Three-dimensional chemical imaging of embedded nanoparticles using atom probe tomography.

Analysis of nanoparticles is often challenging especially when they are embedded in a matrix. Hence, we have used laser-assisted atom probe tomography (APT) to analyze the Au nanoclusters synthesized in situ using ion-beam implantation in a single crystal MgO matrix. APT analysis along with scanning transmission electron microscopy and energy dispersive spectroscopy (STEM-EDX) indicated that the nanoparticles have an average size ~8-12 nm. While it is difficult to analyze the composition of individual nanoparticles using STEM, APT analysis can give three-dimensional compositions of the same. It was shown that the maximum Au concentration in the nanoparticles increases with increasing particle size, with a maximum Au concentration of up to 50%.

Crypt stem cell survival in the mouse intestinal epithelium is regulated by prostaglandins synthesized through cyclooxygenase-1.

Prostaglandins (PGs) are important mediators of epithelial integrity and function in the gastrointestinal tract. Relatively little is known, however, about the mechanism by which PGs affect stem cells in the intestine during normal epithelial turnover, or during wound repair. PGs are synthesized from arachidonate by either of two cyclooxygenases, cyclooxygenase-1 (Cox-1) or cyclooxygenase-2 (Cox-2), which are present in a wide variety of mamalian cells. Cox-1 is thought to be a constitutively expressed enzyme, and the expression of Cox-2 is inducible by cytokines or other stimuli in a variety of cell types. We investigated the role of PGs in mouse intestinal stem cell survival and proliferation following radiation injury. The number of surviving crypt stem cells was determined 3.5 d after irradiation by the microcolony assay. Radiation injury induced a dose-dependent decrease in the number of surviving crypts. Indomethacin, an inhibitor of Cox-1 and Cox-2, further reduced the number of surviving crypts in irradiated mice. The indomethacin dose response for inhibition of PGE2 production and reduction of crypt survival were similar. DimethylPGE2 reversed the indomethacin-induced decrease in crypt survival. Selective Cox-2 inhibitors had no effect on crypt survival. PGE2, Cox-1 mRNA, and Cox-1 protein levels all increase in the 3 d after irradiation. Immunohistochemistry for Cox-1 demonstrated localization in epithelial cells of the crypt in the unirradiated mouse, and in the regenerating crypt epithelium in the irradiated mouse. We conclude that radiation injury results in increased Cox-1 levels in crypt stem cells and their progeny, and that PGE2 produced through Cox-1 promotes crypt stem cell survival and proliferation.

Phosphatidylcholine hydrolysis and c-myc expression are in collaborating mitogenic pathways activated by colony-stimulating factor 1.

Stimulation of diglyceride production via phospholipase C (PLC) hydrolysis of phosphatidylcholine was an early event in the mitogenic action of colony-stimulating factor 1 (CSF-1) in the murine macrophage cell line BAC1.2F5 and was followed by a second phase of diglyceride production that persisted throughout the G1 phase of the cell cycle. Addition of phosphatidylcholine-specific PLC (PC-PLC) from Bacillus cereus to the medium of quiescent cells raised the intracellular diglyceride concentration and stimulated [3H]thymidine incorporation, although PC-PLC did not support continuous proliferation. PC-PLC treatment did not induce tyrosine phosphorylation or turnover of the CSF-1 receptor. The major protein kinase C (PKC) isotype in BAC1.2F5 cells was PKC-delta. Diglyceride production from PC-PLC did not target PKC-delta, since unlike phorbol esters, PC-PLC treatment neither decreased the electrophoretic mobility of PKC-delta nor increased the amount of GTP bound to Ras, and PC-PLC was mitogenically active in BAC1.2F5 cells in which PKC-delta was downregulated by prolonged treatment with phorbol ester. PC-PLC mimicked CSF-1 action by elevating c-fos and junB mRNAs to 40% of the level induced by CSF-1; however, PC-PLC induced c-myc mRNA to only 5% of the level in CSF-1-stimulated cells. PC-PLC addition to CSF-1-dependent BAC1.2F5 clones that constitutively express c-myc increased [3H]thymidine incorporation to 86% of the level evoked by CSF-1 and supported slow growth in the absence of CSF-1. Therefore, PC-PLC is a component of a signal transduction pathway leading to transcription of c-fos and junB that collaborates with c-myc and is independent of PKC-delta and Ras activation.

Comparison of 1-O-alkyl-, 1-O-alk-1'-enyl-, and 1-O-acyl-2-acetyl-sn-glycero-3-phosphoethanolamines and -3-phosphocholines as agonists of the platelet-activating factor family.

Four naturally occurring platelet-activating factor (PAF) analogs, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2-acetyl-sn-glycero-3-phosphocholine, 1-octadecanoyl-2-acetyl-sn-glycero-3-phosphocholine, and 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine, stimulated human neutrophils (PMN) to mobilize Ca2+, degranulate, and produce superoxide anion. They were, respectively, 5-, 300-, 500-, and 4000-fold weaker than PAF in each assay; inhibited PMN-binding of [3H]PAF at concentrations paralleling their biological potencies; and showed sensitivity to the inhibitory effects of PAF antagonists. PAF and the analogs, moreover, desensitized PMN responses to each other but not to leukotriene B4 and actually increased (or primed) PMN responses to N-formyl-MET-LEU-PHE. Finally, 5-hydroxyeicosatetraenoate-enhanced PMN responses to PAF and the analogs without enhancing the actions of other stimuli. It stereospecifically raised each analog's potency by as much as 100-fold and converted a fifth natural analog, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine from inactive to a weak stimulator of PMN. PAF and its analogs thus represent a structurally diverse family of cell-derived phospholipids which can activate, prime, and desensitize neutrophils by using a common, apparently PAF receptor-dependent mechanism.

Stimulated neutrophils produce an ethanolamine plasmalogen analog of platelet-activating factor.

Human neutrophils stimulated by ionophore A23187 incorporate [3H]acetate into platelet-activating factor and an additional product which is chromatographically similar to phosphatidylethanolamine and accounts for approximately 25% of the [3H]acetate-containing lipids. Three general approaches indicated the sn-1 moiety of the unknown phospholipid is primarily alk-1'-enyl-linked: 1) approximately 80% of the intact phospholipid as well as its derivatives was highly sensitive to hydrolysis by HCl, 2) 80% of the product which resulted from treating the unknown with phospholipase C and acetylating the free hydroxyl group at the sn-3 position, chromatographed with authentic 1-O-alk-1'-enyl-2,3-diacetylglycerol, and 3) catalytic hydrogenation of the diacetylglycerol product described in 2) resulted in a product which chromatographed with alkyldiacetylglycerol and was not sensitive to strong acid. Treatment of the intact phospholipid with phospholipase A2 resulted in the release of 88% of the radiolabel into the acidified aqueous phase of the extraction mixture, indicating the moiety in the sn-2 position remained as acetate and had not been elongated to fatty acid. The head group was determined to be phosphoethanolamine based upon its complete conversion to the dinitro- and trinitrophenyl derivatives by the amine-derivatizing reagents fluorodinitrobenzene and trinitrobenzenesulfonic acid, respectively. From these data is was concluded that the unknown product is 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (80%), and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine (10%). Sonicates prepared from neutrophils stimulated with ionophore A23187 contained an acetyltransferase activity capable of utilizing 1-O-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine and [14C]acetyl-CoA to produce the product identified as 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine.

Overexpression of MDR1 in an intestinal cell line results in increased cholesterol uptake from micelles.

The multiple drug resistance protein, MDR1, is highly expressed on the apical surface of intestinal epithelial cells. The physiologic substrate of this protein remains unclear. Several studies using compounds known to act as MDR1 inhibitors have suggested that MDR1 may be involved in the transport of cholesterol from the plasma membrane to the endoplasmic reticulum where it is esterified. To examine the role of MDR1 in cholesterol uptake by intestinal cells, the rat intestinal epithelial cell line IEC-18, was stably transfected with human MDR1. MDR1-transfected cells exhibited increased expression of MDR1 protein, reduced accumulation of vinblastine and increased uptake of [(3)H]cholesterol from cholesterol/monolein/taurocholate micelles. These studies provide the first direct evidence that the level of MDR1 expression in intestinal cells can influence the amount of cholesterol taken up by those cells. This is also the first demonstration that a multiple drug resistance protein can function in the net uptake, rather than efflux, of a substrate.

Selective deacylation of arachidonate-containing ethanolamine-linked phosphoglycerides in stimulated human neutrophils.

The involvement of the ethanolamine-linked phosphoglyceride fraction (PE) in neutrophil signal transduction is suggested by the stimulus-induced release of arachidonic acid from PE (Chilton, F. H., and Connell, T. R. (1988) J. Biol. Chem. 263, 5260-5265) and by the synthesis of acetylated PE species, predominantly 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (alkenylacetyl-GPE; Tessner, T. G., and Wykle, R. L. (1987) J. Biol. Chem. 262, 12660-12664) in stimulated cells. In the studies reported here, we investigated the relationship between arachidonic acid release from PE and generation of the lysophospholipid precursor required in the biosynthesis of alkenylacetyl-GPE. In order to follow these reactions, we prelabeled neutrophils with 1-O-[3H]alk-1'-enyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (alkenyl-acyl-GPE). We also followed the hydrolysis of endogenous PE by analysis as the dinitrophenyl derivative using a high pressure liquid chromatography method we developed. Our results coupled with those of Chilton et al. (Chilton, F. H., Ellis, J. M., Olson, S. C., and Wykle, R. L. (1984) J. Biol. Chem. 259, 12014-12019) indicate that in human neutrophils the metabolism of alkenylacyl-GPE and alkylacyl-sn-glycero-3-phosphocholine (GPC) are strikingly similar with regard to arachidonate metabolism. When added to neutrophils, both 1-O-[3H]alkenyl-2-lyso-GPE and 1-O-[3H]alkyl-2-lyso-GPC are acylated predominantly with arachidonic acid, and the resulting arachidonoyl-containing phospholipids are extensively deacylated upon stimulation. However, hydrolysis of PE in the neutrophil differs from hydrolysis of choline-containing phosphoglycerides in that stimulation leads to a greater accumulation of the ethanolamine-linked lysophospholipid. A comparison of the molecular species of endogenous PE (based on molar concentrations measured as the dinitrophenyl derivative) from resting and stimulated neutrophils indicated that only those species which contain arachidonate are significantly hydrolyzed.

Colony-stimulating factor 1 regulates CTP: phosphocholine cytidylyltransferase mRNA levels.

Growth factor regulation of phosphatidylcholine (PtdCho) metabolism during the G1 stage of the cell cycle was investigated in the colony-stimulating factor 1 (CSF-1)-dependent murine macrophage cell-line BAC1.2F5. The transient removal of CSF-1 arrested the cells in G1. Incorporation of [3H]choline into PtdCho was stimulated significantly 1 h after growth factor addition to quiescent cells. Metabolic labeling experiments pointed to CTP:phosphocholine cytidylyltransferase (CT) as the rate-controlling enzyme for PtdCho biosynthesis in BAC1.2F5 cells. The amount of CT mRNA increased 4-fold within 15 min of CSF-1 addition and remained elevated for 2 h. The rise in CT mRNA levels was accompanied by a 50% increase in total CT specific activity in cell extracts within 4 h after the addition of CSF-1. CSF-1-dependent elevation of CT mRNA content was neither attenuated nor superinduced by the inhibition of protein synthesis with cycloheximide. The rate of CT mRNA turnover decreased in the presence of CSF-1 indicating that message stabilization was a key factor in determining the levels of CT mRNA. These data point to increased CT mRNA abundance as a component in growth factor-stimulated PtdCho synthesis.

Lipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-mediated mechanism.

BACKGROUND & AIMS: The bone marrow and the intestine are the major sites of radiation-induced injury. The cellular response to radiation injury in the intestine or bone marrow can be modulated by agents given before irradiation. Lipopolysaccharide is known to be radioprotective in the bone marrow, but its effect on the intestine is not known. We sought to determine if lipopolysaccharide is radioprotective in the intestine and, if so, to determine the mechanism of its radioprotective effects. METHODS: Mice were treated with parenteral lipopolysaccharide or vehicle and then irradiated (14 Gy total body irradiation in a cesium irradiator). The number of surviving intestinal crypts was assessed 3.5 days after irradiation using a clonogenic assay. RESULTS: Parenteral administration of lipopolysaccharide 2-24 hours before irradiation resulted in a 2-fold increase in the number of surviving crypts 3.5 days after irradiation. The radioprotective effects of lipopolysaccharide could be eliminated by coadministration of a selective inhibitor of cyclooxygenase 2. Lipopolysaccharide was radioprotective in wild-type mice but not in mice with a disrupted cyclooxygenase 2. Parenteral administration of lipopolysaccharide resulted in increased production of prostaglandins in the intestine and in the induction of cyclooxygenase 2 expression in subepithelial fibroblasts and in villous, but not crypt, epithelial cells. CONCLUSIONS: Lipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-dependent pathway.

Stimulation of platelet-activating factor synthesis by a nonmetabolizable bioactive analog of platelet-activating factor and influence of arachidonic acid metabolites.

Platelet-activating factor (PAF) is a potent neutrophil agonist operating through specific receptors located on the cell surface. Binding of PAF to its receptor may also stimulate further PAF synthesis, thus providing a means of amplifying the PAF signal for the cell of origin and/or other responsive cells. In this report we demonstrate that 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (C-PAF), a nonmetabolizable bioactive analog of PAF, stimulates human neutrophils to synthesize PAF, as detected by [3H]acetate incorporation into PAF. This approach allowed us to conclude that [3H]acetate-labeled PAF was formed from endogenous precursor rather than mere turnover of the stimulatory dose of PAF. PAF's ability to initiate further PAF synthesis was confirmed by measuring the PAF-stimulated conversion of 1-O-[3H]alkyl-2-acylglycerophosphocholine to 1-O-[3H]alkyl-2-acetylglycerophosphocholine by prelabeled human neutrophils and by determining the molecular species of 1-O-alkyl-2-[3H]acetylglycerophosphocholine produced by cells stimulated with a single molecular species of PAF (C15:0). Degradation of exogenously added [3H]PAF was not inhibited by C-PAF/5-hydroxyeicosatetraenoic acid treatment. Thus, inhibition of PAF degradation was ruled out as the mechanism accounting for the appearance of labeled PAF in the stimulated cells. Synthesis of PAF in response to C-PAF was not dependent on cytochalasin B pretreatment but was dramatically potentiated by 5-hydroxyeicosatetraenoic acid, which alone was without effect. Additionally, we have demonstrated that another major arachidonate metabolite of neutrophils, leukotriene B4, stimulates PAF production. Thus, at least three products of activated neutrophils, including PAF itself, can promote PAF synthesis by these cells. This positive feedback effect may amplify autacoid production and the final cellular response.

Prostaglandins prevent decreased epithelial cell proliferation associated with dextran sodium sulfate injury in mice.

BACKGROUND & AIMS: Although dextran sodium sulfate (DSS)-induced colitis is a commonly used model of colonic injury, the mechanism of this model is not understood. The aim of this study was to determine the contribution of prostaglandins to the mechanism of DSS-induced epithelial injury. METHODS: Mice were treated with 3% DSS in the drinking water for 5 days followed by water only (recovery). Tissue prostaglandin E2 (PGE2) levels were measured, proliferating cells per cecal crypt were determined by bromodeoxyuridine labeling, and the cellular localization of cyclooxygenase (COX)-1 and COX-2 was determined by immunohistochemistry. RESULTS: DSS decreased the number of proliferating epithelial cells per crypt by approximately 90% and decreased the height of cecal crypts by 40%. Administration of dimethyl PGE2 with DSS reversed the effect of DSS on proliferation but not its effect on crypt shortening. COX-1 was expressed in the crypt epithelium and lamina propria mononuclear cells; DSS treatment down-regulated COX-1 expression only in the epithelium. Dimethyl PGE2 reversed the effect of DSS on COX-1 expression. Recovery was associated with a return to normal COX-1 expression in the epithelium. COX-2 was expressed in lamina propria mononuclear cells. CONCLUSIONS: Epithelial cell proliferation in the presence of DSS contains a PGE2-sensitive component.

Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease.

BACKGROUND & AIMS: Prostaglandins are synthesized by cyclooxygenases (COX)-1 and -2. The expression and cellular localization of COX-1 and COX-2 in normal human colon and inflammatory bowel disease (IBD) surgical resections were studied. METHODS: COX-1 and COX-2 protein expression and cellular localization were assessed by Western blotting and immunohistochemistry. RESULTS: COX-1 protein was expressed at equal levels in normal, Crohn's disease, and ulcerative colitis colonic epithelial cells. COX-2 protein was not detected in normal epithelial cells but was detected in Crohn's disease and ulcerative colitis epithelial cells. Immunohistochemistry of normal, Crohn's colitis, and ulcerative colitis tissue showed equivalent COX-1 expression in epithelial cells in the lower half of the colonic crypts. COX-2 expression was absent from normal colon, whereas in Crohn's colitis and ulcerative colitis, COX-2 was observed in apical epithelial cells and in lamina propria mononuclear cells. In Crohn's ileitis, COX-2 was present in the villus epithelial cells. In ulcerative colitis, colonic epithelial cells expressing COX-2 also expressed inducible nitric oxide synthase. CONCLUSIONS: COX-1 was localized in the crypt epithelium of the normal ileum and colon, and its expression was unchanged in IBD. COX-2 was undetectable in normal ileum or colon, but it was induced in apical epithelial cells of inflamed foci in IBD.

The gene for murine CTP:phosphocholine cytidylyltransferase (Ctpct) is located on mouse chromosome 16.

CTP:phosphocholine cytidylyltransferase is the rate-controlling enzyme in phosphatidylcholine biosynthesis and is essential for the survival of eukaryotic cells. The murine cDNA for the cytidylyltransferase was cloned and sequenced. A genomic clone was isolated and the chromosomal location of the Ctpct locus determined by Southern blot hybridization of DNAs from a panel of interspecific backcross progeny derived from matings of [(C57BL/6J x Mus spretus)F1 x C57BL/6J] mice. These data place the Ctpct gene on mouse chromosome 16 between the Smst and Stf-1 genes.