UNIPROT:P43026 - FACTA Search

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Query: UNIPROT:P43026 (lipopolysaccharide)
62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A synthetic compound (506), beta (1-6) D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated at 2'-amino and 3'-hydroxyl groups with (R)-3-dodecanoyloxytetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl groups, respectively, and has (R)-3-hydroxytetradecanoyl groups at 2-amino and 3-hydroxyl groups, exhibited full endotoxic activities identical to or sometimes stronger than those of a reference lipid A from an Escherichia coli Re-mutant (strain F515). Endotoxic activities tested include pyrogenicity and leukopenia-inducing activity in rabbits, body weight-decreasing toxicity in normal mice, lethal toxicity in galactosamine-sensitized mice and chicken embryos, and the preparation and provocation of the local Shwartzman reaction in rabbits. Compound 406, a synthetic counterpart of a biosynthetic precursor of lipid A molecule, showed by contrast only weak activities in all of the above assay systems except for the lethality in galactosamine-loaded mice. This finding strongly suggests that the presence of acyloxyacyl groups at the C-2' and C-3' positions of the disaccharide backbone is one of the most important determinant structures of the lipid A molecule for exhibition of strong biological activities characteristic of lipopolysaccharide and its lipid A moiety. The activities of the corresponding 4'-monophosphate (compound 504) and 1-monophosphate (505) analogs were considerably less than those of the parent molecule 506 and the reference F515 lipid A. Regarding other biological activities, not only compound 506 but also compounds 504, 505, and 406 showed definite activities, sometimes comparable to those of F515 lipid A and other reference natural products. These are the activation of Tachypleus tridentatus amoebocyte clotting enzyme cascade and human complement via the classical pathway, mitogenic and polyclonal B-cell activation of murine splenocytes, stimulation of peritoneal macrophages in a guinea pig, enhancement of migration of human blood polymorphonuclear leukocytes, and induction of a serum factor that is cytostatic and cytocidal to L-929 cells in Mycobacterium bovis BCG-primed mice. Relative potencies of test synthetic compounds depended on the assay systems and varied from one system to another. Dephospho-compound 503 lacked most of the biological activities that were definitely observed with phosphorylated compounds, probably because of its insolubility. This study demonstrates the successful chemical synthesis of an E. coli-type lipid A.
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PMID:Synthetic lipid A with endotoxic and related biological activities comparable to those of a natural lipid A from an Escherichia coli re-mutant. 389 27

Synthetic lipid A analogs (compounds 404 through 406) were examined for their immunopharmacological activities. These compounds had two amide-bound and two ester-bound (R)-3-hydroxytetradecanoyl groups at the C-2 and C-2' and the C-3 and C-3' positions, respectively, of beta (1-3)glucosamine disaccharide. In all of the in vitro assays, these synthetic compounds exhibited high activities comparable to those of a reference lipid A prepared from Escherichia coli O8:K27 Re-mutant strain F515. The compounds activated the clotting enzyme cascade of the horseshoe crab, activated the human complement via the classical pathway, caused polyclonal B-cell activation, stimulated the phagocytosis of sheep erythrocytes by murine peritoneal macrophages, and enhanced the migration of human polymorphonuclear leukocytes. They also increased the thymidine uptake of splenocytes of BALB/c nu/nu and C3H/HeN mice but not those of C3H/HeJ (a nonresponder to lipopolysaccharide). A dephosphorylated derivative, compound 403, was barely active in all of the above assays except for the enhancement of polymorphonuclear leukocyte migration. However, compounds 404 through 406 were feeble in pyrogenicity and could not prepare the local Shwartzman reaction, although they were very lethal to galactosamine-loaded mice. Therefore, synthetic lipid A analogs described here were fully immunopharmacologically active in in vitro assays, but all of them were far less active than natural E. coli F515 lipid A regarding the biological activities characteristic of endotoxic lipopolysaccharides and lipid A's. The high lethal toxicity of compound 406 (1,4'-bisphosphate) to the galactosamine-loaded mice may not reflect its real toxicity to normal mice. In all activities examined, compound 406 was quite comparable to a biosynthetic lipid A precursor, a natural counterpart of compound 406. The immunopharmacological activities of these newly synthesized lipid A analogs, especially compound 406, were much stronger than those of compounds that had been synthesized earlier by using the originally proposed model of the lipid A structure. The findings described in this report justify the acylation pattern of a disaccharide backbone of lipid A, revised on the basis of recent analytical studies. The low in vivo endotoxic activities of the present lipid A analogs are most probably due to the fact that the kinds of acyl groups were different from those of the complete lipid A from E. coli, although there were no differences in the acylation positions on the disaccharide backbone.
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PMID:Immunopharmacological activities of a synthetic counterpart of a biosynthetic lipid A precursor molecule and of its analogs. 398 84

Studies of the lipopolysaccharide of Pseudomonas alcaligenes strain BR 1/2 were extended to the polysaccharide moiety. The crude polysaccharide, obtained by mild acid hydrolysis of the lipopolysaccharide, was fractionated by gel filtration. The major fraction was the phosphorylated polysaccharide, for which the approximate proportions of residues were; glucose (2), rhamnose (0.7), heptose (2-3), galactosamine (1), alanine (1), 3-deoxy-2-octulonic acid (1), phosphorus (5-6). The heptose was l-glycero-d-manno-heptose. The minor fractions from gel filtration contained free 3-deoxy-2-octulonic acid, P(i) and PP(i). The purified polysaccharide was studied by periodate oxidation, methylation analysis, partial hydrolysis, and dephosphorylation. All the rhamnose and part of the glucose and heptose occur as non-reducing terminal residues. Other glucose residues are 3-substituted, and most heptose residues are esterified with condensed phosphate residues, possibly in the C-4 position. Free heptose and a heptosylglucose were isolated from a partial hydrolysate of the polysaccharide. The location of galactosamine in the polysaccharide was not established, but either the C-3 or C-4 position appears to be substituted and a linkage to alanine was indicated. In its composition, the polysaccharide from Ps. alcaligenes resembles core polysaccharides from other pseudomonads: no possible side-chain polysaccharide was detected.
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PMID:Studies of the polysaccharide fraction from the lipopolysaccharide of Pseudomonas alcaligenes. 436 26

Epitopes for Pseudomonas aeruginosa O11-specific human monoclonal antibodies (mAbs) and O11 serotyping antisera have been characterized. These mAbs recognized the O-polysaccharide portion of the lipopolysaccharide. The structure of the O-polysaccharide of O11 has been reported to be comprised of trisaccharide repeating-units as follows: -->3)-alpha-L-FucpNAc-(1-->1)-beta-D-FucpNAc-(1--> 2)-beta-D-Glcp-(1-->. (FucpNAc, 2-acetamido-2,6-dideoxygalactopyranoside.) Data from inhibition studies of binding in enzyme-linked immunosorbent assays and cell-agglutination assays, using monosaccharides and periodate-oxidized O-polysaccharide showed that the glucose residue, especially the C-3-C-6 segment and the beta-anomeric configuration, in the polysaccharide is essential for the epitopes of all anti-O11 mAbs; however, the detailed epitope specificities were different from one another. Furthermore, epitopes for serotyping antisera of O11 seemed to be similar to those for the human mAbs.
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PMID:Epitopes for human monoclonal antibodies and serotyping antisera against the O-specific polysaccharide of Pseudomonas aeruginosa O11. 752 26

Escherichia coli O111, of various H types and virulence factors, causes enteritis throughout the world, especially in young children. This O type is found rarely in healthy individuals. Serum antibodies to the O-specific polysaccharide of O111 lipopolysaccharide (LPS) protect mice and dogs against infection with this E. coli serotype. The O111 O-specific polysaccharide is composed of a pentasaccharide repeat unit with two colitoses bound to the C-3 and C-6 of glucose in a trisaccharide backbone; this structure is identical to that of Salmonella adelaide (O35), another enteric pathogen. Nonpyrogenic O111 O-specific polysaccharide was prepared by treatment of its LPS with acetic acid (O-SP) or the organic base hydrazine (DeA-LPS). The O-SP had a reduced concentration of colitose. These products were derivatized with adipic acid dihydrazide (ADH) or thiolated with N-succinimidyl-3(2-pyridyldithio) propionate (SPDP). The four derivatives were covalently bound to tetanus toxoid (TT) by carbodiimide-mediated condensation or with SPDP to form conjugates. Immunization of BALB/c and general-purpose mice by a clinically acceptable route showed that DeA-LPS-TTADH, of the four conjugates, elicited the highest level of LPS antibodies. Possible reasons to explain this differential immunogenicity between the four conjugates are discussed.
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PMID:Comparative immunogenicity of conjugates composed of Escherichia coli O111 O-specific polysaccharide, prepared by treatment with acetic acid or hydrazine, bound to tetanus toxoid by two synthetic schemes. 754 31

CDP-6-deoxy-delta 3,4-glucoseen reductase (E3), which catalyzes the reduction of the C-3 deoxygenation step during the formation of CDP-ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis, has been expressed at high level in Escherichia coli (670 times over the wild-type strain). This flavoenzyme, which also contains one plant ferredoxin type [2Fe-2S] cluster, was inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide. In both cases the inactivation followed a pseudo first order kinetics. The second order rate constant for the reaction of DTNB with E3 was 0.25 mM-1 min-1 at 20 degrees C, pH 8.0. Detailed characterization of the inactivated enzyme showed that neither the flavin nor the [2Fe-2S] cluster was altered during inactivation. Since this inactivation was reversible by treating the inactivated enzyme with 1 mM D,L-dithiothreitol (DTT), it was concluded that only cysteine residues were modified during inactivation. Analysis of the inactivation using the method developed by Tsou revealed that two cysteines react with DTNB at similar rates and modification of either one is enough to impair E3's activity. Tryptic digestion of E3 labeled with N-ethyl[2,3-14C]maleimide, followed by fractionation of the digest by high performance liquid chromatography, gave two labeled peptides, both of which were separately isolated as a pair of interconvertible diastereoisomers. Sequence analysis of these labeled peptides allowed the identification of Cys-75 and Cys-296 as the reactive cysteine residues. Interestingly, the C75S and C296S mutant proteins exhibit identical physical and comparable catalytic properties as the wild-type enzyme. Since Cys-296 is a conserved residue in the NAD(P) binding domain of enzymes belonging to the same class, this residue may be involved in stabilizing the charge-transfer complex between E3 and NADH, thus facilitating hydride transfer from the nicotinamide nucleotide to flavin. A chemically modified Cys-75 which is immediately adjacent to the [2Fe-2S] center in E3 may prevent the proper juxtaposition of the redox centers and thus impede electron transfer leading to enzyme inactivation. These results may be useful for placing constraints on the peptide folding comprising the active site of E3 for electron transfer between NADH, FAD, and the [2Fe-2S] center.
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PMID:Mechanistic studies on CDP-6-deoxy-delta 3,4-glucoseen reductase: the role of cysteine residues in catalysis as probed by chemical modification and site-directed mutagenesis. 770 27

The CDP-6-deoxy-delta 3,4-glucoseen reductase (E3) is a NADH-dependent enzyme which catalyzes the key reduction of the C-3 deoxygenation step during the formation of CDP-ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis. This highly purified enzyme is also a NADH oxidase capable of mediating the direct electron transfer from NADH to O2, forming H2O2. While previous work showed that E3 contains no common cofactor, one FAD and one plant ferredoxin type [2Fe-2S] center were found in this study to be associated with each molecule of E3. The iron-sulfur center is essential for E3 activity since bleaching of the [2Fe-2S] center leads to inactive enzyme. These results suggest that E3 employs a short electron-transport chain composed of both FAD and the iron-sulfur center to shuttle electrons from NADH to its acceptor. The order of electron flow, as indicated by EPR measurement with partially reduced E3, starts with hydride reduction of FAD by NADH. The iron-sulfur cluster, receiving electrons one at a time from the reduced flavin, relays the reducing equivalents via another iron-sulfur center in the active site of E1 to its final acceptor, the E1-bound PMP-glucoseen adduct. The participation of a one-electron-carrying iron-sulfur center in this reduction is advantageous since both electrons are dispatched from the same redox state of the prosthetic group, allowing electrons of equal energy to be delivered to the final acceptor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cofactor characterization and mechanistic studies of CDP-6-deoxy-delta 3,4-glucoseen reductase: exploration into a novel enzymatic C-O bond cleavage event. 821 67

Studies of the biosynthesis of ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis V, have shown that the C-3 deoxygenation is a process consisting of two enzymatic steps. The first enzyme involved in this transformation is CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which is a pyridoxamine 5'-phosphate dependent iron-sulfur protein. The second catalyst, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase, formally called CDP-6-deoxy-delta(3,4)-glucoseen reductase (E3), is an NADH dependent plant type [2Fe-2S] containing flavoenzyme. To better understand the electron transfer carried out by these two enzymes, the potentials of the E1 and E3 redox cofactors were determined spectroelectrochemically. At pH 7.5, the midpoint potential of the E3 FAD was found to be -212 mV, with the FADox/FADsq couple (E1o') and the FADsq/FADhq couple (E2o') calculated to be -231 and -192 mV, respectively. However, the E1o' and E2o' of the FAD in E3(apoFeS) at pH 7.5 were estimated to be -215 and -240 mV, respectively, which are quite different from those of the holo-E3, suggesting a significant effect of the iron-sulfur center on the redox properties of the flavin coenzyme. Our data also showed that the midpoint potential of the E3 iron-sulfur is -257 mV and that of the E1 [2Fe-2S] center is -209 mV. These values indicated a thermodynamic barrier to the proposed electron transfer of NADH->FAD=>E3[2Fe-2S]->E1[2Fe-2S] at pH 7.5. Regulation of electron transfer by several mechanisms is possible and experiments were performed to examine ways of overcoming the unfavorable electron transfer energetics in the E1/E3 system. It was found that both binding of E3 with NAD+ and complex formation between E3 and E1 showed no effect on the midpoint potentials of the E3 FAD and iron-sulfur center. Interestingly, the midpoint potential of the E3 FAD shifts dramatically to -273 mV (E1o' approximately -345 mV and E2o' approximately -200 mV) at pH 8.4, with very little semiquinone stabilization (< 5%). The potential of the E3 [2Fe-2S] center at pH 8.4 was also found to undergo a negative shift to -279 mV, and that of the E1 iron sulfur center remained essentially the same at -206 mV. These data indicated that the redox properties of this system may be regulated by pH and the electron transfer between the E3 redox centers may be prototropically controlled. These results also demonstrated that E3 is unique among this class of enzymes.
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PMID:Studies of the redox properties of CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1) and CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3): two important enzymes involved in the biosynthesis of ascarylose. 867 89

Taxol (paclitaxel), a microtubule stabilizer with antitumor activity, mimics the actions of lipopolysaccharide (LPS) on murine macrophages (M phi). In the present study, a variety of synthetic analogs of paclitaxel were examined for their potencies to induce nitric oxide (NO) and tumor necrosis factor (TNF) production by peritoneal M phi from LPS-responsive C3H/HeN, and LPS-hyporesponsive C3H/HeJ mice, and by M phi-like LPS-responsive J774.1 and its mutant LPS-hyporesponsive J7.DEF3 cells. In this structure-activity relationship study, we found that (i) the benzoyl group at the C-3' position of paclitaxel is the most important site to activate C3H/HeN M phi; (ii) the phenyl group at C-3' is not a requisite for the activity; (iii) there is good correlation between NO and TNF production by the M phi in response to compounds, except for the analogs having a tert-butoxycarbonyl (10-acetyldocetaxel) or a thiophene-2-carbonyl group at C-3'-N instead of a benzoyl group, which is more dominant in TNF than in NO production; (iv) the compounds tested induce neither NO nor TNF production by C3H/HeJ M phi; (v) active compounds to C3H/He M phi induce TNF production by J7.DEF3 cells as well as J774.1 cells; and (vi) there is no correlation between the NO/TNF inducibility to C3H/HeN M phi and growth inhibitory activity against M phi-like J774.1 and J7.DEF3 cells. These data also suggest that the binding of taxoid/LPS to tubulin is not essential for the M phi activation.
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PMID:Structural requirements of taxoids for nitric oxide and tumor necrosis factor production by murine macrophages. 885 30

The lipopolysaccharide of Yersinia pseudotuberculosis V includes a 3,6-dideoxyhexose, ascarylose, as the nonreducing end of the O-antigen tetrasaccharide. The C-3 deoxygenation of CDP-6-deoxy-L-threo-D-glycero-4-hexulose is a critical reaction in the biosynthesis of ascarylose. The first half of the reaction is a dehydration catalyzed by CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which is PMP-dependent and contains a redox-active [2Fe-2S] center. The second half is a reduction that requires an additional enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3, formerly known as CDP-6-deoxy-delta 3,4-glucoseen reductase), which has a FAD and a [2Fe-2S] center in the active site. Using NADH as the reductant in the coupled E1-E3 reaction, we have monitored the kinetics of a radical intermediate using both stopped-flow spectrophotometry and rapid freeze-quench EPR under aerobic and hypoxic conditions. In the EPR studies, a sharp signal at g = 2.003 was found to appear at a rate which is kinetically competent, reaching its maximum intensity at approximately 150 ms. Stopped-flow UV-vis analysis of the reaction elucidated a minimum of six optically distinguishable states in the mechanism of electron transfer from NADH to substrate. Interestingly, one of the detected intermediates has a time course nearly identical to that of the radical detected by rapid freeze-quench EPR. The difference UV-vis spectrum of this intermediate displays a maximum at 456 nm with a shoulder at 425 nm. Overall, these results are consistent with an electron transfer pathway that includes a radical intermediate with the unpaired spin localized on the substrate-cofactor complex. Evidence in support of this mechanism is presented in this report. These studies add the PMP-glucoseen radical to the growing list of mechanistically important bioorganic radical intermediates that have recently been discovered.
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PMID:Kinetic characterization of an organic radical in the ascarylose biosynthetic pathway. 896 49

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