Biomedical subjects

E J Walsh

Publications and source records attributed to E J Walsh.

At least 19 recordsLinked to original sources

PubMed2002

Rotifers ingest Giardia cysts.

Seven species of rotifers representing 6 genera, Epiphanes, Plationus, Asplanchna, Philodina species A, Philodina species B. Platyias, and Brachionus, were exposed to Giardia cysts isolated from the feces of experimentally infected holstein calves. Giardia cysts were prestained with a fluorescein isothiocyanate-conjugated monoclonal antibody and mixed with viable rotifers on 3-well Teflon-coated microscope slides. Organisms were observed with phase-contrast, differential interference contrast, and fluorescence microscopy. Five rotifer species, Epiphanes brachionus, Plationus patulus, Philodina (both A and B), and Platyias quadricornis, ingested varying numbers of cysts, which were retained within the rotifers' bodies throughout the observation period. Rotifer ingestion of Giardia cysts may represent a means of reducing water contamination.

Animals↗

PubMed2001

Structural organization of the fibrinogen-binding region of the clumping factor B MSCRAMM of Staphylococcus aureus.

The clumping factor B (ClfB) of Staphylococcus aureus is a surface protein that binds to fibrinogen (Ni Eidhin, D., Perkins, S., Francois, P., Vaudaux, P., Hook, M., and Foster, T. J., 1998 Mol. Microbiol. 30, 245-257). The ligand-binding activity is located in the approximately 500-residue A-region (residues 44-542), which represents the N-terminal half of the MSCRAMM protein. We now hypothesize that the ClfB A-region is composed of three subdomains, which we have named N1, N2, and N3, respectively. To examine this hypothesis, we expressed recombinant forms of the individual putative subdomains, the tandem motifs N12 and N23, and the full-length A-region N123. Far UV circular dichroism spectra showed that each subdomain is composed mainly of beta-sheets with little or no discernible alpha-helices. Heat-induced unfolding of individual subdomains occurred with a single state transition and was reversible, indicating that the subdomains can fold as discreet units. Gel permeation chromatography indicated that N2, N3, and N23 are globular. In contrast, domain N1 appeared to be elongated and conferred a somewhat elongated structure on segments containing this subdomain (i.e. N12 or N123). N123, N12, and N23 all bound to fibrinogen, but N23 had a higher affinity for fibrinogen than that observed for the full-length A-region; N123 or for N12. However, an extended N terminus of N23 was required for ligand binding. A form of N23 that was generated by proteolytic processing and lacked the N-terminal extension was unable to bind fibrinogen. Recombinant forms of individual subdomains did not bind fibrinogen. The addition of recombinant N23 effectively inhibited ClfB-mediated bacterial adherence to fibrinogen, and N123 caused some reduction in bacterial attachment, whereas N12 was essentially inactive. Antibodies raised against the central N2 domain of the A-region were the most effective at inhibiting bacterial adhesion to immobilized fibrinogen, although anti-N3 or anti-N1 antibodies also caused some reduction in ClfB-mediated adherence to fibrinogen.

Adhesins, Bacterial↗

PubMed2001

Loss of clumping factor B fibrinogen binding activity by Staphylococcus aureus involves cessation of transcription, shedding and cleavage by metalloprotease.

The fibrinogen-binding protein clumping factor B (ClfB) of Staphylococcus aureus is present on the surface of cells from the early exponential phase of growth in greater amounts than on cells from late exponential phase and is barely detectable on cells from stationary phase. Expression of a clfB-lacZ fusion indicated that transcription stopped before the end of exponential phase. Mutations in the global regulators agr and sar had no effect on clfB transcription. The loss of ClfB protein from cells in stationary phase was due to expression ending before cells stopped growing, combined with shedding of some of the protein into the growth medium and dilution of those molecules remaining on the cell surface during the two to three cell division events leading to stationary phase. Two forms of the protein occurred on the cell surface, the smaller of which was generated by loss of a domain from the N terminus. The proportion of the smaller form increased as the cultures grew. The metalloprotease aureolysin was shown to be responsible for cleavage of ClfB. Cleavage was inhibited by EDTA and o-phenanthroline and did not occur in an aureolysin-deficient mutant. Purified aureolysin promoted cleavage of cell surface-located ClfB as well as the recombinant A domain of ClfB. Cleavage was detected at two sites, one located between residues Ser(197) and Leu(198) and the other between Ala(199) and Val(200). The truncated form of ClfB did not bind fibrinogen.

Adhesins, Bacterial↗

PubMed2001

Consequences of hypothyroidism on auditory system function in Tshr mutant (hyt) mice.

The otological consequences of hypothyroidism and the outcome of thyroxin (T4) administration during the developmental period preceding the onset of hearing were examined in mice that express a point mutation in the gene encoding the thyrotropin receptor (Tshr), the so-called hyt mouse. Progeny of sires homozygous for the trait and heterozygous dams were injected with T4 or saline placebo from birth through the tenth postnatal day and auditory-evoked brainstem responses (ABRs) to acoustic clicks and tone bursts were recorded from young adults. Mutant (hyt/hyt) mice exhibited a distinctive pattern of sensory pathology that was characterized by their insensitivity to sound, prolonged response latencies, reduced peak amplitudes, and steep latency-intensity curves relative to the phenotypically normal, euthyroid, +/hyt littermates. Following thyroxin treatment, hyt/hyt mice responded to acoustic stimuli more frequently, were more sensitive to tone bursts throughout their audiometric range, and exhibited decreased latencies and increased amplitudes when compared with placebo-treated homozygous mutants. Although thresholds to acoustic stimuli were improved relative to the untreated group, T4-treated homozygotes were less sensitive than normal, euthyroid individuals. In addition, energy consumption by auditory brainstem nuclei, measured by 2-deoxyglucose (2-DG) uptake, was significantly lower in hyt/hyt mice compared with heterozygotes, and T4 treatment increased the level of 2-DG utilization. Moreover, mean ages for eye-opening and pinna-raising were delayed in animals that were homozygous for the hyt allele. When T4 was administered to hyt/hyt animals, pinna-raising occurred earlier than in untreated animals. A subset of homozygotes exhibited circling behavior, indicative of vestibular and/or motor dysfunction, even though all individuals assumed a normal righting reflex. These findings, including recruitment-like behavior and the restoration of response magnitude at high levels but not low, suggest that the cochlear amplifier is the primary locus of an enduring otological defect associated with hypothyroidism in the Tshr mouse.

Animals↗

PubMed2001

Development of auditory brainstem responses (ABRs) in Tshr mutant mice derived from euthyroid and hypothyroid dams.

Developmental changes in auditory brainstem responses (ABRs) to clicks and tone bursts were studied in genetically hypothyroid Tshr mutant mice that were homozygous for the hypothyroid trait (hyt/hyt), as well as in euthyroid individuals that were heterozygous for the trait (+/hyt). The developmental role of maternal thyroid hormones was determined by comparing homozygotes that were offspring of euthyroid (hyt/hyt(c)) or hypothyroid (hyt/hyt(h)) dams; all heterozygotes were born to euthyroid dams (+/hyt(e)). Clear responses to high-level stimuli were recorded from heterozygotes on postnatal day 12 (P12) for most stimulus conditions, and thresholds, response amplitudes, interpeak intervals, and latencies developed normally, achieving nearly adult properties by P21. Most hyt/hyt(h) animals were unresponsive to acoustic stimulation throughout the period of study. Grossly immature responses to high-level stimuli were observed in many hyt/hyt(e) pups on P15; however, clear, low-amplitude responses were not routinely observed until P21. Thresholds improved with age in +/hyt(e) and hyt/hyt(e) individuals, and latency-level curves were relatively steep in young animals and developed normally in +/hyt(e) mice with the most significant changes occurring between P15 and P21. In general, hyt/hyt(e) mice exhibited prolonged latencies, interpeak intervals, and central conduction times throughout the age range studied, and slopes of latency-level curves remained abnormally steep through P28. Response amplitudes were generally larger in heterozygotes than in hyt/hyt(e) mice, regardless of level. Replacement of thyroxin during the first 10 postnatal days in hyt/hyt(h) pups had little to no effect on the development of auditory function, although more animals from this group were responsive at very high stimulation levels. We conclude that auditory function is impaired in hypothyroid Tshr animals throughout development and that impairment is profound when individuals are not exposed to maternal thyroid hormone, i.e., a clear thyroxin-dependent critical prenatal period exists in the Tshr mutant mouse.

Aging↗

PubMed2001

Prevention of auditory dysfunction in hypothyroid Tshr mutant mice by thyroxin treatment during development.

Based on previous work, it is clear that genetically hypothyroid Tshr(hyt) mutant mice are congenitally deaf [O'Malley et al. (1995) Hear. Res. 88: 181-189, Sprinkle et al. 2001b, J. Assoc. Res. Otolaryngol. DOI: 10.1007/s101620010077]. However, the extent to which auditory development is dependent on the availability of thyroxin (T4) during specific developmental stages is unknown. The aim of this study was to determine the relative importance of prenatal and postnatal thyroxin on the ontogeny of hearing in the hyt mouse. Experimental hypothyroid subjects were offspring of hyt/hyt breeders implanted with T4 or placebo controlled-release pellets 14 days prior to mating. Pups received T4 or saline placebo injections from birth through postnatal day 14 (P14) or the time of testing on P28. In the absence of exogenous T4 replacement, very high stimulus levels (>80 dB SPL) were required to elicit responses. Remarkably, T4 treatment confined to the postnatal period failed to significantly improve auditory function relative to untreated animals, while response thresholds, latencies, and amplitudes of mice born to dams that received T4 during pregnancy were significantly improved relative to both of the untreated groups. Response thresholds were improved somewhat when maternal T4 replacement was followed by treatment during the first 14 days of life, and animals treated throughout prenatal and postnatal life were comparable to those of age-matched euthyroid individuals. Findings from this study show that treatment of hyt/hyt mice with exogenous T4 significantly attenuates hypothyroid-induced otopathology in a develop-mental-stage-dependent manner. In addition, we demonstrate that postnatal development is critically dependent on prenatal exposure to thyroxin and that the critical window of T4 dependence extends throughout development.

Aging↗

PubMed2000

Afferent innervation of outer and inner hair cells is normal in neonatally de-efferented cats.

It has been hypothesized that normal pruning of exuberant branching of afferent neurons in the developing cochlea is caused by the arrival of the olivocochlear efferent neurons and the resulting competition for synaptic sites on hair cells. This hypothesis was supported by a report that afferent innervation density on mature outer hair cells (OHCs) is elevated in animals deefferented at birth, before the olivocochlear system reaches the outer hair cell area (Pujol and Carlier [1982] Dev. Brain Res. 3:151-154). In the current study, this claim was evaluated quantitatively at the electron microscopic level in four cats that were de-efferented at birth and allowed to survive for 6-11 months. A semiserial section analysis of 156 OHCs from de-efferented and normal ears showed that, although de-efferentation essentially was complete in all four cases, the number and distribution of afferent terminals on OHCs was indistinguishable from normal, and the morphology of afferent synapses was normal in both the inner hair cell area and the OHC area. Thus, the postnatal presence of an efferent system is not required for the normal development of cochlear afferent innervation, and the synaptic competition hypothesis is not supported.

Afferent Pathways↗

PubMed2000

The animal enterprise protection act: a scientist's perspective.

The Animal Enterprise Protection Act gives courts latitude in sentencing animal rights terrorists, but remains largely unused by prosecutors. The author, himself a victim of animal rights terrorism, comments on the Act's strengths and weaknesses, and challenges the lab animal community to unify in its response.

Journal Article↗

PubMed2000

Lewis X structures in the O antigen side-chain promote adhesion of Helicobacter pylori to the gastric epithelium.

Helicobacter pylori NCTC11637 expresses a lipopolysaccharide (LPS) that comprises an O antigen side-chain with structural homology to the human blood group antigen Lewis X (Le(x)). The role of this molecule in adhesion of H. pylori to gastric epithelial cells was investigated. Mutants expressing truncated LPS structures were generated through insertional mutagenesis of rfbM and galE; genes encode GDP mannose pyrophosphorylase and galactose epimerase respectively. Compositional and structural analysis revealed that the galE mutant expressed a rough LPS that lacked an O antigen side-chain. In contrast, an O antigen side-chain was still synthesized by the rfbM mutant, but it lacked fucose and no longer reacted with anti-Le(x) monoclonal antibodies (Mabs). The ability of these mutants to bind to paraffin-embedded sections from the antrum region of a human stomach was assessed. Adhesion of the wild type was characterized by tropic binding to the apical surface of mucosal epithelial cells and cells lining gastric pits. In contrast, both the rfbM and galE mutants failed to demonstrate tropic binding and adhered to the tissue surface in a haphazard manner. These results indicate that LPS and, more specifically, Le(x) structures in the O antigen side-chain play an important role in targeting H. pylori to specific cell lineages within the gastric mucosa. The role of Le(x) in this interaction was confirmed by the tropic binding of synthetic Le(x), conjugated to latex beads, to gastric tissue. The observed pattern of adhesion was indistinguishable from that of wild-type H. pylori.

Bacterial Adhesion↗

PubMed2000

Contributions of ion conductances to the onset responses of octopus cells in the ventral cochlear nucleus: simulation results.

The onset response pattern displayed by octopus cells has been attributed to intrinsic membrane properties, low membrane impedance, and/or synaptic inputs. Although the importance of a low membrane impedance generally is acknowledged as an essential component, views differ on the role that ion channels play in producing the onset response. In this study, we use a computer model to investigate the contributions of ion channels to the responses of octopus cells. Simulations using current ramps indicate that, during the "ramp-up" stage, the membrane depolarizes, activating a low-threshold K(+) channel, K(LT), which increases membrane conductance and dynamically increases the current required to evoke an action potential. As a result, the model is sensitive to the rate that membrane potential changes when initiating an action potential. Results obtained when experimentally recorded spike trains of auditory-nerve fibers served as model inputs (simulating acoustic stimulation) demonstrate that a model with K(LT) conductance as the dominant conductance produces realistic onset response patterns. Systematically replacing the K(LT) conductance by a h-type conductance (which corresponds to a hyperpolarization-activated inward rectifier current, I(h)) or by a leakage conductance reduces the model's sensitivity to rate of change in membrane potential, and the model's response to "acoustic stimulation" becomes more chopper-like. Increasing the h-type conductance while maintaining a large K(LT) conductance causes an increase in threshold to both current steps and acoustic stimulation but does not significantly affect the model's sensitivity to rate of change in membrane potential and the onset response pattern under acoustic stimulation. These findings support the idea that K(LT), which is activated during depolarization, is the primary membrane conductance determining the response properties of octopus cells, and its dynamic role cannot be provided by a static membrane conductance. On the other hand, I(h), which is activated during hyperpolarization, does not play a large role in the basic onset response pattern but may regulate response threshold through its contribution to the membrane conductance.

Acoustic Stimulation↗

PubMed1998

Long-term effects of sectioning the olivocochlear bundle in neonatal cats.

The olivocochlear bundle (OCB) was cut in neonatal cats to evaluate its role in the development of normal cochlear function. Approximately 1 year after deefferentation, acute auditory nerve fiber (ANF) recordings were made from lesioned animals, lesion shams, and normal controls. The degree of deefferentation was quantified via light microscopic evaluation of the density of OCB fascicles in the tunnel of Corti, and selected cases were analyzed via electron microscopy. In the most successful cases, the deefferentation was virtually complete. ANFs from successfully lesioned animals exhibited significant pathophysiology compared with normals and with other animals in which the surgery failed to interrupt the OCB. Thresholds at the characteristic frequency (CF), the frequency at which ANFs are most sensitive, were elevated across the CF range, with maximal effects for CFs in the 10 kHz region. Frequency threshold or tuning curves displayed reduction of tip-to-tail ratios (the difference between CF and low-frequency "tail" thresholds) and decreased sharpness of tuning. These pathological changes are generally associated with outer hair cell (OHC) damage. However, light microscopic histological analysis showed minimal hair cell loss and no significant differences between normal and deefferented groups. Spontaneous discharge rates (SRs) were lower than normal; however, those fibers with the highest SRs remained more sensitive than those with lower SRs. Findings suggest that the interaction between OC efferents and OHCs early in development may be critical for full expression of active mechanical processes.

Age Factors↗

PubMed1998

Ultrastructural, physiological, and molecular defects in the inner ear of a gene-knockout mouse model for autosomal Alport syndrome.

The cochleae from a COL4A3-deficient mouse line were examined for defects that might shed light on the molecular mechanism of otopathology observed in humans with Alport syndrome. At the light microscopic level no obvious defects were observed. Immunohistochemical analysis using antibodies specific for the basement membrane collagen chains revealed the absence of the COL4A3 and COL4A4 chains throughout the membranous labyrinth. The COL4A5 chain was absent from all cochlear basement membranes except those in the vessels of the stria vascularis. Expression of the COL4A1 and COL4A2 chains was unchanged in the mutant. Electron microscopic examination of the cochlear basement membranes revealed significant thinning of the basement membrane running from the spiral limbus, down the inner sulcus, across the basilar membrane and up to the spiral prominence. Basement membranes that normally ensheathe the root cells were not detectable. In contrast, the basement membranes surrounding the vessels of the stria vascularis were significantly thickened in the mutant. This was associated with endothelial cell swelling and a marked decrease in internal capillary diameter. In severe cases, pathology was observed in the marginal cells with a loss of basolateral infoldings. Immunohistochemical analysis of the strial vessels revealed an increase in entactin and collagen COL4A1 and COL4A2 chains. Auditory-evoked brainstem response measurements suggest a small increase in thresholds across all frequencies when successive measurements on individual mutant mice were examined between 6 and 8 postnatal weeks. Combined, these results illustrate changes in the basement membranes of the strial vessels that bear resemblance to Alport glomerular basement membrane pathology. A closer look at this compartment in human Alport biopsy specimen may be warranted.

Animals↗

PubMed1998

Paradoxical relationship between frequency selectivity and threshold sensitivity during auditory-nerve fiber development.

The acquisition of adult-like frequency selectivity is generally assumed to be the tightly coupled to improvements in threshold sensitivity during cochlear development. In this study, frequency versus threshold (tuning) curves obtained from 1108 auditory-nerve fibers were used to investigate the relationship between tuning and threshold at characteristic frequency (CF) during postnatal development in kittens. At the earliest ages included in this study, sharpness was within the adult range, but thresholds were significantly higher than adult values. Tuning and thresholds improved along different exponential time courses that varied with CF. For units with CFs below 1 kHz, tuning curve slopes below CF matured earliest, followed by CF threshold, and then by slopes above CF. In contrast, for CFs above 1 kHz, the high-frequency slopes matured first, followed by threshold and then by slope below CF. One interpretation of these results is that tuning and thresholds are not tightly coupled in immature animals. Paradoxically, however, high-frequency slopes were correlated with threshold for individual units at all ages, suggesting that the relationship between tuning and threshold is maintained during development. This contradiction can be resolved by a developmental model that features a functional separation between cochlear nonlinearities and mechanical/electrical conversion.

Animals↗

PubMed1998

Metabotropic glutamate receptor activation modulates sound level processing in the cochlear nucleus.

The principal role of ionotropic glutamate receptors in the transmission and processing of information in the auditory pathway has been investigated extensively. In contrast, little is known about the functional contribution of the G-protein-coupled metabotropic glutamate receptors (mGluRs), although their anatomic location suggests that they exercise a significant influence on auditory processing. To investigate this issue, sound-evoked responses were obtained from single auditory neurons in the cochlear nuclear complex of anesthetized cats and gerbils, and metabotropic ligands were administered locally through microionophoretic pipettes. In general, microionophoresis of the mGluR agonists, (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid or (2S,1'S, 2'S)-2-(carboxycyclopropyl)glycine, initially produced a gradual increase in spontaneous and sound-evoked discharge rates. However, activation and recovery times were significantly longer than those observed for ionotropic agonists, such as N-methyl--aspartate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, consistent with the recruitment of a second-messenger system. The efficacy of mGluR agonists was diminished after administration of the mGluR antagonist, (+)-alpha-methyl-4-carboxyphenylglycine, consistent with a selective action at metabotropic recognition sites. In contrast, two distinct changes were observed after the mGluR agonist had been discontinued for several minutes. Approximately 50% of neurons exhibited a chronic depression of sound-evoked discharge rate reminiscent of long-term depression, a cellular property observed in other systems. Approximately 30% of neurons exhibited a long-lasting enhancement of the sound-evoked response similar to the cellular phenomenon of long-term potentiation. These findings suggest that mGluR activation has a profound influence on the gain of primary afferent driven activity in the caudal cochlear nucleus.

Acoustic Stimulation↗

PubMed1997

A simple program for simulating the responses of neurons with arbitrarily structured and active dendritic trees.

We describe a simple program to simulate neural responses. This program is based on the compartmental approach, in which all compartments of a neuron (i.e. axon, soma or dendrite) are represented by the same basic electrical structure. A parameter file is used to store the model parameters, including the nonlinear channel characteristics of each compartment. The model is then automatically configured according to the values specified in the parameter file. The computation of the conductance of each active channel over time is handled by a unique subroutine optimized according to the kinetics of each channel. The equations for arbitrarily structured trees are solved implicitly using a simple algorithm similar to that of Hines (Hines, M. (1984) Int. J. Biomed. Comput., 15:69-76). The output of the model uses PostScript format. The advantage of this program is that it is small in size, simple to use, efficient, and is platform independent.

Algorithms↗

PubMed1997

Hearing loss in the RBF/DnJ mouse, a proposed animal model of Usher syndrome type IIa.

The Usher syndromes (US) are a group of inherited disorders that feature autosomal recessive neurosensory hearing loss or deafness with retinitis pigmentosa (RP). Moderate to severe non-progressive high frequency hearing loss with RP and normal vestibular function describes Usher syndrome type IIa, which has been localized to 1q41. Severe retinal degeneration in the inbred mouse strain RBF/DnJ is caused by rd3, a recessive gene located on mouse chromosome 1 distal to akp1 in a region which is orthologous to human 1q32-q42. We evaluated rd3 as a candidate for orthology with USH2A by first reducing and refining the relatively broad region in which rd3 is thought to reside. DNA of offspring from an RBF/DnJ x MOLF/Ei backcross was genotyped with PCR markers closely flanking the predicted location of rd3. Our haplotype analysis re-positioned rd3 to a 3.6 cM region between markers D1Mit273 (cen) and D1Mit209 (tel), consistent with the expected position of an USH2A murine orthologue. Consequently, rd3 is a positional candidate for Usher type IIa. Next we assessed the rd3/rd3 audiological phenotype to see how closely it paralleled that of Usher IIa. Audiological evaluation of mice at various ages revealed evidence of high frequency progressive hearing loss, previously unreported in the RBF/DnJ strain. However, this newly discovered hearing deficit was observed to be inherited independently of rd3, establishing that a completely different gene is responsible.

Animals↗

PubMed1997

Influence of medium composition on the growth and antigen expression of Helicobacter pylori.

An evaluation of the ability of various solid and liquid media to support both growth and antigen expression, particularly lipopolysaccharide (LPS) expression, by Helicobacter pylori culture collection strains and clinical isolates was performed. Liquid-based basal media (brain heart infusion, Brucella broth, Mueller-Hinton broth and tryptone soya broth) supported the growth of strains, whereas solid basal media of the same formulation did not support growth. Optimal growth of all strains was obtained on solid and in liquid media containing blood. Supplemented solid media containing supplements other than blood supported growth but only to a small extent. In liquid media excluding blood, serum supplements enhanced growth and horse serum was found to be superior to fetal calf serum. In general, beta-cyclodextrin did not increase growth. Mueller-Hinton broth or tryptone soya broth containing horse serum and a nitrogen source such as yeast extract or proteose peptone no. 3 were found to give optimal growth of H. pylori in a blood-free environment. Strains after cultivation in liquid media, irrespective of composition, maintained production of high-molecular weight (mol. wt) LPS with an O side chain independent of medium composition, whereas subculturing on solid media resulted in production of low-mol. wt LPS. Expression of proteins differed in liquid and on solid media, particularly proteins of 57 and 60 kDa, but qualitatively no differences were observed upon supplementation of basal media.

Antigens, Bacterial↗

PubMed1997

Structural characterization of the lipid A component of Helicobacter pylori rough- and smooth-form lipopolysaccharides.

The chemical structure of free lipid A isolated from rough- and smooth-form lipopolysaccharides (R-LPS and S-LPS, respectively) of the human gastroduodenal pathogen Helicobacter pylori was elucidated by compositional and degradative analysis, nuclear magnetic resonance spectroscopy, and mass spectrometry. The predominant molecular species in both lipid A components are identical and tetraacylated, but a second molecular species which is hexaacylated is also present in lipid A from S-LPS. Despite differences in substitution by acyl chains, the hydrophilic backbone of the molecules consisted of beta(1,6)-linked D-glucosamine (GlcN) disaccharide 1-phosphate. Because of microheterogeneity, nonstoichiometric amounts of ethanolamine-phosphate were also linked to the glycosidic hydroxyl group. In S-LPS, but not in R-LPS, the hydroxyl group at position 4' was partially substituted by another phosphate group. Considerable variation in the distribution of fatty acids on the lipid A backbone was revealed by laser desorption mass spectrometry. In tetraacyl lipid A, the amino group of the reducing GlcN carried (R)-3-hydroxyoctadecanoic acid (position 2), that of the nonreducing GlcN carried (R)-3-(octadecanoyloxy)octadecanoic acid (position 2'), and ester-bound (R)-3-hydroxyhexadecanoic acid was attached at position 3. Hexaacyl lipid A had a similar substitution by fatty acids, but in addition, ester-bound (R)-3-(dodecanoyloxy)hexadecanoic acid or (R)-3(tetradecanoyloxy)hexadecanoic acid was attached at position 3'. The predominant absence of ester-bound 4'-phosphate and the presence of tetraacyl lipid A with fatty acids of 16 to 18 carbons in length differentiate H. pylori lipid A from that of other bacterial species and help explain the low endotoxic and biological activities of H. pylori LPS.

Ethanolamine↗