Nilofer Qureshi

Neutrophil Activation by Bacterial Lipoprotein Versus Lipopolysaccharide: Differential Requirements for Serum and CD14

Neutrophil activation plays an important role in the inflammatory response to Gram-negative bacterial infections. LPS has been shown to be a major mediator of neutrophil activation which is accompanied by an early down-regulation of L-selectin and up-regulation of CD1lb/CD18. In this study, we investigated whether lipoprotein (LP), the most abundant protein in the outer membrane of bacteria from the family Enterobacteriaceae, can activate neutrophils and whether this activation is mediated by mechanisms that differ from those used by LPS or Escherichia coli diphosphoryl lipid A (EcDPLA). Neutrophil activation was assessed by measuring down-regulation of L-selectin and up-regulation of CD11b/CD18. When comparing molar concentrations of LP vs EcDPLA, LP was more potent (four times) at activating neutrophils. In contrast to LPS/EcDPLA, LP activation of neutrophils was serum independent. However, LP activation of neutrophils was enhanced by the addition of soluble CD14 and/or LPS-binding protein. In the presence of serum, LP activation of neutrophils was inhibited by different mAbs to CD14. This inhibition was significantly reduced or absent when performed in the absence of serum. Diphosphoryl lipid A from Rhodobacter spheroides (RaDPLA) completely inhibited LPS/EcDPLA activation of neutrophils but only slightly inhibited LP activation of neutrophils. These results suggest that LP activation of human neutrophils can be mediated by a mechanism that is different from LPS activation and that LP is a potentially important component in the development of diseases caused by Gram-negative bacteria of the family Enterobacteriaceae.

Diphosphoryl Lipid A from Rhodobacter sphaeroides Blocks the Binding and Internalization of Lipopolysaccharide in RAW 264.7 Cells

Diphosphoryl lipid A derived from the nontoxic LPS of Rhodobacter sphaeroides (RsDPLA) has been shown to be a powerful LPS antagonist in both human and murine cell lines. In addition, RsDPLA also can protect mice against the lethal effects of toxic LPS. In this study, we complexed both the deep rough LPS from Escherichia coli D31 m4 (ReLPS) and RsDPLA with 5- and 30-nm colloidal gold and compared their binding to the RAW 264.7 cell line by electron microscopy. Both ReLPS and RsDPLA bound to the cells with the following observations. First, binding studies revealed that pretreatment with RsDPLA completely blocked the binding and thus internalization of ReLPS-gold conjugates to these cells at both 37°C and 4°C. Second, ReLPS was internalized via micropinocytosis (noncoated plasma membrane invaginations) involving formation of caveolae-like structures and leading to the formation of micropinocytotic vesicles, macropinocytosis (or phagocytosis), formation of clathrin-coated pits (receptor mediated), and penetration through plasma membrane into cytoplasm. Third, in contrast, RsDPLA was internalized predominantly via macropinocytosis. These studies show for the first time that RsDPLA blocks the binding and thus internalization of LPS as observed by scanning and transmission electron microscopy.

Structural analysis of the lipid A derived from the lipopolysaccharide of Brucella abortus

Lipopolysaccharide (LPS) of Brucella abortus strain 45/20 was purified using a novel method. Monophosphoryl lipid A (MPLA) was prepared from this LPS, methylated, and purified by high performance liquid chromatography. Chemical, mass spectral, and nuclear magnetic resonance analyses showed that MPLA consists of heptaacyl lipid As with molecular weights of 2095, 2123, 2151 and 2179. They contained the β-1,6-linked 2,3-diamino-2,3,-dideoxy-glucose disaccharide backbone and a phosphate group at the 4 position. Bisphosphoryl lipid A was also prepared and completely O-deacylated. It contained an additional phosphate group, and either 2 hydroxyhexadecanoic, 1 hydroxytetradecanoic, 1 hydroxydodecanoic acids or 2 hydroxyhexadecanoic and 2 hydroxydodecanoic acids, all in amide linkage. The predominant ester-linked fatty acyl group in acyloxyacyl linkage was hexadecanoate. The purified LPS, bisphosphoryl lipid A, and MPLA from B. abortus showed about 14%, 3% and 1%, respectively, of the B cell mitogen activity of ReLPS from Escherichia coli at 1.0 μg/ml.

Lipopolysaccharide (LPS) binding to 73-kDa and 38-kDa surface proteins on lymphoreticular cells: preferential inhibition of LPS binding to the former by Rhodopseudomonas sphaeroides lipid A

Using a photoactivable, radioiodinated lipopolysaccharide probe, [125I]ASD-LPS (derivatized from purified E. coli 0111:B4 S-LPS), we earlier reported the presence of a 73-kDa (p73) predominant LPS-binding protein on mouse lymphocytes and macrophages with specificity for the lipid A region of LPS. Both Re-LPS from Salmonella minnesota and purified lipid A will inhibit the binding of LPS to the p73 LPS receptor. In the studies reported here, we have found that non-toxic diphosphoryl lipid A purified from Rhodo-pseudomonas sphaeroides has the capability to inhibit the binding of [125I]ASD-LPS to the p73 protein. However, using the same LPS probe and photoaffinity cross-linking techniques, our data suggest that a less dominant 38-kDa (p38) LPS-specific binding protein identified on mouse splenocytes, J774.1 macrophage-like cell line, and 70Z/3 pre B-cell line by SDS-PAGE is not inhibited by purified lipid A, even at a concentration in 50-fold excess of that of [125]ASD-LPS. The binding of the LPS probe to the 38 protein could be inhibited in a dose-dependent manner by underivatized native S. minnesota Re-LPS (composed only of Kdo and lipid A). We speculate that this p38 LPS-binding protein may manifest a specificity for inner core oligosaccharide determinants on LPS.

Effects of brewer's grain and other cereals on lipid metabolism in chickens

Abstract The effect of brewers grain, its fraction high protein flour, wheat-germ and oats on cholesterogenesis was studied in six-week-old male chickens. These cereals are rich sources of tocopherols and tocotrienols. The enzymatic activity of β -hydroxy- β -methylglutaryl Coenzyme A (HMG-CoA) reductase (the rate-limiting enzyme of cholesterol biosynthesis) was suppressed significantly (34.3–49.7%) with all cereals as compared to a corn-based diet. The enzymatic activity of cholesterol 7 α -hydroxylase was significantly increased with oats and BBHPF and slightly increased with wheat-germ and brewers grain as compared to the control diet. The supplementation of brewers grain, oats and BBHPF caused a significant decrease (22.0–35.7%) in HMG-CoA synthase activity as compared to the control. The wheat-germ had little effect on this enzyme. The levels of serum total cholesterol and low density lipoprotein (LDL)-cholesterol were decreased wheat-germ (7.2%, 18.5%), oats (8.5, 24.6%) brewers grain (10.8%, 22.7%) and BBHPF (15.4%, 30.8%), respectively, as compared to the controls. These results clearly indicate the presence of cholesterol inhibitor(s) in brewers grain, BBHPF, wheat-germ and oats.

The enzymatic conversion of cis-[14C]phytofluene, trans-[14C]phytofluene, and trans-ζ-[14C]carotene to poly-cis-acyclic carotenes by a cell-free preparation of tangerine tomato fruit plastids

Abstract The conversion of cis -[ 14 C]phytofluene to trans -[ 14 C]phytofluene and the conversion of the latter to trans -ζ-[ 14 C]carotene by a soluble enzyme system obtained from plastids of tangerine tomato fruits is reported. Each of these compounds is also converted to cis -ζ-carotene, proneurosporene, prolycopene, neurosporene, lycopene, and γ- and β-carotenes. [ 14 C]Prolycopene was also incubated with the above enzyme system. No conversion of this compound to trans -lycopene or cyclic carotenes was observed. Proof for the formation of the above carotenes from each of the substrates mentioned above was obtained by cochromatography with authentic samples on an alumina column. A close correspondence between radioactivity and light absorbance of each carotene was observed. Further proof for the formation of acyclic and cyclic carotenes from the above radioactive substrates was obtained by gas-liquid chromatography of the hydrogenated products. Coincidence between mass and radioactivity was observed in each case.

The enzymatic conversion of cis-[14C]phytofluene, trans-[14C]phytofluene, and trans-ζ-[14C]carotene to more unsaturated acyclic, monocyclic, and dicyclic carotenes by a cell-free preparation of red tomato fruits☆☆☆

Abstract This paper reports the conversion of cis -[ 14 C]phytofluene to trans -[ 14 C|phytofluene and the conversion of the latter compound to trans -ζ-[ 14 C]carotene by a soluble enzyme system obtained from the plastids of red tomato fruits. Each of these radioactive compounds was also converted to labeled neurosporene, lycopenc, α-carotene, and β-carotene by the same enzyme system. The incorporation of each substrate into more unsaturated carotenes was carried out under nitrogen at pH 7.5–8.2 (borate buffer), at 25 °C in the dark. Proof of the formation of the above carotenes from each of the three radioactive substrates was demonstrated by cochromatography with authentic nonradioactive carotenes on an alumina chromatographic column. A close correspondence between radioactivity and light absorbance for each carotene was observed. Confirmation of these conversions was achieved by cochromatography with authentic samples on thinlayer plates. Final proof for the formation of the acyclic and cyclic carotenes from the above radioactive substrates was obtained by gas-liquid chromatography of the hydrogenated products. Coincidence between mass and radioactivity was observed. Maximum conversion of cis - and trans -phytofluenes to more unsaturated carotenes by the red tomato fruit enzyme system appears to be dependent upon the presence of NADP + , FAD, and Tween 80. The formation of the carotenes is also increased in the presence of Mg 2+ or Mn 2+ ions.

The isolation, purification, and characterization of cis-ζ-carotene and the demonstration of its conversion to acyclic, monocyclic, and dicyclic carotenes by a soluble enzyme system obtained from the plastids of tangerine tomato fruits

Abstract Cis -ζ-carotene was isolated, purified in several chromatographic systems, and then identified as an intermediate in the biosynthesis of poly- cis -carotenes. The structure of cis -ζ-carotene was tentatively established from its visible light-absorption spectrum, and by a comparison of the infrared spectrum with that of trans -ζ-carotene. Confirmation of the identity of this compound was obtained by high resolution mass spectroscopy. The presence of the cis -configuration was indicated by a bathochromic shift of 6–10 nm in the visible spectrum when the compound was subjected to iodinecatalyzed photoisomerization. The infrared spectrum also showed characteristic peaks for the cis -configuration. Proof of the conversion of cis -ζ-[ 14 C]carotene to trans -ζ-carotene, proneurosporene, prolycopene, neurosporene, lycopene, β- and γ carotenes was obtained on incubation with soluble enzyme systems obtained from plastids of fruits of two different tangerine varieties of tomato. Proof for the formation of each of the carotenes was provided by column and thin-layer chromatography A close correspondence of radioactivity and optical density was observed for each carotene. Additional proof was obtained by gas-liquid chromatography of each hydrogenated carotene. A coincidence of mass and radioactivity was observed for each carotene.

Modulation of Humoral and Cell-Mediated Immune Responses by a Structurally Established Nontoxic Lipid A

In 1954 Westphal and his associates reported on the isolation of a moiety of bacterial endotoxin which they liberated by means of hydrolysis in dilute acetic or hydrochloric acid solutions (25). The water soluble phase of the hydrolysis reaction contained a haptenic polysaccharide which no longer retained the ability to stimulate physiological responses characteristic of the starting material (3,4). On the other hand, the hydrolysis products extractable with organic solvents did retain some of the endotoxic properties of the original substance, leading Westphal and coworkers to postulate that the “endotoxic” activities of LPS were attributable to a lipidic component, which they designated lipid A (5).

Rhodobacter sphaeroides diphosphoryl lipid A inhibits interleukin-6 production in CD14-negative murine marrow stromal ST2 cells stimulated with lipopolysaccharide or paclitaxel (taxol)

Paclitaxel (taxol), a microtubule stabilizer with anticancer activity, mimics the actions of lipopolysaccharide (LPS) on murine macrophages in vitro. Recent studies have shown that the Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA) inhibits both LPS- and paclitaxel-induced activation of murine macrophages, and have suggested that LPS, RsDPLA, and paclitaxel share the same receptor site on murine macrophages. To analyze this receptor site, the present study focused on the interactions between LPS, RsDPLA and paclitaxel in the activation of ST2 cells derived from murine bone marrow stroma. The ST2 cells did not express CD14 mRNA. The cells produced IL-6 molecules and expressed IL-6 mRNA in response to LPS, but did not produce TNF and nitric oxide. Paclitaxel induced IL-6 mRNA expression in ST2 cells. RsDPLA inhibited both LPS- and paclitaxel-induced IL-6 mRNA expression in a dose-dependent manner. These results suggest that LPS, RsDPLA, and paclitaxel are recognized by the same receptor complex on ST2 cells, and that the receptor functions without membrane CD14.