Bioactive chitin derivatives. Activation of mouse-peritoneal macrophages by O-(carboxymethyl)chitins

The effect of O-(carboxymethyl)chitins (CM-chitins) on the activation of mouse-peritoneal macrophages in vivo and their mitogenic activity on mouse spleen-cells were investigated. The induction of cytotoxic macrophages is enhanced by an increase of negative charge at O-6 and decreased by further modification at O-3 of the GlcNAc residue. CM-Chitins had a minor effect on mitogenic activity that was independent of the site of modification; partially N-deacetylated chitins had little activity. Although there was remarkable enhancement of accessibility to lysozyme upon modification at O-6 of the GlcNAc residue, the accessibility was decreased by further substitution at O-3.     

Efficient microwave assisted access to chiral O-(alpha-protected-aminoacyl)steroids

Chiral O-(alpha-protected-aminoacyl)steroids 4a-f, 6a-b, 8 and 4a+4d and O-(alpha-protected-dipeptidoyl)steroids 12a,b are conveniently prepared under microwave irradiation in isolated yields of 65-96%, with complete chirality retention. The reaction utilized readily available N-(Z-alpha-aminoacyl)benzotriazoles 2a-f and Z-dipeptidoylbenzotriazole 11, with naturally occurring steroidal alcohols 3,5,7,9.     

O-(Acridinium)hydroxylamine (AHA): a reagent for the preparation of chemiluminescent acridinium oxime (AO)-steroid conjugates

O-(Acridinium)hydroxylamine (AHA) reacted with a representative sample of oxo-steroids (6-oxoestradiol, estrone, norethindrone, cortisol, progesterone, digoxin dialdehyde, and digitoxin dialdehyde) to produce chemiluminescent acridinium oxime (AO) conjugates in a single step in 37-68% yield after preparative HPLC.     

Differential O-3/O-4 regioselectivity in the glycosylation of alpha and beta anomers of 6-O-substituted N-dimethylmaleoyl-protected D-glucosamine acceptors

An assessment of the relative O-3/O-4 reactivities of both methyl alpha- and beta-d-glycosides of N-dimethylmaleoyl (DMM) d-glucosamine acceptors protected at O-6 with benzoyl (Bz), benzyl (Bn), and tert-butyldiphenylsilyl (TBDPS) groups is presented using per-O-benzoylated beta-d-galactofuranosyl and per-O-acetylated alpha-d-galactopyranosyl trichloroacetimidates as glycosyl donors. Using the former donor, the alpha anomer of the 6-O-benzoylated compound gave exclusive substitution at O-3, whereas the other two compounds with alpha-configuration kept this site as preferential. The beta anomer of the 6-O-benzoylated compound gave the same amounts of reaction products on O-3 and O-4, whereas the other beta analogs carried a more reactive O-4. The same reactions were carried out using as donor the less-reactive per-O-acetylated alpha-d-galactopyranosyl trichloroacetimidate. Although the same trend was found to occur, the O-4 was always relatively more reactive with the pyranosyl donor than with the furanosyl donor, when keeping the remaining factors constant. Furthermore, the beta anomers of the acceptor gave almost exclusive substitution at O-4. These observations confirm and extend the utility of these 'matching' donor and acceptor reactivities.     

Chiral O-(Z-alpha-aminoacyl) sugars: convenient building blocks for glycopeptide libraries

1,2:3,4-Di-O-isopropylidene-alpha-D-galactopyranose (2), 1,2:5,6-di-O-isopropylidene-d-glucose (5), and 2,3:5,6-di-O-isopropylidene-alpha-D-mannofuranose (7) are efficiently O-acylated in 78-96% yields with readily available N-(Z-alpha-aminoacyl)benzotriazoles 1a-e, 1d+1d' under microwave irradiation to give chiral 3a-d, 4, 6a-d, 8a,b and diastereomeric mixtures (3d+3d'), (6a+6a'), and (6d+6d'). The original chirality was retained as evidenced by HPLC. The diisopropylidene protecting groups were removed from compounds 3a,d, 6d to give the free O-(Z-alpha-aminoacyl) sugars 9a,b, 10.     

Syntheses of 19-[O-(carboxymethyl)oxime] haptens of epipregnanolone and pregnanolone

O-(Carboxymethyl)oximes 1 and 2 derived from two epimeric 5beta-pregnanolones (3beta-hydroxy-5beta-pregnan-20-one and 3alpha-hydroxy-5beta-pregnan-20-one) in position 19 were prepared. Two synthetic routes were employed, both using protection of the 20-keto group after reduction into the (20R)-alcohol in the form of acetate. In the first route, (20R)-19-hydroxy-5beta-pregnan-3beta,20-diyl diacetate (3) was transformed into the corresponding 19-[O-(carboxymethyl)oxime] methyl ester 6, then deacetylated by acid and partially silylated with tert-butyldimethylsilyl chloride. The desired 3-O-silylated derivative 8 was separated, oxidized to the 20-ketone and protecting groups were sequentially removed to give the first title hapten 1. The second route started from (20R)-19-hydroxy-3-oxopregn-4-en-20-yl acetate (11), which was hydrogenated in the presence of base to the 5beta-pregnan-3-one derivative 12, protected in position 19 with tert-butyldimethylsilyl group and reduced with borohydride. The prevailing 3alpha-alcohol 15 was separated, protected in position 3 with a methoxymethyl group, deprotected in position 19 and transformed into the 19-[O-(carboxymethyl)oxime] 19. After deacetylation, esterification with diazomethane and oxidation in position 20, the pregnanolone skeleton was regenerated. Final deprotection steps gave the second title hapten 2. Both haptens, i.e., (19E)-3beta- and -3alpha-hydroxy-20-oxo-5beta-pregnan-19-al 19-[O-(carboxymethyl)oxime], were designed for the development of immunoassays of the corresponding parent neuroactive steroids.     

The Neisseria meningitidis serogroup A capsular polysaccharide O-3 and O-4 acetyltransferase

Neisseria meningitidis serogroup A capsular polysaccharide (CPS) is composed of a homopolymer of O-acetylated, alpha1-->6-linked ManNAc 1-phosphate that is distinct from the capsule structures of the other meningococcal disease-causing serogroups, B, C, Y, and W-135. The serogroup A capsule biosynthetic genetic cassette consists of four open reading frames, mynA-D (sacA-D), that are specific to serogroup A, but the functions of these genes have not been well characterized. mynC was found to encode an inner membrane-associated acetyltransferase that is responsible for the O-acetylation of the CPS of serogroup A. The wild-type CPS as revealed by 1H NMR had 60-70% O-acetylated ManNAc residues that contained acetyl groups at O-3, with some species acetylated at O-4 and at both O-3 and O-4. A non-polar mynC mutant generated by introducing an aphA-3 kanamycin resistance cassette produced CPS with no O-acetylation. A serogroup A capsule-specific monoclonal antibody was shown to recognize the wild-type O-acetylated CPS, but not the CPS of the mynC mutant, which lacked O-acetylation. MynC was C-terminally His-tagged and overexpressed in Escherichia coli to obtain the predicted approximately 26-kDa protein. The acetyltransferase activity of purified MynC was demonstrated in vitro using [14C]acetyl-CoA. MynC O-acetylated the O-acetylated CPS of the mynC mutant and further acetylated the wild-type CPS of serogroup A meningococci, but not the CPS of serogroup B or C meningococci. Genetic complementation of the mynC mutant confirmed the function of MynC as the serogroup A CPS O-3 and O-4 acetyltransferase. MynC represents a new subclass of O-acetyltransferases that utilize acetyl-CoA to decorate the D-mannosamine capsule of N. meningitidis serogroup A.     

Lucigenin: redox potential in aqueous media and redox cycling with O-(2) production

The use of lucigenin luminescence as a measure of ?O-(2) has been questioned because lucigenin has been shown to be capable of mediating the production of O-(2). This being the case, lucigenin can signal the presence of O-(2) even in systems not producing it in the absence of lucigenin. The reduction potential of lucigenin should be in accord with its ability to mediate O-(2) production; but it has not heretofore been measured in aqueous media. The problems facing such measurement are the insolubility of the divalently reduced form, which deposits on the electrode, and the slow conformational transition that follows the second electron transfer and which interferes with reversibility. We have now used rapid scan cyclic voltammetry to determine that the reduction potential for lucigenin is -0.14 +/- 0.02 V versus the normal hydrogen electrode. This value applies to both the first and the second electron transfers to lucigenin and it is in accord with the facile mediation of O-(2) production by this compound.     

Synthesis and properties of diastereoisomers of adenosine 5'-(O-1-thiotriphosphate) and adenosine 5'-(O-2-thiotriphosphate).

The chemical synthesis of adenosine 5'-(O-1-thiotriphosphate) (ATPalphaS) and adenosine 5'-(O-2-thiotriphosphate) (ATPbetaS) is described. Both exist as a pair of diastereomers, A and B. The isomers of ATPalphaS can be distinguished on the basis of their different reaction rates with myokinase as well as nucleoside diphosphate kinase. With both enzymes, isomer A reacts fast whereas isomer B reacts considerably more slowly. Phosphorylation of a mixture of isomers of ADPalphaS with pyruvate or acetate kinase yields ATPalphaS, isomer A, whereas the phosphoryl transfer with creatine or arginine kinase yields isomer B. The isomers of ATPbetaS differ in their reactivity with myosin. Isomer A is readily hydrolyzed, whereas isomer B is not. However, isomer B reacts faster with nucleoside diphosphate kinase and ADP than isomer A. Phosphoryl transfer with pyruvate kinase onto ADPbetaS yields ATPbetaS, isomer A, with acetate kinase, isomer B.     

Arrangement of proteins O-8 and O-9 in outer membrane of Escherichia coli K-12. Existence of homotrimers and heterotrimers.

1. The molecular arrangement of major outer membrane proteins O-8 and O-9 that exist as trimers has been studied by means of cross-linking with dimethylsuberimidate. 2. The cross-linked samples were examined on a urea/sodium dodecyl sulfate/polyacrylamide gel which was developed to separate cross-linked trimer and dimer of O-8 from those of O-9. 3. Cells simultaneously synthesizing both O-8 and O-9 formed heterotrimers (trimers containing both proteins) as well as homotrimers. 4. Quantitative analyses revealed that there was no discrimination between O-8 and O-9 in the assembly process to form trimers. 5. When cells were grown sequentially under two different sets of conditions so that the cells synthesized either one of the two proteins in the first stage and the other in the second stage of growth, no heterotrimers were formed. This result indicates that subunit exchange did not take place between trimers which had been incorporated into the outer membrane.     

Interaction of O-(undec-10-en)-yl-D-glucose derivatives with the Plasmodium falciparum hexose transporter (PfHT)

All-O-undec-en-10-yl derivatives of d-glucose have been prepared and their affinities for the Plasmodium falciparum hexose transporter (PfHT) determined; the O-2 derivative displays a good apparent affinity for PfHT (K(I)=2 microM) with no significant interaction with the mammalian transporter GLUT1. This selectivity points to position -2 of glucose as an appropriate substitution site for the development of inhibitors of P. falciparum glucose uptake.     

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.     

Tyrosine nitration by simultaneous generation of (.)NO and O-(2) under physiological conditions. How the radicals do the job

Radiation chemical experiments demonstrate that the reaction of tyrosyl radical (TyrO(.)) with (.)NO(2) yields 45 +/- 3% 3-nitrotyrosine and that a major product of the reaction of TyrO(.) with (.)NO is 3,3'-dityrosine. Radiolysis was used to generate (.)NO and O-(2) in the presence of tyrosine and bicarbonate at pH 7.5 +/- 0.1. The nitration yield was found to be dose rate-dependent, and the yield per radical produced by pulse radiolysis was identical to that obtained with authentic peroxynitrite. The proposed mechanism that accounts for the data is as follows: (i) In the presence of CO(2) the reaction of (.)NO with O-(2) yields 33% (.)NO(2) and CO-(3), where the latter reacts rapidly with tyrosine to form TyrO(.); (ii) The formation of 3-nitrotyrosine takes place via the reaction of (.)NO(2) with TyrO(.), which is the main process at high dose rates; and (iii) Under continuous generation of (.)NO and O-(2), the formation of 3-nitrotyrosine is strongly suppressed because of efficient scavenging of (.)NO(2) by tyrosine. The proposed model shows that the highest nitration yield is obtained for similar fluxes of (.)NO and O-(2) and is completely inhibited upon excess production of O-(2) because of efficient scavenging of TyrO(.) by O-(2). The biological implications of these findings are discussed.     

A facile Er(OTf)3-catalyzed synthesis of 2,3-unsaturated O- and S-glycosides

Er(OTf)(3) is a useful catalyst for the Ferrier rearrangement furnishing high yields of O- and S-glycosides. The transformation has wide applicability, cleaner reaction profiles, mild reaction conditions, and high stereoselectivity and the catalyst, which is also commercially available, can be recovered and reused.     

Interactions of outer membrane proteins O-8 and O-9 with peptidoglycan sacculus of Escherichia coli K-12.

The outer membrane proteins O-8 and O-9 were specifically bound to the peptidoglycan sacculus in sodium dodecyl sulfate (SDS) solution. Other cellular proteins failed to interact with the peptidoglycan sacculus under the same conditions. When the outer membrane was preheated in SDS solution, the binding did not take place. Optimum binding was observed at pH 8 in the presence of 5 mM Mg2+. A high concentration of sodium chloride strongly inhibited the binding. The effects of these factors on the bindings of O-8 and O-9 required neither the bound nor the free form of Braun's lipoprotein, nor was the binding of either protein necessary for the binding of the other. Proteins O-8 and O-9 were also found in the peptidoglycan sacculus when it was prepared from cells in SDS solution at 60 degrees. A dilution experiment showed that the complex was not an artifact. The mode of interaction between these proteins and peptidoglycan in the preparation was similar to that in the reassembled O-8-O-9-peptidoglycan complex, as judged from the sensitivity to sodium chloride and temperature. The physiological importance of the complex is discussed in relation to the assembly of the outer membrane on the cell surface.     

Synthesis of 2-(4-trifluoroacetamidophenyl)ethyl O-(L-glycero-alpha-D-manno-heptopyranosyl)-(1----7)-O-(L-glycero-alpha- D-manno- heptopyranosyl)-(1----3)-L-glycero-alpha-D-manno-heptopyranoside, corresponding to the heptose region of the Salmonella Ra core structure.

The title trisaccharide was synthesized from methyl 2,3,4-tri-O-benzyl-L-glycero-alpha-D-manno-heptopyranoside by acetolysis, followed by conversion into ethyl thioglycosides and also glycosyl bromides, which were both used in glycosylation reactions. In glycosylations using thioglycosides as glycosyl donors, N-iodosuccinimide-silver triflate and dimethyl(methylthio)sulfonium triflate were used as promoters, and in glycosylations with glycosyl bromides silver triflate was used. The protecting groups introduced into intermediates during the synthesis of the title trisaccharide were designed to allow later glycosylation at O-3' to give larger oligosaccharide fragments of the Salmonella LPS core region, and also to allow the introduction of phosphate groups at O-4 and O-4', a structural element that is suggested to be present in the Ra core.     

The crystal structures of Man(alpha1-3)Man(alpha1-O)Me and Man(alpha1-6)Man(alpha1-O)Me in complex with concanavalin A.

The crystal structures of concanavalin A in complex with Man(alpha1-6)Man(alpha1-O)Me and Man(alpha1-3)Man(alpha1-O)Me were determined at resolutions of 2.0 and 2.8 A, respectively. In both structures, the O-1-linked mannose binds in the conserved monosaccharide-binding site. The O-3-linked mannose of Man(alpha1-3)Man(alpha1-O)Me binds in the hydrophobic subsite formed by Tyr-12, Tyr-100, and Leu-99. The shielding of a hydrophobic surface is consistent with the associated large heat capacity change. The O-6-linked mannose of Man(alpha1-6)Man(alpha1-O)Me binds in the same subsite formed by Tyr-12 and Asp-16 as the reducing mannose of the highly specific trimannose Man(alpha1-3)[Man(alpha1-6)]Man(alpha1-O)Me. However, it is much less tightly bound. Its O-2 hydroxyl makes no hydrogen bond with the conserved water 1. Water 1 is present in all the sugar-containing concanavalin A structures and increases the complementarity between the protein-binding surface and the sugar, but is not necessarily a hydrogen-bonding partner. A water analysis of the carbohydrate-binding site revealed a conserved water molecule replacing O-4 on the alpha1-3-linked arm of the trimannose. No such water is found for the reducing or O-6-linked mannose. Our data indicate that the central mannose of Man(alpha1-3)[Man(alpha1-6)]Man(alpha1-O)Me primarily functions as a hinge between the two outer subsites.     

Transmembrane potential variations accompanying the PMA-triggered O-2 and H2O2 release by mouse peritoneal macrophages

Stimulation by PMA of Streptococci-elicited macrophages induced a transient membrane depolarization preceding the onset of detectable O-2 production. Mice-resident peritoneal macrophages were unresponsive to PMA for both activities. The PMA-triggered membrane depolarization seemed to be independent from O-2 production because inhibition of membrane depolarization by EGTA had no effect on rates of O-2 or H2O2 release and rate of antimycin A insensitive O2 uptake by Streptococci-elicited macrophages. The portion of O2 uptake recovered as O-2 was found to be 1/3. The rate of O-2 release was twice the rate of H2O2 production (1.1 nmol H2O2.min-1 X 10(6) macrophages-1).     

Antigenic characteristics of standard strains of Providencia rettgeri (O- and H-antigens)

Additions and changes have been introduced into the existing antigenic diagnostic scheme of P. rettgeri on the basis of the study of the antigenic structure of standard strains from foreign collections: new, formerly unknown varieties of somatic and flagellar antigens (O35, O36, H27, H28) have been discovered, the complex of antigenic factors for H-antigens 7, 10, 23, 27 has been discovered. Strains 958 (36 : 28) and 979 (16 : 27a, 23b, 2a), previously classified with the genus Morganella, have been identified by O- and H-antigens.     

Identification of Salmonella with the O-1 Bacteriophage

The O-1 bacteriophage test of Cherry et al. (1954) for the presumptive identification of salmonellae in the diagnostic laboratory was investigated. A phage lysate with a titer of 10(12) plaque-forming units per ml was found to be optimal. This preparation lysed 98.2% of Salmonella strains tested, while maintaining its high specificity for salmonellae. Gram-negative organisms other than salmonellae were resistant to the O-1 phage; however, 5.9% of Escherichia coli strains tested were susceptible. The O-1 phage test is a simple, rapid, inexpensive, sensitive, and specific procedure for the identification of salmonellae in the diagnostic laboratory. A presumptive identification is obtained 1 day earlier than with conventional biochemical tests.