Jace W. Jones

Human Toll-like receptor 4 responses to P. gingivalis are regulated by lipid A 1- and 4′-phosphatase activities

Signal transduction following binding of lipopolysaccharide (LPS) to Toll‐like receptor 4 (TLR4) is an essential aspect of host innate immune responses to infection by Gram‐negative pathogens. Here, we describe a novel molecular mechanism used by a prevalent human bacterial pathogen to evade and subvert the human innate immune system. We show that the oral pathogen, Porphyromonas gingivalis, uses endogenous lipid A 1‐ and 4′‐phosphatase activities to modify its LPS, creating immunologically silent, non‐phosphorylated lipid A. This unique lipid A provides a highly effective mechanism employed by this bacterium to evade TLR4 sensing and to resist killing by cationic antimicrobial peptides. In addition, lipid A 1‐phosphatase activity is suppressed by haemin, an important nutrient in the oral cavity. Specifically, P. gingivalis grown in the presence of high haemin produces lipid A that acts as a potent TLR4 antagonist. These results suggest that haemin‐dependent regulation of lipid A 1‐dephosphorylation can shift P. gingivalis lipid A activity from TLR4 evasive to TLR4 suppressive, potentially altering critical interactions between this bacterium, the local microbial community and the host innate immune system.

Sustained virologic control in SIV+ macaques after antiretroviral and α4β7 antibody therapy

Antibodies sustain viral control For many infected individuals, antiretroviral therapy (ART) means that an HIV-1 diagnosis is no longer a death sentence. But the virus persists in treated individuals, and complying with the intense drug regimen to keep virus loads down can be challenging for patients. Seeking an alternative, Byrareddy et al. treated ART-suppressed monkeys with antibodies targeting α4β7 integrin. When ART was halted in the antibody-treated animals, viral loads stayed undetectable, and normal CD4 T cell counts were maintained for over 9 months—and persisted—even after stopping the antibody therapy. Science, this issue p. 197 Update: An Editorial Expression of Concern has been published here Combining short-term antiretroviral therapy with specific anti-integrin treatment sustains low viral loads in monkeys. Antiretroviral drug therapy (ART) effectively suppresses replication of both the immunodeficiency viruses, human (HIV) and simian (SIV); however, virus rebounds soon after ART is withdrawn. SIV-infected monkeys were treated with a 90-day course of ART initiated at 5 weeks post infection followed at 9 weeks post infection by infusions of a primatized monoclonal antibody against the α4β7 integrin administered every 3 weeks until week 32. These animals subsequently maintained low to undetectable viral loads and normal CD4+ T cell counts in plasma and gastrointestinal tissues for more than 9 months, even after all treatment was withdrawn. This combination therapy allows macaques to effectively control viremia and reconstitute their immune systems without a need for further therapy.

Unique structural modifications are present in the lipopolysaccharide from colistin-resistant strains of Acinetobacter baumannii.

ABSTRACT Acinetobacter baumannii is a nosocomial opportunistic pathogen that can cause severe infections, including hospital-acquired pneumonia, wound infections, and sepsis. Multidrug-resistant (MDR) strains are prevalent, further complicating patient treatment. Due to the increase in MDR strains, the cationic antimicrobial peptide colistin has been used to treat A. baumannii infections. Colistin-resistant strains of A. baumannii with alterations to the lipid A component of lipopolysaccharide (LPS) have been reported; specifically, the lipid A structure was shown to be hepta-acylated with a phosphoethanolamine (pEtN) modification present on one of the terminal phosphate residues. Using a tandem mass spectrometry platform, we provide definitive evidence that the lipid A isolated from colistin-resistant A. baumannii MAC204 LPS contains a novel structure corresponding to a diphosphoryl hepta-acylated lipid A structure with both pEtN and galactosamine (GalN) modifications. To correlate our structural studies with clinically relevant samples, we characterized colistin-susceptible and -resistant isolates obtained from patients. These results demonstrated that the clinical colistin-resistant isolate had the same pEtN and GalN modifications as those seen in the laboratory-adapted A. baumannii strain MAC204. In summary, this work has shown complete structure characterization including the accurate assignment of acylation, phosphorylation, and glycosylation of lipid A from A. baumannii, which are important for resistance to colistin.

Hidden Histidine Radical Rearrangements upon Electron Transfer to Gas-Phase Peptide Ions. Experimental Evidence and Theoretical Analysis

Protonated peptides containing histidine or arginine residues and a free carboxyl group (His-Ala-Ile, His-Ala-Leu, Ala-His-Leu, Ala-Ala-His-Ala-Leu, His-Ala-Ala-Ala-Leu, and Arg-Ala-Ile) form stable anions upon collisional double electron transfer from Cs atoms at 50 keV kinetic energies. This unusual behavior is explained by hidden rearrangements occurring in peptide radical intermediates formed by transfer of the first electron. The rearrangements occur on a approximately 120 ns time scale determined by the radical flight time. Analysis of the conformational space for (His-Ala-Ile + H)(+) precursor cations identified two major conformer groups, 1a(+)-1m(+) and 5a(+)-5h(+) , that differed in their H-bonding patterns and were calculated to collectively account for 39% and 60%, respectively, of the gas-phase ions. One-electron reduction in 1a(+) and 5a(+) triggers exothermic hydrogen atom migration from the terminal COOH group onto the His imidazole ring, forming imidazoline radical intermediates. The intermediate from 5a is characterized by its charge and spin distribution as a novel cation radical-COO(-) salt bridge. The intermediate from 1a undergoes spontaneous isomerization by imidazoline N-H migration, re-forming the COOH group and accomplishing exothermic isomerization of the initial (3H)-imidazole radical to a (2H)-imidazole radical. An analogous unimolecular isomerization in simple imidazole and histidine radicals requires activation energies of 150 kJ mol(-1), and its occurrence in 1a and 5a is due to the promoting effect of the proximate COOH group. The rearrangement is substantially reduced in Ala-Leu-His due to an unfavorable spatial orientation of the imidazole and COOH groups and precluded in the absence of a free carboxyl group in His-Ala-Leu amide. In contrast to His-Ala-Ile and Arg-Ala-Ile, protonated Lys-Ala-Ile does not produce stable anions upon double electron transfer. The radical trapping properties of histidine residues are discussed.

Determination of pyrophosphorylated forms of lipid A in Gram-negative bacteria using a multivaried mass spectrometric approach

Lipid A isolated from several bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, and various strains of Yersinia) showed abundant formation of pyrophosphate anions upon ion dissociation. Pyrophosphate [H3P2O7]− and/or [HP2O6]− anions were observed as dominant fragments from diphosphorylated lipid A anions regardless of the ionization mode (matrix-assisted laser desorption ionization or electrospray ionization), excitation mode (collisional activation or infrared photoexcitation), or mass analyzer (time-of-flight/time-of-flight, tandem quadrupole, Fourier transform–ion cyclotron resonance mass spectrometry). Dissociations of anions from model lipid phosphate, pyrophosphate, and hexose diphosphates confirmed that pyrophosphate fragments were formed abundantly only in the presence of an intact pyrophosphate group in the analyte molecule and were not due to intramolecular rearrangement upon ionization, ion-molecule reactions, or rearrangement following activation. This indicated that pyrophosphate groups are present in diphosphorylated lipid A from a variety of Gram-negative bacteria.

Generic lamotrigine versus brand-name Lamictal bioequivalence in patients with epilepsy: A field test of the FDA bioequivalence standard

To test the current U.S. Food and Drug Administration (FDA) bioequivalence standard in a comparison of generic and brand‐name drug pharmacokinetic (PK) performance in “generic‐brittle” patients with epilepsy under clinical use conditions.

A Mollusk Retinoic Acid Receptor (RAR) Ortholog Sheds Light on the Evolution of Ligand Binding

Nuclear receptors are transcription factors that regulate networks of target genes in response to small molecules. There is a strong bias in our knowledge of these receptors because they were mainly characterized in classical model organisms, mostly vertebrates. Therefore, the evolutionary origins of specific ligand-receptor couples still remain elusive. Here we present the identification and characterization of a retinoic acid receptor (RAR) from the mollusk Nucella lapillus (NlRAR). We show that this receptor specifically binds to DNA response elements organized in direct repeats as a heterodimer with retinoid X receptor. Surprisingly, we also find that NlRAR does not bind all-trans retinoic acid or any other retinoid we tested. Furthermore, NlRAR is unable to activate the transcription of reporter genes in response to stimulation by retinoids and to recruit coactivators in the presence of these compounds. Three-dimensional modeling of the ligand-binding domain of NlRAR reveals an overall structure that is similar to vertebrate RARs. However, in the ligand-binding pocket (LBP) of the mollusk receptor, the alteration of several residues interacting with the ligand has apparently led to an overall decrease in the strength of the interaction with the ligand. Accordingly, mutations of NlRAR at key positions within the LBP generate receptors that are responsive to retinoids. Altogether our data suggest that, in mollusks, RAR has lost its affinity for all-trans retinoic acid, highlighting the evolutionary plasticity of its LBP. When put in an evolutionary context, our results reveal new structural and functional features of nuclear receptors validated by millions of years of evolution that were impossible to reveal in model organisms.

Synthesis, modeling, and pharmacological evaluation of UMB 425, a mixed μ agonist/δ antagonist opioid analgesic with reduced tolerance liabilities.

Opioid narcotics are used for the treatment of moderate-to-severe pain and primarily exert their analgesic effects through μ receptors. Although traditional μ agonists can cause undesired side effects, including tolerance, addition of δ antagonists can attenuate said side effects. Herein, we report 4a,9-dihydroxy-7a-(hydroxymethyl)-3-methyl-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7(7aH)-one (UMB 425) a 5,14-bridged morphinan-based orvinol precursor synthesized from thebaine. Although UMB 425 lacks δ-specific motifs, conformationally sampled pharmacophore models for μ and δ receptors predict it to have efficacy similar to morphine at μ receptors and similar to naltrexone at δ receptors, due to the compound sampling conformations in which the hydroxyl moiety interacts with the receptors similar to orvinols. As predicted, UMB 425 exhibits a mixed μ agonist/δ antagonist profile as determined in receptor binding and [(35)S]GTPγS functional assays in CHO cells. In vivo studies in mice show that UMB 425 displays potent antinociception in the hot plate and tail-flick assays. The antinociceptive effects of UMB 425 are blocked by naloxone, but not by the κ-selective antagonist norbinaltorphimine. During a 6-day tolerance paradigm, UMB 425 maintains significantly greater antinociception compared to morphine. These studies thus indicate that, even in the absence of δ-specific motifs fused to the C-ring, UMB 425 has mixed μ agonist/δ antagonist properties in vitro that translate to reduced tolerance liabilities in vivo.

Comprehensive Structure Characterization of Lipid A Extracted from Yersinia pestis for Determination of its Phosphorylation Configuration

We report on comprehensive structure characterization of lipid A extracted from Yersinia pestis (Yp) for determination of its phosphorylation configuration that was achieved by combining the methods of molecular biology with high-resolution tandem mass spectrometry. The phosphorylation pattern of diphosphorylated lipid A extracted from Yp has recently been found to be a heterogeneous mixture of C-1 and C-4′ bisphosphate, C-1 pyrophosphate, and C-4′ pyrophosphate (Proc. Natl. Acad. Sci.2008,105, 12742). To reduce the inherent phosphate heterogeneity of diphosphorylated lipid A extracted from Yp, we incorporated specific C-1 and C-4′ position phosphatases into wild type KIM6+ Yp grown at 37°C. Comprehensive high-resolution tandem mass spectrometric analyses of lipid A extracted from Yp modified with either the C-1 or C-4′ phosphatase allowed for unambiguous structure assignment of monophosphorylated and diphosphorylated lipid A and distinction of isomeric bisphosphate and pyrophosphate forms. The prevalent aminoarabinose modification was determined to be exclusively attached to the lipid A disaccharide via a phospho-diester linkage at either or both the C-1 and C-4′ positions.

All-Trans Retinoic Acid Activity in Acute Myeloid Leukemia: Role of Cytochrome P450 Enzyme Expression by the Microenvironment

Differentiation therapy with all-trans retinoic acid (atRA) has markedly improved outcome in acute promyelocytic leukemia (APL) but has had little clinical impact in other AML sub-types. Cell intrinsic mechanisms of resistance have been previously reported, yet the majority of AML blasts are sensitive to atRA in vitro. Even in APL, single agent atRA induces remission without cure. The microenvironment expression of cytochrome P450 (CYP)26, a retinoid-metabolizing enzyme was shown to determine normal hematopoietic stem cell fate. Accordingly, we hypothesized that the bone marrow (BM) microenvironment is responsible for difference between in vitro sensitivity and in vivo resistance of AML to atRA-induced differentiation. We observed that the pro-differentiation effects of atRA on APL and non-APL AML cells as well as on leukemia stem cells from clinical specimens were blocked by BM stroma. In addition, BM stroma produced a precipitous drop in atRA levels. Inhibition of CYP26 rescued atRA levels and AML cell sensitivity in the presence of stroma. Our data suggest that stromal CYP26 activity creates retinoid low sanctuaries in the BM that protect AML cells from systemic atRA therapy. Inhibition of CYP26 provides new opportunities to expand the clinical activity of atRA in both APL and non-APL AML.