Presence Of Phosphoethanolamine Research Articles

Mapping phosphate modifications of substituted lipid A via a targeted MS3 CID/UVPD strategy.

Structural characterization of lipid A from Gram-negative bacteria remains a significant challenge, especially with respect to localizing modifications of the phosphate groups typically found on the reducing and non-reducing ends of the β-1',6-linked glucosamine disaccharide backbone of lipid A. As reported here, combining traditional collisional activated dissociation (CAD) and ultraviolet photodissociation (UVPD) in a hybrid MS3 approach facilitates identification and localization of substituents of the phosphate groups. The focus is on rapid identification and characterization of substituted lipid A species with specific emphasis on the modifications on the 1 and 4' phosphate moieties. Mapping these modifications, typically ones that modify the surface charges of lipopolysaccharides, is particularly important owing to the impact of these types of modifications on antibiotic resistance. The presence of phosphoethanolamine, aminoarabinose, and galactosamine moieties in hexaacylated and heptaacylated lipid A species, including ones from Enterobacter cloacae and Acinetobacter baumannii, are characterized using a targeted MS3 strategy to identify glycosidic product ions (1,5X1 and 0,4A2, typically) which allow localization of the substituents.

Pool of phosphoethanolamine and phosphoserine in the brain of the snail Lymnaea stagnalis L. in summer and before winter dormancy

Small amounts of phospholipid metabolites, phosphoethanolamine and phosphoserine, were discovered at a ratio of 1:9 in the brain of a freshwater mollusk, the pond snail Lymnaea stagnalis L., collected both in summer and autumn. The phosphoethanolamine pool increased by 15% in autumn relative to the summer level (up to 625 ± 44 nmol per g of wet tissue), although this value still constituted 6% of the total pool of free amino acids and ninhydrin-positive substances. These findings are in striking contrast with our previous results that showed adaptive modifications of the amino acid and phosphoethanolamine pools in the brain of eurythermal freshwater fish at low temperatures. At the same time, these data demonstrate the presence of phosphoethanolamine and phosphoserine in the central nervous system at comparatively early stages of its evolution.

Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis Are Correlated with Pathogenicity and Carriage

The degree of phosphorylation and phosphoethanolaminylation of lipid A on neisserial lipooligosaccharide (LOS), a major cell-surface antigen, can be correlated with inflammatory potential and the ability to induce immune tolerance in vitro. On the oligosaccharide of the LOS, the presence of phosphoethanolamine and sialic acid substituents can be correlated with in vitro serum resistance. In this study, we analyzed the structure of the LOS from 40 invasive isolates and 25 isolates from carriers of Neisseria meningitidis without disease. Invasive strains were classified as groups 1-3 that caused meningitis, septicemia without meningitis, and septicemia with meningitis, respectively. Intact LOS was analyzed by high resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Prominent peaks for lipid A fragment ions with three phosphates and one phosphoethanolamine were detected in all LOS analyzed. LOS from groups 2 and 3 had less abundant ions for highly phosphorylated lipid A forms and induced less TNF-α in THP-1 monocytic cells compared with LOS from group 1. Lipid A from all invasive strains was hexaacylated, whereas lipid A of 6/25 carrier strains was pentaacylated. There were fewer O-acetyl groups and more phosphoethanolamine and sialic acid substitutions on the oligosaccharide from invasive compared with carrier isolates. Bioinformatic and genomic analysis of LOS biosynthetic genes indicated significant skewing to specific alleles, dependent on the disease outcome. Our results suggest that variable LOS structures have multifaceted effects on homeostatic innate immune responses that have critical impact on the pathophysiology of meningococcal infections.

O-antigen structure of Shigella flexneri serotype Yv and effect of the lpt-O gene variation on phosphoethanolamine modification of S. flexneri O-antigens

Shigella flexneri is the major human pathogen causing shigellosis. O-antigens of all S. flexneri serotypes (except for serotype 6) share the →2)-α-l-Rhap(III)-(1 → 2)-α-l-Rhap(II)-(1 → 3)-α-l-Rhap(I)-(1 → 3)-β-d-GlcpNAc-(1→ basic O-unit, whereas differences between the serotypes are conferred by phage-encoded glucosylation and/or O-acetylation at various positions. Recently, in serotype X and 4a variants called Xv and 4av, respectively, O-antigen modification with phosphoethanolamine (PEtN) has been identified, which is encoded by a plasmid-borne gene (lpt-O) for a PEtN-transferase and confers the monoclonal antibody IV-1(MASF IV-1) determinant to the bacteria. In this study, we elucidated the O-antigen structure of serotype Yv, another MASF IV-1-positive novel variant of S. flexneri. The serotype Yv O-antigen has the same basic carbohydrate backbone structure as that of the "classical" serotype Y, but differs in the presence of PEtN at position 3 of Rha(III) (major) or both Rha(II) and Rha(III) (minor). This pattern is similar to that of serotype 4av, but different from the pattern of serotype Xv, which is characterized by major PEtN modification on Rha(II). In serotype Yv, mono- and bisphosphorylated O-units generate a block-copolymeric structure, the former being partially O-acetylated at position 6 of GlcNAc and the latter lacking O-acetylation. Functional analysis revealed a correlation between the serotype-specific PEtN modification pattern and the lpt-O variation in different serotypes: lpt-O(RII) in serotype Xv is better tuned for phosphorylation of Rha(II) and lpt-O(RIII) in serotypes Yv and 4av for phosphorylation of Rha(III). These data enhance our knowledge of S. flexneri serotype conversion mechanisms and help to understand the biosynthesis process of the new O-antigen variants.

Requirements for the binding of C-reactive protein to complement factor H (134.60)

Abstract Human C-reactive protein (CRP) protects mice from lethality following infection with Streptococcus pneumoniae type 3. Because both CRP and type 3 pneumococci are reported to bind to complement regulatory protein factor H, we propose that the factor H-binding capability of CRP may contribute to anti-pneumococcal function of CRP. In this study, we explored interactions between CRP and factor H with the aim to generate a CRP mutant incapable of binding to factor H. Surprisingly, we found that freshly-purified CRP did not bind human factor H. But, in the presence of phosphoethanolamine (PEt), freshly-purified CRP bound to human factor H efficiently in a dose-dependent manner. In contrast, and as reported previously, when an old preparation of CRP was thawed and used, CRP bound to human factor H even in the absence of PEt. Identical results were obtained with murine factor H. We also found that the mutations at the C1q-binding site on CRP did not affect the binding of PEt-complexed CRP to factor H, indicating separate binding sites on CRP for C1q and factor H. The mechanism of action of PEt in the binding of CRP to factor H is unknown; however, it seems that PEt modifies CRP in a way similar to storage-induced modification of CRP. Our findings raise the possibility that CRP, once in contact with a PEt-containing surface, gains factor H-binding specificity to modulate the complement attack on factor H-bound pneumococci. NIH R01HL071233

Deep zone articular chondrocytes in vitro express genes that show specific changes with mineralization.

We have developed a method to form reconstituted mineralized articular cartilagenous tissue in vitro from isolated deep zone chondrocytes. The aim of this study was to characterize further these cultures prior to and during mineralization. Histologic examination of the cells up to 8 days in culture showed that the chondrocytes had formed cartilagenous tissue. Similar to the in vivo cartilage, the chondrocytes expressed aggrecan, types II, I, and X collagens, osteopontin, and alkaline phosphatase (ALP). No osteocalcin mRNA expression was detected in either the in vivo cartilage or in vitro-generated tissue. Addition of beta-glycerophosphate (beta-GP) to the medium on day 5 induced mineralization and changes in gene expression. Expression of type X collagen, type II collagen, aggrecan core protein, and ALP were inhibited significantly between 2 h and 24 h after the addition of beta-GP. At 72 h, expression of these genes were still significantly depressed. These changes correlated with a decrease in collagen and proteoglycan synthesis, and ALP activity. Osteopontin expression increased within 8 h but returned to constitutive levels by 72 h. No change in type I collagen expression was detected. The changes in gene expression were not due to a direct effect of beta-GP itself, because similar gene changes occurred in the presence of phosphoethanolamine, another agent which induces mineralization. No changes in gene expression were seen in nonmineralizing cultures. In summary, articular chondrocytes grown on filter culture show expression of similar genes to the chondrocytes in the deep zone of articular cartilage and that changes in expression of specific genes were observed during tissue mineralization, suggesting that it is a suitable model to use to study the mechanism(s) regulating the localized mineralization of articular cartilage.

Phosphoethanolamine enhances high-affinity choline uptake and acetylcholine synthesis in dissociated cell cultures of the rat septal nucleus.

Dissociated rat septal nucleus cells cultured in defined medium exhibited twofold increases in the maximal rates of sodium-dependent, high-affinity choline uptake and acetylcholine formation when grown in the presence of phosphoethanolamine. The effect was concentration-dependent (EC50 = 15 microM) and appeared to be associated with in vitro maturation of cholinergic neurons rather than with enhanced survival. Choline acetyltransferase, acetylcholinesterase, and choline kinase activities were unaffected by this treatment. The effect of phosphoethanolamine was specific for cholinergic neurons, because treatment with this compound did not alter the kinetic constants for high-affinity neuronal uptake of gamma-aminobutyric acid or dopamine. The action appeared to be mediated primarily through activation of the sodium-dependent, high-affinity transport mechanism for choline as opposed to alterations in the storage and release of acetylcholine.

Role of phosphatidylserine in the MI-MIII equilibrium of rhodopsin

Bovine rhodopsin was recombined with various phospholipids in which the lipid acyl chain composition was held constant at that of egg phosphatidylcholine (PC), while the identity of the headgroups was varied. The ratio of MII/MI produced in the recombinant membrane vesicles by an actinic flash was studied as a function of pH, and compared to the photochemical activity observed for rhodopsin in native ROS membranes. MI and MII were found to coexist in a pH-dependent, acid-base equilibrium on the millisecond timescale. Recombinants made with phospholipids containing unsaturated acyl chains were capable of full native-like MII production, but demonstrated titration curves with different pK values. The presence of phosphoethanolamine or phosphoserine headgroups increased the amount of MII produced. In the case of phosphatidylserine this may result from alteration of the membrane surface potential, leading to an increase in the local H+ activity. The results indicate that the Gibbs free energies of the MI and MII conformational states are influenced by the membrane bilayer environment, suggesting a possible role of lipids in visual excitation.

Purification and Characterization of a Central Cholinergic Enhancing Factor from Rat Brain: Its Identity as Phosphoethanolamine

A compound that can enhance the apparent synthesis of acetylcholine in cultured explants of the medial septal nucleus has been purified from rat brain and identified as phosphoethanolamine. Acetylcholine synthesis is stimulated two- to threefold in cultures grown for 5 days in the presence of phosphoethanolamine, ethanolamine, or cytidine 5'-diphosphoethanolamine at concentrations above 100 microM. This effect appears to result from an increase in the accumulation of choline via the high-affinity, sodium-dependent uptake mechanism. The development of choline acetyltransferase activity is not affected. Phosphoethanolamine and ethanolamine seem to enhance the ability of developing cholinergic neurons to utilize choline accumulated via the sodium-dependent high-affinity choline uptake mechanism for the preferential production of acetylcholine without increasing the general metabolism of the cultures. Choline itself and its related derivatives are not stimulatory for these effects.

Orthopaedic problems in adult hypophosphatasia: a report of two cases

Hypophosphatasia in adults is rare. Two elderly sisters presenting with pathological fractures of the femur are reported to illustrate the difficulties in orthopaedic management of this disease. All patients with a history of repeated fractures, especially from minor trauma and with generalised radiological bony abnormality, should be screened for this rare disease. A consistently low level of serum alkaline phosphatase with the presence of phosphoethanolamine in the urine is diagnostic.