31P nuclear magnetic resonance spectroscopy of lipopolysaccharides from pseudomonas aeruginosa

Intact lipopolysaccharide antigens isolated from seven different immunotypes of Pseudomonas aeruginosa have been examined by ³¹P-NMR spectroscopy. These macromolecular complexes contain phosphorus covalently attached to the carbohydrate residues present in the lipid A moiety and the ‘core’ oligosaccharide region. The spectral signals for various ortho- and pyro-phosphoric esters were observed. All phosphate groups appeared to be mono-esterified. Certain shifts characteristic for phosphate diester groups, observed in lipopolysaccharide complexes from other Gram-negative bacteria, were absent. Furthermore, no evidence was found to indicate that phosphate groups are involved in the covalent linkage of individual lipopolysaccharide complexes to form dimers or trimers.     

Interaction of lipopolysaccharide with detergents and its possible role in the detergent resistance of the outer membrane of gram-negative bacterua

In the presence of MgCl₂, amounts of detergents which disrupted phospholipid vesicles caused lipopolysaccharide I from Proteus mirabilis to aggregate and form vesicular, membrane-like structures. Vesicle formation with P. mirabilis lipopolysaccharide II containing longer O-polysaccharide chains was extremely poor. Lipopolysaccharides of Salmonella minnesota R mutants (chemotypes Ra, Rc and Re) displayed a growing tendency for vesicle formation with increasing deficiency of the R core polysaccharide. Lipopolysaccharides of chemotypes Rc and Re produced vesicles even in the absence of MgCl₂ and detergent. Spherical aggregates consisting of P. mirabilis lipopolysaccharide I, MgCl₂ and detergent were unable to either entrap or retain [¹⁴C]-sucrose, [³H]inulin or [³H]dextran. On the other hand, S. minnesota R mutant lipopolysaccharides of chemotypes Rc and Re could entrap all three saccharides and retain them for at least short periods of time. Leakage of [³H]-inulin out of Re-lipopolysaccharide vesicles was greatly retarded by addition of MgCl₂ to the vesicle system. Incorporation of P. mirabilis lipopolysaccharide I or S. minnesota Rc lipopolysaccharide into phospholipid vesicles protected these model membranes from disruption by detergent. This suggested a similar protective function of lipopolysaccharide in the outer membrane of enteric bacteria against the action of surfactants occuring in their normal intestinal habitat.     

Determination of molecular size of O-(2-hydroxyethyl)cellulose (HEC) and its relationship to the mechanism of enzymatic hydrolysis by cellulases

Z-average root mean square end-to-end distance 〈r_o²〉_z^1/2 and radius of gyration 〈s_o²〉_z^1/2 for 13 samples of O-(2-hydroxyethyl)cellulose (HEC) of different molecular weights were derived from Gel Permeation Chromatography and intrinsic viscosity measurements with water as a solvent. At 40 °C and pH 4.5, contraction of chain dimensions is observed, compared with the sizes observed under neutral conditions at room temperature. The effect is lower for samples with higher molecular weights. Values of 〈r_o²〉₄₀/DP_z also indicate that chain flexibility increases at higher temperature and acidic conditions. From the analysis of molecular weight dependence of 〈s_o²〉 _z^1/2, Flory exponent υ was derived at 40 °C and pH 4.5. A value of υ = 0.70 ± 0.02 was recorded, which indicates that a relatively stiff chain is present under these conditions. Finally, different equations to calculate persistence length L_p were evaluated. Values in the range of 260-400 Å were derived for persistence length. Implications of chain conformation in the enzymatic action of cellulases are also discussed.     

Block of the Kir2.1 Channel Pore by Alkylamine Analogues of Endogenous Polyamines

Inward rectification induced by mono- and diaminoalkane application to inside-out membrane patches was studied in Kir2.1 (IRK1) channels expressed in Xenopus oocytes. Both monoamines and diamines block Kir2.1 channels, with potency increasing as the alkyl chain length increases (from 2 to 12 methylene groups), indicating a strong hydrophobic interaction with the blocking site. For diamines, but not monoamines, increasing the alkyl chain also increases the steepness of the voltage dependence, at any concentration, from a limiting minimal value of ∼1.5 (n = 2 methylene groups) to ∼4 (n = 10 methylene groups). These observations lead us to hypothesize that monoamines and diamines block inward rectifier K⁺ channels by entering deeply into a long, narrow pore, displacing K⁺ ions to the outside of the membrane, with this displacement of K⁺ ions contributing to “extra” charge movement. All monoamines are proposed to lie with the “head” amine at a fixed position in the pore, determined by electrostatic interaction, so that zδ is independent of monoamine alkyl chain length. The head amine of diamines is proposed to lie progressively further into the pore as alkyl chain length increases, thus displacing more K⁺ ions to the outside, resulting in charge movement (zδ) increasing with the increase in alkyl chain length.     

Ter mutation and susceptibility to phi X174 phage in E. coli K12.

The Ter-15 mutant derived from E. coli K12 W2252-11U^? RC^str (wild type I) is found to be sensitive to φx174 phage infection. Lipopolysaccharide extracted from this mutant inactivates the phage, and has core oligosaccharides identical in amounts to those in the lipopolysaccharide from wild type cells.In contrast, the Ter-21 mutant derived from E. coli K12 W2252-11U^? RC^rel (wild type II) is not sensitive to this phage infection, and its lipopolysaccharide does not inactivate the phage. Its lipopolysaccharide sugars are found to be D-glucose and D-ribose, thus differing from the lipopolysaccharide sugars of the wild type cells.     

Factors affecting the metal ion-hydroxamate interactions II: effect of the length of the connecting chain on the Fe(III), Mo(VI) and V(V) complexation of some new desferrioxamine B (DFB) model dihydroxamic acids

Three new dihydroxamic acids (HO(CH₃)NCO-(CH₂)₂-CO-NH-(CH₂)_x-CON(CH₃)OH where the x values are 4; 3 and 2, and the compounds are abbreviated as 2,4-DIHA, 2,3-DIHA and 2,2-DIHA), containing the peptide group in a certain position to one of the two functional groups and in different distances to the other one, were synthesized and their complexation with Fe(III), Mo(VI) and V(V) was studied by pH-potentiometric, spectrophotometric and in some cases by CV methods to evaluate the redox behaviour of the Fe(III) complexes and assess their potential biological activity as siderophore models. All these compounds are structural models for the natural siderophore, desferrioxamine B (DFB). The results were compared to those of the complexes of 2,5-DIHA having the same connecting chain structure and length as DFB has, and the effects of the length of the connecting chain on the co-ordination mode and on the stability of the complexes formed were evaluated.Very similar stability of the mono-chelated complexes formed with all these dihydroxamic acids was found. All the results obtained suggest that one dihydroxamic acid (even the 2,2-DIHA) is able to complete the four coordination sites of a MoO₂ ²⁺ core forming simple mononuclear complexes. Favoured monomeric structures of the bis-chelated complexes of these dihydroxamic acids are also suggested with V(V) having the smallest ionic radius among the three metal ions studied. In the case of iron(III), however, clear indication was obtained for the slightly different complexation behaviour of 2,2-DIHA. Namely, the formation of the mononuclear bis-chelated complex with this shortest ligand seems to have sufficient strain to induce the formation of bimetallic species such as [Fe(2,2-DIHA)₂Fe)]²⁺.     

Guanidino groups greatly enhance the action of antimicrobial peptidomimetics against bacterial cytoplasmic membranes

Antimicrobial peptides or their synthetic mimics are a promising class of potential new antibiotics. Herein we assess the effect of the type of cationic side chain (i.e., guanidino vs. amino groups) on the membrane perturbing mechanism of antimicrobial α-peptide–β-peptoid chimeras. Langmuir monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG) were used to model cytoplasmic membranes of both Gram-positive and Gram-negative bacteria, while lipopolysaccharide Kdo2-lipid A monolayers were mimicking the outer membrane of Gram-negative species. We report the results of the measurements using an array of techniques, including high-resolution synchrotron surface X-ray scattering, epifluorescence microscopy, and in vitro antimicrobial activity to study the molecular mechanisms of peptidomimetic interaction with bacterial membranes. We found guanidino group-containing chimeras to exhibit greater disruptive activity on DPPG monolayers than the amino group-containing analogues. However, this effect was not observed for lipopolysaccharide monolayers where the difference was negligible. Furthermore, the addition of the nitrobenzoxadiazole fluorophore did not reduce the insertion activity of these antimicrobials into both model membrane systems examined, which may be useful for future cellular localization studies.     

Fe2+-catalyzed non-enzymatic glycosylation alters collagen conformation during AGE-collagen formation in vitro

Advanced glycation end-products (AGEs) are one of the major factors of hyperglycemia related complications for diabetic patients. We studied the formation of AGEs in type I collagen after Fe²⁺-catalyzed non-enzymatic glycosylation in vitro. Type I collagen isolated from rat tail tendon was incubated with glucose and increasing concentrations of iron ions Fe²⁺. After 4 weeks incubation, cytotoxity of AGEs was indicated by the cytotoxity assay of primary human umbilical vein endothelial cells and primary human monocytes cultured with glycosylated collagen AGEs. Fourier transform infrared spectroscopy analysis revealed that structural changes of functional groups in glycosylated collagen are accelerated by the catalyst Fe²⁺. Using two-dimensional Fourier-transform infrared correlation spectroscopy analyses, for the first time, we demonstrated that the order of structural changes of these functional groups is -CH- > Amide I > Amide II > Amide III > ν(CO) the carboxylic group of Asn, Gln or polyproline amino acid residue in the course of AGE-collagen formation. Knowing the positions of these functional groups in collagen, this order of changes indicates that during glycation of collagen, the structure of the main chain residues in collagen changed first, and then the side chain changed gradually, which may lead to more carboxylic groups exposed to glucose for further formation of AGE-collagen irreversibly. The findings presented may support the design of new therapeutic strategies to prevent or slow down the Fe²⁺-catalyzed glycosylation of collagen and other matrix proteins.     

Lipopolysaccharide Engineering in Neisseria meningitidis: STRUCTURAL ANALYSIS OF DIFFERENT PENTAACYL LIPID A MUTANTS AND COMPARISON OF THEIR MODIFIED AGONIST PROPERTIES*

Engineering the lipopolysaccharide (LPS) biosynthetic pathway offers the potential to obtain modified derivatives with optimized adjuvant properties. Neisseria meningitidis strain H44/76 was modified by expression of the pagL gene encoding lipid A 3-O-deacylase from Bordetella bronchiseptica and by inactivation of the lgtB gene encoding the terminal oligosaccharide galactosyltransferase. Mass spectrometry analysis of purified mutant LPS was used for detailed compositional analysis of all present molecular species. This determined that the modified LPS was mainly pentaacylated, demonstrating high efficiency of conversion from the hexaacyl to the 3-O-deacylated form by heterologous lipid A 3-O-deacylase (PagL) expression. MS analyses also provided evidence for expression of only one major oligosaccharide glycoform, which lacked the terminal galactose residue as expected from inactivation of the lgtB gene. The immunomodulatory properties of PagL-deacylated LPS were compared with another pentaacyl form obtained from an lpxL1⁻ mutant, which lacks the 2′ secondary acyl chain. Although both LPS mutants displayed impaired capacity to induce production of the pro-inflammatory cytokine IL-6 in the monocytic cell line Mono Mac 6, induction of the Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β-dependent chemokine interferon-γ-induced protein 10 was largely retained only for the lgtB⁻/pagL⁺ mutant. Removal of remaining hexaacyl species exclusively present in lgtB⁻/pagL⁺ LPS demonstrated that these minor species potentiate but do not determine the activity of this LPS. These results are the first to indicate a qualitatively different response of human innate cells to pentaacyl lpxL1⁻ and pagL⁺ LPS and show the importance of detailed structure-function analysis when working with modified lipid A structures. The pagL⁺ LPS has significant potential as immune modulator in humans.     

NMR-based Structural Analysis of the Complete Rough-type Lipopolysaccharide Isolated from Capnocytophaga canimorsus

We here describe the NMR analysis of an intact lipopolysaccharide (LPS, endotoxin) in water with 1,2-dihexanoyl-sn-glycero-3-phosphocholine as detergent. When HPLC-purified rough-type LPS of Capnocytophaga canimorsus was prepared, ¹³C,¹⁵N labeling could be avoided. The intact LPS was analyzed by homonuclear (¹H) and heteronuclear (¹H,¹³C, and ¹H,³¹P) correlated one- and two-dimensional NMR techniques as well as by mass spectrometry. It consists of a penta-acylated lipid A with an α-linked phosphoethanolamine attached to C-1 of GlcN (I) in the hybrid backbone, lacking the 4′-phosphate. The hydrophilic core oligosaccharide was found to be a complex hexasaccharide with two mannose (Man) and one each of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), Gal, GalN, and l-rhamnose residues. Position 4 of Kdo is substituted by phosphoethanolamine, also present in position 6 of the branched Man^I residue. This rough-type LPS is exceptional in that all three negative phosphate residues are “masked” by positively charged ethanolamine substituents, leading to an overall zero net charge, which has so far not been observed for any other LPS. In biological assays, the corresponding isolated lipid A was found to be endotoxically almost inactive. By contrast, the intact rough-type LPS described here expressed a 20,000-fold increased endotoxicity, indicating that the core oligosaccharide significantly contributes to the endotoxic potency of the whole rough-type C. canimorsus LPS molecule. Based on these findings, the strict view that lipid A alone represents the toxic center of LPS needs to be reassessed.     

Increased expression of Interleukin-18 receptor in blood cells of subjects with Mild Cognitive Impairment and Alzheimer’s disease

Inflammation has been proposed as a leading force in neurodegeneration and Interleukin (IL)-18 is a pro-inflammatory cytokine which is suggested to be implicated in Alzheimer’s disease (AD). However, the meaning of the IL-18 participation in this disease is still unclear.Since IL-18 activity is mediated by its heterodimeric receptor complex IL-18Rα/β, we evaluated the presence of both IL-18R chains on peripheral blood cells of AD patients, as well as in individuals with Mild Cognitive Impairment (MCI), at increased risk to develop AD. More specifically, we compared the levels of CD14⁺ monocytes and CD3⁺ T-lymphocytes bearing IL-18Rα and β chains in the two groups of patients with those in healthy control subjects, both before and after in vitro cell treatment with lipopolysaccharide (LPS).While no differences in the levels of monocytes and T-lymphocytes bearing IL-18Rα chain were found among the three groups, either in untreated and LPS-treated conditions, the IL-18Rβ chain expression appeared differently regulated in MCI and AD patients, as compared to controls. In particular, the amount of IL-18Rβ-bearing monocytes was similar among the three groups at unstimulated conditions, while after LPS treatment it was increased in MCI vs. controls. A significant increase of IL-18Rβ-bearing T-lymphocytes was also observed in MCI and AD vs. controls, both in untreated and LPS-stimulated conditions.Our findings indicate that the expression of IL-18R complex on blood cells is perturbed in AD and even more markedly in its preclinical state of MCI, confirming that an increased peripheral activity of IL-18 may be involved in the early phase of AD pathophysiology.     

Two new isoflavonoid glucosides from the roots of Achyranthes bidentata and their activities against nitric oxide production

Two new isoflavonoid glucosides, Achyranthosides A and B, were isolated from the roots of Achyranthes bidentata. Their structures were established through extensive analyses using ¹H, ¹³C NMR, HSQC, HMBC, NOESY, and HREISMS spectroscopic data. The structures of the new compounds are characterized by the methoxyl groups substituted on the specific positions of the phenyl rings. The two compounds were evaluated for their anti-inflammatory activities on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 murine macrophage cells. The compounds showed significant inhibition on LPS-induced NO production.     

Two different 1D-chains in the structures of the copper(I) coordination polymers based on bidentate Schiff-base building units and thiocyanate anions as bridging ligands

The reaction of the bidentate Schiff-base ligands (3,4,5-MeO-ba)₂en (L¹) and (4-Me-ba)₂en (L²) with Cu(SCN) in CH₃CN yielded two copper(I) coordination polymers [Cu(L¹)(SCN)]_n (1) and [Cu(L²)(SCN)]_n (2), which have been characterized by elemental analyses, IR- and ¹H NMR-spectroscopy, and X-ray crystallography. The non-centrosymmetric structures of both Cu(I) complexes consist of an one-dimensional polymeric chain in which copper(I) ions are bridged by two thiocyanate groups bonding in an end-to-end fashion. The Cu(I)?Cu(I) separation is 5.604 Å in 1 and 5.706 Å in 2.     

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive ¹³C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive ¹³C-methylation is a chemical reaction performed under mild conditions that modifies a protein''s primary amino groups (lysine ε-amino groups and the N-terminal α-amino group) to ¹³C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive ¹³C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein ¹³C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of ¹³C-labeling at each primary amino group. One limitation of the method arises when the protein''s N-terminal residue is a lysine because the α- and ε-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the ¹³C-dimethylamines associated with both the N-terminal α-amine and the side chain ε-amine. The NMR signals of the N-terminal α-dimethylamine and the side chain ε-dimethylamine of hen egg white lysozyme, Lys1, are identified in ¹H-¹³C heteronuclear single-quantum coherence spectra.     

Chemical characterization of the O-specific chain of Sphaerotilus natans ATCC 13338 lipopolysaccharide

The chemical structure of the O-specific chain of the Sphaerotilus natans lipopolysaccharide was established. The isolated polysaccharide moiety contained neutral sugars (rhamnose, glucose, and l-glycero-d-manno-heptose), 3-deoxy-d-manno-octulosonic acid (Kdo), phosphate and ethanolamine. Alditol acetate and methylation analyses showed that the O-specific chain contained linear pentasaccharide repeating units, composed of four units of rhamnose, substituted at 3-position, and one unit of glucose, substituted at 4-position. Oxidation by chromium trioxide showed that all sugars were α-linked. The structure proposed for the O-specific chain has been confirmed by periodate oxidation, and by ¹H-and ¹³C-NMR spectroscopic studies for the original and the Smith-degraded PS moiety. The O-specific unit of the S. natans LPS has the following structure: $$\begin{gathered} [ \to 4) - Glup - (1\xrightarrow{a}3) - Rhap - (1\xrightarrow{a}3) - Rhap - \hfill \\ - (1\xrightarrow{a}3) - Rhap - (1\xrightarrow{a}3) - Rhap - (1]_n \xrightarrow{a} \hfill \\ \end{gathered} $$      

Variations in the pathways of malate oxidation and phosphorylation in different species of Mycobacteria

Mycobacterium tuberculosis H₃₇ Rv, the slow-growing human pathogenic strain of tubercle bacilli and Mycobacterium smegmatis and Mycobacterium phlei, the fast-growing saprophytes, have shown variations regarding the type of dehydrogenase that initiates malate oxidation in the respiratory chain.M. tuberculosis H₃₇Rv is characterized by having a malate oxidase system (designated MAL_NAD pathway) in which malate oxidation is mediated by the NAD⁺_? dependent malate dehydrogenase (EC 1.1.1.37) but not by FAD-dependent malatevitamin K reductase. M. smegmatis possesses a different malate oxidase system (designated MAL_FAD pathway) in which malate oxidation is exclusively carried out by the FAD-dependent malate-vitamin K reductase because NAD⁺-dependent malate dehydrogenase is absent in this organism. M. phlei has a mixed system of malate oxidase (designated MAL_NAD+FAD pathways) in which both the NAD⁺_? and FAD-dependent dehydrogenases take part. In all the three systems, the rest of the electron transport chain is common.     

Electrochemical survey of the chain length influence in phytochelatins competitive binding by cadmium

Multivariate curve resolution with alternating least squares (MCR-ALS) was applied to voltammetric data obtained in the analysis of the competitive binding of glutathione (GSH) and phytochelatins [(γGlu-Cys)_n-Gly, PC_n, n = 2-5] by Cd²⁺. The displacements between ligands and chain length influence on the competitive binding of PC_n toward Cd²⁺ were investigated. The analysis of the resulting pure voltammograms and concentration profiles of the resolved components suggests that ligands containing more thiol groups are able to displace the shortest chain ligands from their metal complexes, whereas the opposite does not happen. However, when the length of the chain surpasses that of PC₃, the binding capacity of the molecule still increases (i.e., it can bind more metal ions), but the position and shape of the voltammetric signals practically rest unchanged. This suggests that at this level, the stability of metal binding could depend more on the nature of the binding sites separately than on the quantity of the sites (i.e., the chain length).     

Endocannabinoids Are Expressed in Bone Marrow Stromal Niches and Play a Role in Interactions of Hematopoietic Stem and Progenitor Cells with the Bone Marrow Microenvironment

Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB₁ and CB₂. The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB₁ receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10⁷ cells), and 2-AG (75.2 ng/10⁷ cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10⁷ cells) and 2-AG (98.8 ng/10⁷ cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB₁ agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1^−/− showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)^−/− mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1^−/−, as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.     

Epigenetic Control of Salmonella enterica O-Antigen Chain Length: A Tradeoff between Virulence and Bacteriophage Resistance

The Salmonella enterica opvAB operon is a horizontally-acquired locus that undergoes phase variation under Dam methylation control. The OpvA and OpvB proteins form intertwining ribbons in the inner membrane. Synthesis of OpvA and OpvB alters lipopolysaccharide O-antigen chain length and confers resistance to bacteriophages 9NA (Siphoviridae), Det7 (Myoviridae), and P22 (Podoviridae). These phages use the O-antigen as receptor. Because opvAB undergoes phase variation, S. enterica cultures contain subpopulations of opvAB ^OFF and opvAB ^ON cells. In the presence of a bacteriophage that uses the O-antigen as receptor, the opvAB ^OFF subpopulation is killed and the opvAB ^ON subpopulation is selected. Acquisition of phage resistance by phase variation of O-antigen chain length requires a payoff: opvAB expression reduces Salmonella virulence. However, phase variation permits resuscitation of the opvAB ^OFF subpopulation as soon as phage challenge ceases. Phenotypic heterogeneity generated by opvAB phase variation thus preadapts Salmonella to survive phage challenge with a fitness cost that is transient only.