lymphocytes.

Animals↗
PubMed1982

Essential regions of the lipopolysaccharide of Pseudomonas aeruginosa responsible for pyrogenicity and activation of the proclotting enzyme of horseshoe crabs. Comparison with antitumor, interferon-inducing and adjuvant activities.

Regions of lipopolysaccharide derived from Pseudomonas aeruginosa essential for pyrogenicity and activation of the proclotting enzyme of the horseshoe crab were examined. Free lipid A with intact fatty acids showed strong pyrogenicity but showed little activation of the proclotting enzyme. Chemical modification of the polysaccharide portion and deacylation of the lipopolysaccharide diminished activation of the proclotting enzyme. The native-protein portion attached to the lipopolysaccharide also inhibited the activation of proclotting enzyme by lipopolysaccharide, but not pyrogenicity. These results indicate that free lipid A is sufficient for pyrogenicity, whereas the complete lipopolysaccharide is the strongest activator of the proclotting enzyme. The lipopolysaccharide of P. aeruginosa, which showed the strongest activation of proclotting enzyme, showed the weakest pyrogenicity of all the lipopolysaccharides tested here. All these results demonstrate that there is not correlation between pyrogenicity and proclotting enzyme activation induced by lipopolysaccharides.

Animals↗
PubMed1979

Regions of the lipopolysaccharide of Pseudomonas aeruginosa essential for antitumor and interferon-inducing activities.

Resistance against ascites tumor development and interferon-inducing activity were demonstrated in lipopolysaccharide derived from the protein-lipopolysaccharide complex obtained from an autolysate of Pseudomonas aeruginosa. Lipid A obtained from the lipopolysaccharide was sufficient to induce interferon in vitro but no antitumor activity was found if lipid A or the polysaccharide derived from lipopolysaccharide was injected into the animal. Chemical modification of the polysaccharide portion or deacylation of the lipopolysaccharide also diminished antitumor activity. In contrast, interferon was induced by these incomplete lipopolysaccharides. These results indicate that both the lipid A portion and covalently linked polysaccharide are necessary for the inhibition of ascites tumor development, whereas incomplete lipid A with amide-linked fatty acids is sufficient to induce interferon in vitro.

Amino Acids↗
PubMed1978

Effectiveness of immunization with single and multi-component vaccines prepared from a common antigen (OEP), protease and elastase toxoids of Pseudomonas aeruginosa on protection against hemorrhagic pneumonia in mink due to P. aeruginosa.

Toxoids of protease and elastase of Pseudomonas aeruginosa were successfully prepared by treatment with 8% formalin plus 0.2 m lysine and by 4% formalin respectively. The two toxoids proved sufficiently potent to elicit high antibody titers as estimated by both the enzyme-neutralizing and passive hemagglutination tests. The effectiveness of immunizing minks with a single component-vaccine consisting of the common antigen (OEP) of P. aeruginosa or the protease toxoid (PT) or the elastase toxoid (ET) and with the two (PT and ET) or the three (OEP, PT and ET) component-mixed vaccines, on hemorrhagic pneumonia in minks due to the bacteria was investigated. Female Sapphire minks, 3.5 months old, were used in two experiments performed in 1975 and 1976. Minks were immunized three times in one month with a total of 1 mg of each of the three antigens in the case of a single component vaccine and with a total of 2 or 3 mg (equal amounts of each component) in the case of the two or three component vaccines. Two or three weeks after the last immunization, challenge exposure with strain No. 5 was carried out by intranasally inoculating an inoculum containing serial dilutions of 10(3) -10(10) of live bacteria. Summarizing the results of the two experiments, in the case of controls, nonimmunized minks and minks immunized with potassium aluminum sulfate (potash alum) alone, the LD50 values were approximately 10(3) -10(4) with no significant difference between the two. In the case of OEP-vaccinated minks, the LD50 value was about 10(6) and thus clearly differed from those of the controls. In minks immunized with the three-component-vaccine, however, the LD50 value was about 10(8) -10(9), which indicated that the three-component-mixed vaccine was remarkably more effective than the single OEP vaccine component. In minks immunized with either PT or ET or both, the LD50 values were about 10(8) -10(9). The effectiveness of the vaccine made with ET or PT alone is discussed in the text. The pathological findings of the minks which died or survived are described.

Animals↗