Overall symptom severity as a patient-reported outcome measure for chronic rhinosinusitis: what it reflects and how to measure it.

BACKGROUND: The objective of this study was to identify how - and to what extent - overall symptom severity (OSS) score reflects individual chronic rhinosinusitis (CRS) symptoms and whether it can be measured using alternatives to the standard visual analog scale (VAS). METHODS: CRS patients from four sites across three continents rated their OSS scores, severities of nasal obstruction, nasal drainage, decreased sense of smell, facial pain/pressure and sleep disturbance using a standard VAS, VAS with labeled tick marks at every 1 centimeter, and by writing down their OSS on a scale of 0 - 100 (which was divided by 10), all of which lead to severity scores ranging from 0 - 10 in 0.1 intervals. Quality of life was measured using the SNOT-22 and EQ-5D VAS. RESULTS: In 311 CRS patients, OSS score was significantly correlated with SNOT-22 and EQ-5D VAS. OSS score was most greatly associated with the mean of all individual symptom severity scores. From individual CRS symptoms, OSS was most greatly associated with nasal obstruction followed by nasal drainage and facial pain/pressure severities. These results held true for participants with and without nasal polyps. Measurement of OSS and individual symptom severity scores using a standard VAS, tick-marked VAS, and write-in option had near-perfect consistency. CONCLUSIONS: We demonstrate for the first time that OSS largely reflects the mean of individual CRS symptom severities, although OSS is=== most weighted by nasal obstruction severity. OSS and individual symptom severity scores can be measured using a standard VAS, tick-marked VAS or write-in prompt with near-perfect consistency.

Effects of Small Temperature Differences Detected in Callosal Circuits of the Anterior Cingulate Cortex.

We measured the sensitivity of cortical circuit activity to small differences in local cortical environments by studying how temperature affects the trajectory of epileptiform events (EEs). EEs evoked via blockade of GABA-A receptors were recorded extracellularly from mouse coronal brain slices containing both hemispheres of anterior cingulate cortex synaptically connected by corpus callosum axons. Preferentially illuminating one hemisphere with the microscope condenser produced temperature differences of 0.1 °C between the hemispheres. The relatively warmer hemisphere typically initiated the EEs that then propagated to the contralateral side, demonstrating temperature directed propagation. Severing the callosum following one hour of EEs showed that the warmer hemisphere possessed a higher rate of EE generation. Further experiments implied that intact callosal circuits were required for the increased EE generation in the warmer hemisphere. We propose a hypothesis whereby callosal circuits can amplify differences in respective hemispheric activity, promoting this directionality in seizure propagation.

Implication of Major Adverse Postoperative Events and Myocardial Injury on Disability and Survival: A Planned Subanalysis of the ENIGMA-II Trial.

BACKGROUND: Globally, >300 million patients have surgery annually, and ≤20% experience adverse postoperative events. We studied the impact of both cardiac and noncardiac adverse events on 1-year disability-free survival after noncardiac surgery. METHODS: We used the study cohort from the Evaluation of Nitrous oxide in Gas Mixture of Anesthesia (ENIGMA-II) trial, an international randomized trial of 6992 noncardiac surgical patients. All were ≥45 years of age and had moderate to high cardiac risk. The primary outcome was mortality within 1 postoperative year. We defined 4 separate types of postoperative adverse events. Major adverse cardiac events (MACEs) included myocardial infarction (MI), cardiac arrest, and myocardial revascularization with or without troponin elevation. MI was defined using the third Universal Definition and was blindly adjudicated. A second cohort consisted of patients with isolated troponin increases who did not meet the definition for MI. We also considered a cohort of patients who experienced major adverse postoperative events (MAPEs), including unplanned admission to intensive care, prolonged mechanical ventilation, wound infection, pulmonary embolism, and stroke. From this cohort, we identified a group without troponin elevation and another with troponin elevation that was not judged to be an MI. Multivariable Cox proportional hazard models for death at 1 year and assessments of proportionality of hazard functions were performed and expressed as an adjusted hazard ratio (aHR) and 95% confidence intervals (CIs). RESULTS: MACEs were observed in 469 patients, and another 754 patients had isolated troponin increases. MAPEs were observed in 631 patients. Compared with control patients, patients with a MACE were at increased risk of mortality (aHR, 3.36 [95% CI, 2.55-4.46]), similar to patients who suffered a MAPE without troponin elevation (n = 501) (aHR, 2.98 [95% CI, 2.26-3.92]). Patients who suffered a MAPE with troponin elevation but without MI had the highest risk of death (n = 116) (aHR, 4.29 [95% CI, 2.89-6.36]). These 4 types of adverse events similarly affected 1-year disability-free survival. CONCLUSIONS: MACEs and MAPEs occur at similar frequencies and affect survival to a similar degree. All 3 types of postoperative troponin elevation in this analysis were associated, to varying degrees, with increased risk of death and disability.

A review of occupationally-linked suicide for dentists.

BACKGROUND AND OBJECTIVES: Suicide rates among dentists and a perceived elevated risk for suicide have been debated in the academic literature. It has filtered into the public psyche that dentists have the highest suicide rate of any occupation. The present review seeks support for both protagonist and antagonist positions from multidisciplinary perspectives. Contemporary risk factors and strategies for intervention and the prevention of suicide in dentistry are explored. METHODS: An online database search for articles and reports, with selected target words, was conducted for peer reviewed publications on suicide in the dental profession, and for factors contributing to dentist suicide. Review guidelines from the American Psychological Association were used to clarify concepts, identify where most work was focussed, and to explore the superiority of any approach to the emotive topic over another. RESULTS: Findings suggest the dominant belief that dentists have an elevated risk of suicide may be historically, but not currently, accurate. Although dentists' suicide is trending down, diversity in methodology means no current consensus is possible. Factors found to be influencing dentists' suicide ranged from known occupational stressors, to toxins and substance abuse, and untreated mental health problems. CONCLUSION: The contemporary position in New Zealand shows dentists per sé are not more likely than other health professionals to commit suicide although they may have been in the past. Dentists should be aware of individual susceptibility to burnout and mental health problems. Future directions are outlined to address this including peer intervention, and programmes available for dentists to cope better with risks leading to suicide.

Varicella zoster virus vasculopathy: analysis of virus-infected arteries.

OBJECTIVE: Varicella zoster virus (VZV) is an under-recognized yet treatable cause of stroke. No animal model exists for stroke caused by VZV infection of cerebral arteries. Thus, we analyzed cerebral and temporal arteries from 3 patients with VZV vasculopathy to identify features that will help in diagnosis and lead to a better understanding of VZV-induced vascular remodeling. METHODS: Normal and VZV-infected cerebral and temporal arteries were examined histologically and by immunohistochemistry using antibodies directed against VZV, endothelium, and smooth muscle actin and myosin. RESULTS: All VZV-infected arteries contained 1) a disrupted internal elastic lamina; 2) a hyperplastic intima composed of cells expressing α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-myosin) but not endothelial cells expressing CD31; and 3) decreased medial smooth muscle cells. The location of VZV antigen, degree of neointimal thickening, and disruption of the media were related to the duration of disease. CONCLUSIONS: The presence of VZV primarily in the adventitia early in infection and in the media and intima later supports the notion that after reactivation from ganglia, VZV spreads transaxonally to the arterial adventitia followed by transmural spread of virus. Disruption of the internal elastic lamina, progressive intimal thickening with cells expressing α-SMA and SM-MHC, and decreased smooth muscle cells in the media are characteristic features of VZV vasculopathy. Stroke in VZV vasculopathy may result from changes in arterial caliber and contractility produced in part by abnormal accumulation of smooth muscle cells and myofibroblasts in thickened neointima and disruption of the media.

Nitrosative stress with HIV dementia causes decreased L-prostaglandin D synthase activity.

BACKGROUND: The prevalence of HIV-associated neurocognitive disorders is increasing as HIV-infected individuals are living longer. The clinical manifestations of the syndrome also continue to evolve under the influence of antiretroviral drugs and comorbidities such as drugs of abuse. However, there are no surrogate markers for the disease, either to identify it de novo or to track its progression, and there is no proven treatment with the exception of antiretroviral drugs. METHODS: Levels of nitric oxide, nitrate, and 3-nitrotyrosine (3-NT)-modified proteins were measured in the CSF of 46 patients with HIV infection stratified according to their neurocognitive status and history of IV drug use (IVD). The 3-NT-modified proteins were isolated and identified by tandem mass spectrometry, and the functional consequence of 3-NT modification of L-prostaglandin D synthase (L-PGDS), the most abundant protein, was determined. RESULTS: 3-NT-modified proteins were significantly elevated in patients with HIV infection who had progressive neurocognitive decline over the next 6 months and in patients with a history of IVD. Thirteen different proteins with 3-NT modification were identified in the CSF of these patients. L-PGDS was the most abundant. 3-NT modification of this protein resulted in loss of its enzymatic activity. CONCLUSIONS: There is increased nitrosative stress in CSF of HIV-infected patients with active dementia and in patients with a history of IV drug use, measurement of which may serve as a surrogate marker for these patients. Nitrosative stress may also have important functional consequences and may impact the pathogenesis of HIV-associated neurocognitive disorders.

Fractalkine (CX3CL1) and brain inflammation: Implications for HIV-1-associated dementia.

Leukocyte migration and activation play an important role in immune surveillance and the pathogenesis of a variety of neurodegenerative disorders, including human immunodeficiency virus (HIV)-1-associated dementia (HAD). A novel chemokine named fractalkine (FKN, CX3CL1), which exists in both membrane-anchored and soluble isoforms, has been proposed to participate in the generation and progression of inflammatory brain disorders. Upon binding to the CX3C receptor one (CX3CR1), FKN induces adhesion, chemoattraction, and activation of leukocytes, including brain macrophages and microglia (MP). Constitutively expressed in the central nervous system (CNS), mainly by neurons, FKN is up-regulated and released in response to proinflammatory stimuli. Importantly, FKN is up-regulated in the brain tissue and cerebrospinal fluid (CSF) of HAD patients. Together, these observations suggest that FKN and its receptor have a unique role in regulating the neuroinflammatory events underlying disease. This review will examine how FKN contributes to the recruitment and activation of CX3CR1-expressing MP, which are critical events in the neuropathogenesis of HAD.

Miniaturized linear time-of-flight mass spectrometer with pulsed extraction.

Improved resolution for a miniaturized instrument is demonstrated at high masses using a pulsed extraction, 3(") linear time-of-flight (TOF) mass analyzer. This illustrates the utility of a small and simple mass spectrometer for biological/medical analyses. Current and future applications suggested by this instrument include rapid mass spectral reading of oligonucleotides that differ in one base (single nucleotide polymorphisms), distinction of biomarker signatures from different species of bacterial spores (biological weapons detection) and point-of-care instruments for proteomics-based diagnostics. We have incorporated a two-stage, pulsed-extraction design that places the focal plane of the ions at the detector channel plate surface. The ions are accelerated to a total energy of 12 keV to enable detection of high-mass proteins in a design that incorporates a floatable flight tube set at the voltage of the front channel plate of the detector. The resultant elimination of post-acceleration at the detector is intended to improve mass resolution by reducing the difference in arrival times between ions and their neutral products. Resolutions of one part in 1200 at m/z 4500 and one part in 600 at m/z 12 000 have been achieved. Proteins with molecular masses up to 66 000 Da, mixtures of oligonucleotides, and biological spores have all been successfully measured, results that increase the potential use of this TOF analyzer.

Lipid A modifications in polymyxin-resistant Salmonella typhimurium: PMRA-dependent 4-amino-4-deoxy-L-arabinose, and phosphoethanolamine incorporation.

Lipid A of Salmonella typhimurium can be resolved into multiple molecular species. Many of these substances are more polar than the predominant hexa-acylated lipid A 1,4'-bisphosphate of Escherichia coli K-12. By using new isolation methods, we have purified six lipid A subtypes (St1 to St6) from wild type S. typhimurium. We demonstrate that these lipid A variants are covalently modified with one or two 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties. Each lipid A species with a defined set of polar modifications can be further derivatized with a palmitoyl moiety and/or a 2-hydroxymyristoyl residue in place of the secondary myristoyl chain at position 3'. The unexpected finding that St5 and St6 contain two l-Ara4N residues accounts for the anomalous structures of lipid A precursors seen in S. typhimurium mutants defective in 3-deoxy-d-manno-octulosonic acid biosynthesis in which only the 1-phosphate group is modified with the l-Ara4N moiety (Strain, S. M., Armitage, I. M., Anderson, L., Takayama, K., Quershi, N., and Raetz, C. R. H. (1985) J. Biol. Chem. 260, 16089-16098). Phosphoethanolamine (pEtN)-modified lipid A species are much less abundant than l-Ara4N containing forms in wild type S. typhimurium grown in broth but accumulate to high levels when l-Ara4N synthesis is blocked in pmrA(C)pmrE(-) and pmrA(C)pmrF(-) mutants. Purification and analysis of selected compounds demonstrate that one or two pEtN moieties may be present. Our findings show that S. typhimurium contains versatile enzymes capable of modifying both the 1- and 4'-phosphates of lipid A with l-Ara4N and/or pEtN groups. PmrA null mutants of S. typhimurium produce lipid A species without any pEtN or l-Ara4N substituents. However, PmrA is not needed for the incorporation of 2-hydroxymyristate or palmitate.

An inner membrane enzyme in Salmonella and Escherichia coli that transfers 4-amino-4-deoxy-L-arabinose to lipid A: induction on polymyxin-resistant mutants and role of a novel lipid-linked donor.

Attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A is required for the maintenance of polymyxin resistance in Escherichia coli and Salmonella typhimurium. The enzymes that synthesize l-Ara4N and transfer it to lipid A have not been identified. We now report an inner membrane enzyme, expressed in polymyxin-resistant mutants, that adds one or two l-Ara4N moieties to lipid A or its immediate precursors. No soluble factors are required. A gene located near minute 51 on the S. typhimurium and E. coli chromosomes (previously termed orf5, pmrK, or yfbI) encodes the l-Ara4N transferase. The enzyme, renamed ArnT, consists of 548 amino acid residues in S. typhimurium with 12 possible membrane-spanning regions. ArnT displays distant similarity to yeast protein mannosyltransferases. ArnT adds two l-Ara4N units to lipid A precursors containing a Kdo disaccharide. However, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single l-Ara4N residue to the 1-phosphate moiety of lipid IV(A), a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesize l-Ara4N-modified lipid A, including Pseudomonas aeruginosa and Yersinia pestis. As shown in the following article (Trent, M. S., Ribeiro, A. A., Doerrler, W. T., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 43132-43144), ArnT utilizes the novel lipid undecaprenyl phosphate-alpha-l-Ara4N as its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane.

Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli: structural characterization and transfer to lipid A in the periplasm.

Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecaprenyl phosphate-alpha-l-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H](-) at m/z 977.5, consistent with undecaprenyl phosphate-alpha-l-Ara4N (M(r) = 978.41). (31)P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional (1)H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-alpha-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.

Sequencing cyclic peptide inhibitors of mammalian ribonucleotide reductase by electrospray ionization mass spectrometry.

Mammalian ribonucleotide reductase (mRR) is a potential target for cancer intervention. A series of lactam-bridged cyclic peptide inhibitors (1-9) of mRR have been synthesized and tested in previous work. These inhibitors consist of cyclic and linear regions, causing their mass spectral characterization to be a challenge. We determined the fragmentation mechanism of cyclic peptides 1-9 using an ion-trap mass spectrometer equipped with an ESI source. Low-energy collision-induced dissociation of sodiated cyclic peptides containing linear branches follows a general pathway. Fragmentation of the linear peptide region produced mainly a and b ions. The ring peptide region was more stable and ring opening required higher collision energy, mainly occurring at the amide bond adjacent to the lactam bridge. The sodium ion, which bound to the carbonyl oxygen of the lactam bridge, acted as a fixed charge site and directed a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues were cleaved sequentially from the C-terminus to the N-terminus. Our findings have established a new way to sequence cyclic peptides containing a lactam bridge based on charge-remote fragmentation. This methodology will permit unambiguous identification of high-affinity ligands within cyclic peptide libraries.

A leucine-rich repeat region is conserved in pollen extensin-like (Pex) proteins in monocots and dicots.

We previously isolated a pollen-specific gene encoding a pollen tube wall-associated glycoprotein with a globular domain and an extensin domain from maize (mPex1). To evaluate which protein domains might be important for function, we isolated a second monocot gene (mPex2) and a dicot gene (tPex). Each gene encodes a signal sequence, an N-terminal globular domain comprised of a variable region, a leucine-rich repeat (LRR) with an adjacent cysteine-rich region, a transition region and an extensin-like C-terminal domain. The LRRs of the maize and tomato Pex proteins are highly conserved. Although the extensin domains in the maize and tomato proteins vary in length and in amino acid sequence, they are likely to be structurally conserved. Additional putative Pex gene sequences were identified by either GenBank search (Arabidopsis) or PCR (sorghum and potato): all encode conserved LRRs. The presence of a conserved LRR in the known and potential Pex proteins strongly suggests that this motif is involved in the binding of a specific ligand during pollen tube growth. Gene expression studies using RNA and protein blotting as well as promoter-reporter gene fusions in transient and stable transformation indicate that the tomato Pex gene is pollen-specific.

Resuscitation of dormant Mycobacterium tuberculosis by phospholipids or specific peptides.

The presence of dormant tubercle bacilli presents a major problem for tuberculosis treatment. The culture supernatant of Mycobacterium tuberculosis was previously shown to resuscitate dormant bacilli in vitro. Here we report identification of active components as phospholipids and a tuberculosis protein Rv1174c. Remarkably, dormant bacilli from a one year old culture which failed to form any colonies could be resuscitated with peptides derived from Rv1174c and formed 10(5-7) colonies/ml. This finding represents the first unambiguous demonstration of resuscitation of dormant tubercle bacilli in vitro and may have implication for the study of mycobacterial dormancy and the design of novel strategies for improved treatment of tuberculosis.

Miniaturized EI/Q/oa TOF mass spectrometer.

A miniaturized orthogonal time-of-flight mass spectrometer with an electron impact ionization ion source and a rf quadrupole ion guide has been developed. A mass resolving power of m/deltam = 5500 has been obtained in a 0.4 m instrument. The addition of helium at pressures of about 4.0 mtorr into the ion source showed collisional focusing taking place in the rf quadrupole. An automated gas chromatograph designed for air monitoring applications has been coupled to the time-of-flight mass analyzer and tested for the detection of simulants of chemical-warfare agents.

Regulation of human immunodeficiency virus type 1 infection, beta-chemokine production, and CCR5 expression in CD40L-stimulated macrophages: immune control of viral entry.

Mononuclear phagocytes (MP) and T lymphocytes play a pivotal role in the host immune response to human immunodeficiency virus type 1 (HIV-1) infection. Regulation of such immune responses can be mediated, in part, through the interaction of the T-lymphocyte-expressed molecule CD40 ligand (CD40L) with its receptor on MP, CD40. Upregulation of CD40L on CD4+ peripheral blood mononuclear cells during advanced HIV-1 disease has previously been reported. Based on this observation, we studied the influence of CD40L-CD40 interactions on MP effector function and viral regulation in vitro. We monitored productive viral infection, cytokine and beta-chemokine production, and beta-chemokine receptor expression in monocyte-derived macrophages (MDM) after treatment with soluble CD40L. Beginning 1 day after infection and continuing at 3-day intervals, treatment with CD40L inhibited productive HIV-1 infection in MDM in a dose-dependent manner. A concomitant and marked upregulation of beta-chemokines (macrophage inhibitory proteins 1alpha and 1beta and RANTES [regulated upon activation normal T-cell expressed and secreted]) and the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) was observed in HIV-1-infected and CD40L-treated MDM relative to either infected or activated MDM alone. The addition of antibodies to RANTES or TNF-alpha led to a partial reversal of the CD40L-mediated inhibition of HIV-1 infection. Surface expression of CD4 and the beta-chemokine receptor CCR5 was reduced on MDM in response to treatment with CD40L. In addition, treatment of CCR5- and CD4-transfected 293T cells with secretory products from CD40L-stimulated MDM prior to infection with a CCR5-tropic HIV-1 reporter virus led to inhibition of viral entry. In conclusion, we demonstrate that CD40L-mediated inhibition of viral entry coincides with a broad range of MDM immune effector responses and the down-modulation of CCR5 and CD4 expression.

A Chlamydia trachomatis UDP-N-acetylglucosamine acyltransferase selective for myristoyl-acyl carrier protein. Expression in Escherichia coli and formation of hybrid lipid A species.

Chlamydia trachomatis lipid A is unusual in that it is acylated with myristoyl chains at the glucosamine 3 and 3' positions. We have cloned and expressed the gene encoding UDP-N-acetylglucosamine 3-O-acyltransferase of C. trachomatis (CtlpxA), the first enzyme of lipid A biosynthesis. C. trachomatis LpxA displays approximately 20-fold selectivity for myristoyl-ACP over R/S-3-hydroxymyristoyl-ACP under standard assay conditions, consistent with the proposed structure of C. trachomatis lipid A. CtLpxA is the first reported UDP-N-acetylglucosamine acyltransferase that prefers a non-hydroxylated acyl-ACP to a hydroxyacyl-ACP. When CtlpxA was expressed in RO138, a temperature-sensitive lpxA mutant of Escherichia coli, five new hybrid lipid A species were made in vivo after 2 h at 42 degrees C, in place of Escherichia coli lipid A. These compounds were purified and analyzed by matrix-assisted laser desorption ionization/time of flight mass spectrometry. In each case, a myristoyl chain replaced one or both of the ester linked 3-hydroxymyristoyl residues of E. coli lipid A. With prolonged growth at 42 degrees C, all the ester-linked 3-hydroxymyristoyl residues were replaced with myristate chains. Re-engineering the structure of E. coli lipid A should facilitate the microbiological production of novel agonists or antagonists of the innate immunity receptor TLR-4, with possible uses as adjuvants or anti-inflammatory agents.

Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine.

Certain strains of Escherichia coli and Salmonella contain lipopolysaccharide (LPS) modified with a phosphoethanolamine (pEtN) group at position 7 of the outer 3-deoxy-d-manno-octulosonic acid (Kdo) residue. Using the heptose-deficient E. coli mutant WBB06 (Brabetz, W., Muller-Loennies, S., Holst, O., and Brade, H. (1997) Eur. J. Biochem. 247, 716-724), we now demonstrate that the critical parameter determining the presence or absence of pEtN is the concentration of CaCl(2) in the medium. As judged by mass spectrometry, half the LPS in WBB06, grown on nutrient broth with 5 mm CaCl(2), is derivatized with a pEtN group, whereas LPS from WBB06 grown without supplemental CaCl(2) is not. Membranes from E. coli WBB06 or wild-type W3110 grown on 5-50 mm CaCl(2) contain a novel pEtN transferase that uses the precursor Kdo(2)-[4'-(32)P]lipid IV(A) as an acceptor. Transferase is not present in membranes of E. coli grown with 5 mm MgCl(2), BaCl(2), or ZnCl(2). Hydrolysis of the in vitro reaction product, pEtN-Kdo(2)-[4'-(32)P]lipid IV(A), at pH 4.5 shows that the pEtN substituent is located on the outer Kdo moiety. Membranes from an E. coli pss knockout mutant grown on 50 mm CaCl(2), which lack phosphatidylethanolamine, do not contain measurable transferase activity unless exogenous phosphatidylethanolamine is added back to the assay system. The induction of the pEtN transferase by 5-50 mm CaCl(2) suggests possible role(s) in establishing transformation competence or resisting environmental stress, and represents the first example of a regulated covalent modification of the inner core of E. coli LPS.