Staining of the O-specific polysaccharide chains of lipopolysaccharides with alkaline bismuth.
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Bismuth↗
Biomedical subjects
N Kido
Publications and source records attributed to N Kido.
Inhibition by succinyl concanavalin A of strong adjuvant activity of lipopolysaccharides which possess mannans as the O-specific polysaccharide chains.
It has been reported that lipopolysaccharides (LPS) from Klebsiella O3 and O5 and Escherichia coli O8 and O9 exhibit extraordinarily strong adjuvant activity in augmenting antibody responses against protein antigens in mice as compared with other kinds of LPS. These four kinds of LPS all possess homopolysaccharides consisting of mannose (mannans) as the O-specific side chains. When these kinds of LPS were mixed in vitro with succinyl concanavalin A (Con A) which is known to bind specifically to alpha-mannoside and alpha-glucoside, their strong adjuvant activity was inhibited. Degree of the inhibition of the adjuvant activity of Klebsiella O3 LPS by succinyl Con A was dependent upon the dose of succinyl Con A. However, phytohemagglutinin, which is known to bind specifically to N-acetyl-D-galactosamine, did not inhibit the adjuvant activity of Klebsiella O3 LPS and O5 LPS. When Klebsiella O3 LPS was mixed with succinyl Con A in the presence of excess amounts of alpha-methyl mannoside or the polysaccharide fraction isolated from Klebsiella O3 LPS, the inhibitory effect of succinyl Con A on the adjuvant activity of Klebsiella O3 LPS was blocked. By contrast, the activity of Klebsiella O3 LPS as a polyclonal B-cell activator was not affected by treatment with succinyl Con A. From these results it is concluded that the mannans, as the O-specific polysaccharide chains of the LPS, significantly contribute to expression of their strong adjuvant activity.
Adjuvants, Immunologic↗
Potent adjuvant action of lipopolysaccharides possessing the O-specific polysaccharide moieties consisting of mannans in antibody response against protein antigen.
It was previously reported that Klebsiella O3 lipopolysaccharide (LPS) exhibits extraordinarily strong adjuvant activity in augmenting antibody response against protein antigens in mice compared with other kinds of LPS, for example, LPS from Escherichia coli O55, O111, and O127 and Salmonella enteritidis. The present study was undertaken to clarify the relationship between the strong adjuvant activity in augmenting antibody response against deaggregated bovine gammaglobulin and the chemical structure of LPS. Among LPS from Klebsiella O1, O4, O5, and O7, only O5 LPS exhibited nearly the same degree of the strong adjuvant activity as did O3 LPS. The adjuvant activity of the other LPS was very weak in a degree similar to that of LPS from E. coli O55 and O127. Even when the natural forms of Klebsiella O3 LPS and O1 LPS were converted to various defined uniform salt forms, their adjuvant activity did not significantly differ from that of the respective natural forms. It is therefore unlikely that the difference in strength of the adjuvant activity between Klebsiella O3 LPS and O1 LPS is due to the difference in their salt forms. The common feature in the structures of Klebsiella O3 LPS and O5 LPS is their O-specific polysaccharide chains consisting of the mannose homopolysaccharides (mannans). LPS from E. coli O8 and O9, the O-specific polysaccharide chains of which consist of the mannans, also exhibited much stronger adjuvant activity than do LPS from E. coli O55 and O127, and the strength of the adjuvant activities of the former two was comparable to that of LPS from Klebsiella O3 and O5. On the other hand, LPS from Klebsiella O3 and O5 and E. coli O8 and O9 showed the ability to activate B lymphocytes polyclonally in vivo in a degree similar to that of the other kinds of LPS. From the present results it can be concluded that LPS possessing the O-specific polysaccharide moieties consisting of the mannans exhibit extraordinarily strong adjuvant activity in augmenting antibody response against protein antigen.
Adjuvants, Immunologic↗
Somatostatin and GABA correlate with cervical autonomic nerve activity.
The mode of inhibitory action of centrally administered SRIF on the efferent activity of autonomic nerves was investigated in the rat by assessing the SRIF-induced change in the activity of the superior laryngeal nerve with or without pretreatment with various drugs. After picrotoxin or bicuculline treatment, the inhibition of nerve activity by SRIF was abolished while reserpine and atropine failed to abolish the SRIF effect. The centrally administered GABA inhibited the activity of the superior laryngeal nerve and the cervical sympathetic trunk. However, SRIF did not affect the sympathetic trunk. Arterial blood pressure was increased by SRIF while GABA produced hypotension. These data provide evidence for a GABAergic system as the mediator of SRIF action in the brain and for the selectivity of SRIF action on the particular intermediary GABAergic neurones.
Animals↗
Adjuvant activity of Klebsiella O3 lipopolysaccharide: inhibition of the adjuvant activity by concanavalin A.
Klebsiella O3 lipopolysaccharide (KO3 LPS) was found to exhibit extraordinarily strong adjuvant activity in augmenting antibody responses and delayed-type hypersensitivity (DTH) to protein antigens in mice. The O-specific polysaccharide chain of KO3 LPS consists of alpha-mannoside. We investigated the effect of concanavalin A (Con A) or succinyl Con A, which is known to bind to alpha-mannoside, on the adjuvant activity of KO3 LPS in augmenting DTH to ovalbumin. When KO3 LPS was mixed with Con A prior to injection, the strong adjuvant activity of KO3 LPS in augmenting DTH was inhibited and the degree of inhibition depended upon the dose of Con A. An equal amount of Con A elicited nearly complete inhibition of the adjuvant activity of KO3 LPS, Con A at 1/10 the amount of LPS elicited partial inhibition, and Con A at 1/100 the amount of LPS showed no inhibition. An equal amount of succinyl Con A, which induced less marked aggregation of KO3 LPS than Con A, elicited inhibition of the adjuvant activity of KO3 LPS to an extent similar to that by Con A. On the other hand, Con A or succinyl Con A bound to KO3 LPS did not impair in any way the lethal toxicity of KO3 LPS for mice which is known to be due to the lipid A moiety. From these findings it is concluded that the strong adjuvant activity of KO3 LPS does not solely depend upon the lipid A moiety but the O-specific polysaccharide moiety plays an important role in expression of the adjuvant activity.
Adjuvants, Immunologic↗
Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella: comparative study of preparations extracted by the phenol-water and the phenol-chloroform-petroleum ether methods.
An R-form lipopolysaccharide (LPS) extracted from Klebsiella strain LEN-111 (O3-:K1-) by the phenol-chloroform-petroleum ether method was compared with that extracted by the phenol-water method in the ability to form a hexagonal assembly. The LPS which was extracted by the phenol-water method and dialyzed against tap water to remove phenol showed ribbon-like structures, and it formed a hexagonal lattice structure with a lattice constant of 14.5 +/- 0.3 nm when it was precipitated by addition of two volumes of 10 mM MgCl2-ethanol. The LPS which was extracted by the phenol-chloroform-petroleum ether method and lyophilized consisted of ribbon-like structures and their fragments and it often formed small pieces of a hexagonal lattice, although the LPS before lyophilization did not form such a lattice. When the LPS extracted by the phenol-chloroform-petroleum ether method was precipitated by addition of two volumes of 10 mM MgCl2-ethanol, it formed essentially the same hexagonal lattice structure as that formed by the LPS extracted by the phenol-water method. From these results it is concluded that the ability of the LPS to form a hexagonal lattice structure does not depend upon the method of its extraction from bacterial cells.
Chemical Phenomena↗
Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella: relationship between lattice formation and uniform salt forms.
Various uniform salt forms of an R-form lipopolysaccharide (LPS) extracted from Klebsiella strain LEN-111 (O3-:K1-) were prepared and their ultrastructure was examined. The LPS, which was extracted by the phenol-water method, freed from contamination with RNA by treatment with RNase, and precipitated by addition of two volumes of 10 mM MgCl2-ethanol, was used as the original preparation for uniform salt forms. The original LPS preparation formed a hexagonal lattice structure with a lattice constant of 14.9 +/- 0.2 nm. The LPS after electrodialysis retained the ability to form a hexagonal lattice structure, although its lattice constant was large (18.7 +/- 0.5 nm) and the lattice structure of the electrodialyzed LPS was labile at pH 8.0 in contrast to that of the original LPS preparation. The magnesium salt form of the LPS formed essentially the same ordered hexagonal lattice structure (lattice constant of 15.0 +/- 0.2 nm) as that of the original LPS preparation. The calcium and ammonium salt forms formed a hexagonal lattice structure, but the lattice constants of the calcium and ammonium salt forms were larger (18.6 +/- 0.6 nm and 19.3 +/- 0.4 nm, respectively) than that of the magnesium salt form. The sodium and potassium salt forms consisted of freely branching ribbon-like structures with an average width of 13 nm and an average thickness of 9 nm. The triethylamine salt form consisted principally of short rods (10 nm X 9-13 nm).
Calcium↗
Excimer formation of N-(1-pyrenesulfonyl)-dipalmitoyl-L-alpha-phosphatidylethanolamine at the lipid-water interface of fat cell plasma membranes.
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Adipose Tissue↗
Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella sp.
We extracted an R-form lipopolysaccharide (LPS) by the phenol-water method from Klebsiella sp. strain LEN-111 (O3-:KI-) and followed the changes in ultrastructure of the LPS during the extraction procedure. When the LPS was obtained from the water phase of an extract by addition of 2 volumes of 10 mM MgCI2-ethanol, it consisted of membrane pieces with a hexagonal lattice structure with a lattice constant of 14 to 15 nm. The lattice structure of the LPS was disrupted into short rods with sodium dodecyl sulfate, but the same hexagonal lattice structure was again formed by precipitation with 2 volumes of 10 mM MgCI2-ethanol. The LPS preparation after two cycles of treatment by the phenol-water method, which contained no detectable amounts of proteins, kept an unaltered ability to form the hexagonal lattice structure. Extensive treatment with pronase and extraction with chloroform did not impair the ability of the LPS preparation to form the lattice structure. When the other salts, NaCI, CaCI2 or Zn(CH3COO)2, were used for precipitation of the LPS with ethanol in place of MgCI2, the LPS did not form the hexagonal lattice structure. However, if the LPS precipitated with NaCI-ethanol was converted to the magnesium salt form after it was electrodialyzed, it formed the same hexagonal lattice structure as the LPS precipitated with MgCI2-ethanol. From these results, it was concluded that the R-form LPS has the ability of in vitro self-assembly into a hexagonal lattice structure in the presence of Mg2+ without the help of other components such as proteins and free lipids from outer membrane.
Bacterial Outer Membrane Proteins↗
Comparative studies on antitumor activity of Klebsiella O3 lipopolysaccharide and its polysaccharide fraction in mice.
The antitumor activity of the polysaccharide fraction (OPS) obtained by the acid hydrolysis of Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from the culture supernatant of the decapsulated mutant strain LEN-1 (03: K1-) against both allogeneic tumor and syngeneic tumor systems in mice was compared with that of KO3 LPS. OPS prolonged the life span of MM2-bearing C3H/He mice by intraperitoneal (i.p.) pre- and post-treatment at the doses of 100 and 1000 mg/kg. However, large amounts of OPS were needed to show the antitumor activity as compared with KO3 LPS. OPS showed no growth inhibitory activity against Meth-A sarcoma in BALB/c mice by i.p., intravenous (i.v.) or intratumoral (i.t.) administration. When 1000 mg/kg of OPS was i.p. administered once a day for 10 days, OPS significantly inhibited the tumor growth of Sarcoma-180 solid type tumor. On the other hand, KO3 LPS significantly suppressed the growth of Meth-A tumor by i.t. administration at the doses of 0.3 and 1.0 mg/kg and showed complete regression in 8 and 9 out of 10 mice, respectively. In MM2 tumor, KO3 LPS also showed complete regression in all mice post-treated by i.p. administration at the dose of 1.0 mg/kg. These results suggest that OPS has antitumor activity on the tumors used in this study, but the activity was less than that of KO3 LPS.
Animals↗
Adjuvant actions of linear mannan-possessing lipopolysaccharide (LPS) in LPS-resistant C3H/HeJ mice.
Immunopotentiation has been demonstrated when Klebsiella O3 lipopolysaccharide (KO3 LPS), which possesses a linear mannan as the O-specific side chain, was injected subcutaneously into endotoxin resistant C3H/HeJ mice together with soluble protein antigens. The LPS exhibited significantly positive adjuvant effects on antibody responses in vivo after secondary antigen challenge and on delayed-type hypersensitivity (DTH) reactions against protein antigens. However, KO3 LPS was not a polyclonal B cell activator (PBA) in C3H/HeJ mice nor mitogenic in cultures of spleen cells of C3H/HeJ. Thus, the activity of the LPS in C3H/HeJ mice is confined to the potentiation of T-dependent immune responses. The contribution of the mannan O side chain to the adjuvant action of KO3 LPS was suggested.
Adjuvants, Immunologic↗
Comparative studies on adjuvanticity of Klebsiella O3 lipopolysaccharide and its lipid A and polysaccharide fractions.
Previously, we found that Klebsiella O3 lipopolysaccharide (KO3 LPS) exhibits very strong adjuvant activity in augmenting the delayed-type hypersensitivity and antibody responses to protein antigens, and also strong ability to enlarge the regional lymph node in mice. By hydrolysis in 1% acetic acid at 100 degrees for 1 hr, KO3 LPS was dissociated into 54-60% polysaccharide fraction and 25-27% lipid A fraction. The lipid A fraction thus prepared retained lethal toxicity for mice, whereas the polysaccharide fraction was essentially non-toxic. However, neither the lipid A fraction nor the polysaccharide fraction could reproduce the strong adjuvanticity of KO3 LPS, and a simple mixture of these two fractions also failed to do so. It is therefore concluded that, for expression of the strong adjuvanticity of KO3 LPS, both the lipid A and polysaccharide moieties are indispensable and must be combined in the form of LPS. By contrast, administration of relatively large amounts of the lipid A fraction alone could elicit enlargement of the regional lymph node to the same degree as that attained by KO3 LPS. The regional lymph node-enlarging activity of the lipid A fraction in lesser amounts was enhanced by addition of the polysaccharide fraction. It is therefore likely that there is no direct correlation between adjuvant activity of the LPS or its derivatives, and their regional lymph node-enlarging activity.
Adjuvants, Immunologic↗
Strong adjuvanticity of bacterial lipopolysaccharides possessing the homopolysaccharides consisting of mannose as the O-specific polysaccharide chains.
The lipopolysaccharides (LPS) from Klebsiella 03 and 05 and Escherichia coli 08 and 09 are unique in having linear homopolysaccharides consisting of mannose as the O-specific polysaccharide chains. All four kinds of LPS were found to exhibit very strong adjuvanticity in induction of delayed-type hypersensitivity to ovalbumin in mice compared with other kinds of LPS from Klebsiella, E. coli and Salmonella. Even if the natural forms of Klebsiella 03 LPS and 01 LPS were converted to various defined uniform salt forms, their adjuvanticity did not differ significantly from that of the respective natural forms. It was concluded therefore that the difference in strength of the adjuvanticity between them is not due to the difference in their salt forms, solubility and physical state. Correspondingly, with strong adjuvanticity of Klebsiella 03 LPS and 05 LPS, their activity in enlarging the regional lymph node was also strong. Various uniform salt forms of Klebsiella 03 LPS caused stronger regional lymph node enlargement than those of Klebsiella 01 LPS. However, the activity of E. coli 08 LPS and 09 LPS in enlarging the regional lymph node was significantly weaker than that of Klebsiella 03 LPS and 05 LPS, and there were other kinds of LPS which showed a capacity to enlarge the regional lymph node similar to that of E. coli 08 LPS and 09 LPS, despite their weak adjuvanticity. Therefore, correlation did not necessarily exist between the degree of adjuvanticity of LPS and its activity in enlarging the regional lymph node.
Adjuvants, Immunologic↗
Correlation between strong adjuvanticity of Klebsiella O3 lipopolysaccharide and its ability to induce interleukin-1 secretion.
Adjuvant activity of Klebsiella O3 lipopolysaccharide (KO3 LPS) in augmenting antibody response and delayed-type hypersensitivity to protein antigens in SMA mice was much stronger than that of LPS from Escherichia coli O55 and O127 (EO55 LPS and EO127 LPS). Relationship between strength of the adjuvant activity and that of the ability to induce interleukin-1 (IL-1) secretion by peritoneal macrophages from C3H/HeN or SMA mice was investigated using these three kinds of LPS. When supernatant samples of macrophages cultured at 37 degrees C for 24 hr in the presence of 5 micrograms/ml LPS were assayed by their mitogenic effect on thymocytes from C3H/HeJ mice, KO3 LPS induced the secretion of about four to six times greater amounts of IL-1 activity than did EO127 LPS. When concentration of LPS used for stimulation of macrophages was varied from 0.1 to 50 micrograms/ml, KO3 LPS induced the secretion of definitely greater amounts of IL-1 activity than did EO55 LPS and EO127 LPS throughout the LPS concentrations tested. Nearly the same amount of IL-1 activity as that produced by 10 micrograms/ml EO55 LPS or 50 micrograms/ml EO127 LPS could be produced by 1.0 microgram/ml or lower concentrations of KO3 LPS.
Adjuvants, Immunologic↗
Ultrastructure of Klebsiella O3 lipopolysaccharide isolated from culture supernatant: comparison with other lipopolysaccharides.
Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from the culture supernatant, which was found to exhibit a very strong adjuvant activity in augmenting antibody response and delayed-type hypersensitivity to protein antigens in mice, was examined by electron microscopy. When negatively stained with uranyl acetate or ammonium molybdate, the KO3 LPS was found to consist principally of flat ribbon-like structures branching freely (average width 16 nm and average thickness 7 nm) and to contain a small proportion of spheres (diameter 20-50 nm), both structures covered with fine hairy structures (average length approximately 10 nm). When the polysaccharide of KO3 LPS was stained by the periodic acid-thiosemicarbazide-silver proteinate procedure, silver granules were deposited on the ribbon-like structures and around the spheres, suggesting that the polysaccharide moiety is located on their surface and that the fine hairy structures consist of the polysaccharide moiety. Comparison by means of preparations stained with uranyl acetate or ammonium molybdate showed that KO3 LPS isolated from the culture supernatant has structural features in common with KO3 LPS isolated from bacterial cells, Escherichia coli O9 LPS isolated from the culture supernatant, and E. coli O127 LPS isolated from bacterial cells. On the basis of the present results, schematic representations of the common physical structure of LPS were drawn; the fine hairy structures attach to the wide surface of the flat ribbon-like structures along their lateral margin.
Escherichia coli↗
Ultrastructure of Klebsiella O3 lipopolysaccharide isolated from culture supernatant: structure of various uniform salt forms.
Various uniform salt forms of Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from culture supernatant were prepared as follows. Basic materials present in KO3 LPS were rigorously removed by electrodialysis and the electrodialyzed KO3 LPS was neutralized with NaOH, KOH, NH4OH, Ca(OH)2, tris(hydroxymethyl)aminomethane, or triethylamine. The ultrastructure of the uniform salt forms of KO3 LPS was examined using preparations stained with uranyl acetate. The sodium, potassium, ammonium, and trisaminomethane salt forms were structurally very similar to the natural form of KO3 LPS which consisted of a mixture of flat ribbon-like structures (average width of 16 nm and average thickness of 7 nm) and spheres with various diameters, both covered with fine hairy structures. When KO3 LPS was converted to the triethylamine salt form, the ribbon-like structures were disrupted into very small granules (7-9 nm X 9-15 nm). The calcium salt form consisted of particles and rods of various sizes and ribbon-like structures which were markedly extended (maximum width of 50 nm) and presented irregular shapes. When converted to the calcium salt form, the ribbon-like structures were extended and eventually divided into particles and rods. For reasons still unknown, the sodium salt of KO3 LPS was mostly stained positively with uranyl acetate in contrast to the natural form and the other uniform salt forms which were always negatively stained. In the positively stained preparation of the sodium salt form, it was clearly shown that the ribbon-like structures consisted of a bilayer.
Klebsiella↗
Adjuvant activity of Klebsiella O3 lipopolysaccharide: comparative study using defined uniform salt forms.
Previously we showed that Klebsiella O3 lipopolysaccharide (KO3 LPS) is much more potent than other kinds of LPS including Escherichia coli O127 LPS (EO127 LPS) in adjuvant activity in augmenting antibody response and delayed-type hypersensitivity to protein antigens and in the ability to enlarge the regional lymph node. Various defined uniform salt forms, the triethylamine, sodium, potassium, ammonium, tris(hydroxymethyl)aminomethane, and calcium salt forms, of KO3 LPS and EO127 LPS were prepared by removing basic materials present in LPS preparations by electrodialysis and neutralizing the electrodialyzed LPS preparations with various kinds of alkali. The triethylamine salt form showed the best solubility and consisted of the smallest granules and, on the other hand, the calcium salt form showed the lowest solubility, compared with the natural form and the other uniform salt forms. Even if the natural forms of KO3 LPS and EO127 LPS were converted to the defined uniform salt forms, adjuvanticity of KO3 LPS and EO127 LPS in augmenting delayed-type hypersensitivity to ovalbumin and the ability to enlarge the regional lymph node did not significantly differ from those of the respective natural forms. From these results it is concluded that the difference in strength of the adjuvanticity between KO3 LPS and EO127 LPS is not due to the difference in their salt forms, solubility or physical state. Moreover, there were no significant differences in lethal toxicity for mice by the intraperitoneal route among the natural form and all the uniform salt forms of KO3 LPS tested.
Adjuvants, Immunologic↗
Intact and subcellular form of thymocytes of rats are exceptionally immunogenic in mice for inducing anti-Thy-1 T cell-independent class 2 antibody responses.
Results of the present study show that the primary anti-Thy-1.1 antibody response to rat antigen in Thy-1.2 mice is induced exclusively by thymocyte antigen. Thy-1 antigens of brain and bone marrow, which expressed much Thy-1 antigen, were poorly immunogenic if at all. Brain Thy-1 antigen considerably inhibited the immunogenicity of thymocyte Thy-1. Moreover, we found that subcellular form of thymocytes induce as high antibody responses as intact thymocytes do. The subcellular thymocyte Thy-1 antigen behaved as TI-2 antigen, inducing a good response in athymic nude mice but not in CBA/N mice with a B cell defect. The significance of these findings is discussed in relation to the possible physiological activity of Thy-1 or Thy-1-linked molecules on thymocytes specifically mediating lymphocyte differentiation.
Animals↗