INTERSTELLAR ALCOHOLS

We have investigated the gas-phase chemistry in dense cores where ice mantles containing ethanol and other alcohols have been evaporated. Model calculations show that methanol, ethanol propanol, and butanol drive a chemistry leading to the formation of several large ethers and esters. Of these molecules, methyl ethyl ether (CH3OC2H5) and diethyl ether [(C2H5)(2)O] attain the highest abundances and should be present in detectable quantities within cores rich in ethanol and methanol. Gas-phase reactions act to destroy evaporated ethanol and a low observed abundance of gas-phase C2H5OH does not rule out a high solid-phase abundance. Grain surface formation mechanisms and other possible gas-phase reactions driven by alcohols are discussed, as are observing strategies for the detection of these large interstellar molecules.

Elimination kinetic of 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate ester metabolites in calves' urine

Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C-18 SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CC alpha) for steroids were below 0.1 mu g L-1, except for 19-norandrosterone (CC alpha = 0.7 mu g L-1) and estrone (CC alpha = 0.3 mu g L-1). Kinetics of elimination of the administered 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate were established by monitoring 17 beta-estradiol, 17 alpha-estradiol, estrone and 17 beta-nandrolone, 17 alpha-nandrolone, 19-noretiocholanolone, 19-norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17 alpha-estradiol and 17 alpha-nandrolone (> 20 and 2 mg L-1, respectively after 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate administration) whereas 17 beta-estradiol, estrone, 17 beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the mu g L-1 level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17 beta form. (c) 2008 Elsevier Ltd. All rights reserved.

Intracellular survival of Burkholderia cepacia complex isolates in the presence of macrophage cell activation

Strains of the Burkholderia cepacia complex have emerged as a serious threat to patients with cystic fibrosis due to their ability to infect the lung and cause, in some patients, a necrotizing pneumonia that is often lethal. It has recently been shown that several strains of the B. cepacia complex can escape intracellular killing by free-living amoebae following phagocytosis. In this work, the ability of two B. cepacia complex strains to resist killing by macrophages was explored. Using fluorescence microscopy, electron microscopy and a modified version of the gentamicin-protection assay, we demonstrate that B. cepacia CEP021 (genomovar VI), and Burkholderia vietnamiensis (previously B. cepacia genomovar V) CEP040 can survive in PU5-1.8 murine macrophages for a period of at least 5 d without significant bacterial replication. Furthermore, bacterial entry into macrophages stimulated production of tumour necrosis factor and primed them to release toxic oxygen radicals following treatment with phorbol myristoyl acetate. These effects were probably caused by bacterial LPS, as they were blocked by polymyxin B. Infected macrophages primed with interferon gamma produced less nitric oxide than interferon-gamma-primed uninfected cells. We propose that the ability of B. cepacia to resist intracellular killing by phagocytic cells may play a role in the pathogenesis of cystic fibrosis lung infection. Our data are consistent with a model where repeated cycles of phagocytosis and cellular activation without bacterial killing may promote a deleterious inflammatory response causing tissue destruction and decay of lung function.

Treatment of intestinal Behçet's syndrome with chimeric tumour necrosis factor alpha antibody

Few patients with Behçet's syndrome have gastrointestinal ulceration. Such patients are difficult to treat and have a higher mortality. Faced with refractory symptoms in two patients with intestinal Behçet's, we used the tumour necrosis factor alpha (TNF-alpha) monoclonal antibody infliximab to induce remission. Both women (one aged 27 years, the other 30 years) presented with orogenital ulceration, pustular rash, abdominal pain, bloody diarrhoea due to colonic ulceration, weight loss, and synovitis. One had thrombophlebitis, digital vasculitis, perianal fistula, and paracolic abscess; the other had conjunctivitis and an ulcer in the natal cleft. Treatment with prednisolone, methyl prednisolone, and thalidomide in one and prednisolone, colchicine, and cyclosporin in the other was ineffective. After full discussion, infliximab (3 mg/kg, dose reduced because of recent sepsis in one, and 5 mg/kg in the other) was administered. Within 10 days the ulcers healed, with resolution of bloody diarrhoea and all extraintestinal manifestations. A second infusion of infliximab was necessary eight weeks later in one case, followed by sustained (>15 months) remission on low dose thalidomide. Remission was initially sustained for 12 months in the other but thalidomide had to be stopped due to intolerance, and a good response to retreatment lasted only 12 weeks without immunosuppression, before a third infusion. The cause of Behçet's syndrome is unknown but peripheral blood CD45 gammadelta T cells in Behçet's produce >50-fold more TNF-alpha than controls when stimulated with phorbol myristate acetate and anti-CD3. Infliximab could have a role for inducing remission in Behçet's syndrome.

Age-related changes in non-receptor dependent generation of reactive oxygen species from phagocytes of healthy adults

Several authors have shown that neutrophil generation of reactive oxygen species (ROS) declines with advancing age. Similar changes have also been suggested in monocytes. In both cases alterations in second messenger activity have been implicated as the most likely explanation for these observations. The aim of this study was to investigate the effect of age on phagocyte ROS generation, stimulated by the direct activation of protein kinase C (PKC). Venous blood was drawn from normal healthy subjects, cells were separated on a double density gradient into mononuclear and polymorphonuclear (pmn) cells. Phorbol myristate acetate (PMA) was employed as a cell stimulus. Superoxide generation was measured by cytochrome c reduction and myeloperoxidase (MPO) products by measurement of peak luminol chemiluminescence (CL). Fifty-eight subjects, 25 males and 33 females, were studied, median age 49 years (range 26-88 years). Polymorphonuclear cell superoxide generation was significantly higher in males and there was a trend towards higher pmn MPO product generation in males. Using Spearman's ranked correlation coefficient, monocyte superoxide generation was negatively correlated with age (r = -0.473, P <0.001). No changes in the generation of MPO products was found. There were also trends towards a negative correlation of pmn cytochrome c reduction and peak luminol CL with age in males but not females. Since PMA directly activates protein kinase C, reduced monocyte superoxide generation with increasing age appears to be related to alterations in the ROS generating system downstream of the cell receptor. Impaired monocyte superoxide generation may have implications for non-specific defence against certain infections and early tumour growth in the elderly. Factors underlying these changes in monocyte function therefore require further study.

Isotope dilution gas chromatography/mass spectrometry method for the determination of methionine sulfoxide in protein

We have developed a new technique for quantifying methionine sulfoxide (MetSO) in protein to assess levels of oxidative stress in physiological systems. In this procedure, samples are hydrolyzed with methanesulfonic acid (MSA) in order to avoid the conversion of MetSO to methionine (Met) that occurs during hydrolysis of protein in HCl. The hydrolysate is fractionated on a cation exchange column to remove the nonvolatile MSA from amino acids, and the amino acids are then derivatized as their trimethylsilyl esters for analysis by selected ion monitoring-gas chromatography/mass spectrometry. The limit of detection of the assay is 200 pmol of MetSO per analysis, and the interassay coefficient of variation is 5.8%. Compared to current methods, the SIM-GC/MS assay avoids the potential for conversion of Met to MetSO during sample preparation, requires less sample preparation time, has lower variability, and uses mass spectrometry for sensitive and specific analyte detection.

Activation of MAPK by modified low-density lipoproteins in vascular smooth muscle cells

A high concentration of circulating low-density lipoproteins (LDL) is a major risk factor for atherosclerosis. Native LDL and LDL modified by glycation and/or oxidation are increased in diabetic individuals. LDL directly stimulate vascular smooth muscle cell (VSMC) proliferation; however, the mechanisms remain undefined. The extracellular signal-regulated kinase (ERK) pathway mediates changes in cell function and growth. Therefore, we examined the cellular effects of native and modified LDL on ERK phosphorylation in VSMC. Addition of native, mildly modified (oxidized, glycated, glycoxidized) and highly modified (highly oxidized, highly glycoxidized) LDL at 25 microg/ml to rat VSMC for 5 min induced a fivefold increase in ERK phosphorylation. To elucidate the signal transduction pathway by which LDL phosphorylate ERK, we examined the roles of the Ca(2+)/calmodulin pathway, protein kinase C (PKC), src kinase, and mitogen-activated protein kinase kinase (MEK). Treatment of VSMC with the intracellular Ca(2+) chelator EGTA-AM (50 micromol/l) significantly increased ERK phosphorylation induced by native and mildly modified LDL, whereas chelation of extracellular Ca(2+) by EGTA (3 mmol/l) significantly reduced LDL-induced ERK phosphorylation. The calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (40 micromol/l) significantly decreased ERK phosphorylation induced by all types of LDL. Downregulation of PKC with phorbol myristate acetate (5 micromol/l) markedly reduced LDL-induced ERK phosphorylation. Pretreatment of VSMC with a cell-permeable MEK inhibitor (PD-98059, 40 micromol/l) significantly decreased ERK phosphorylation in response to native and modified LDL. These findings indicate that native and mildly and highly modified LDL utilize similar signaling pathways to phosphorylate ERK and implicate a role for Ca(2+)/calmodulin, PKC, and MEK. These results suggest a potential link between modified LDL, vascular function, and the development of atherosclerosis in diabetes.

Metabolism of very low- and low-density lipoproteins isolated from normolipidaemic type 2 (non-insulin-dependent) diabetic patients by human monocyte-derived macrophages

The very low- and low-density lipoprotein fractions were isolated from 16 normolipidaemic Type 2 (non-insulin-dependent) diabetic patients in good to fair glycaemic control and from corresponding age-, sex-, and race-matched, non-diabetic control subjects. Rates of cholesteryl ester synthesis averaged 268 +/- 31 vs 289 +/- 40 pmol 14C-cholesteryl oleate.mg cell protein-1.20 h-1 for very low- and 506 +/- 34 vs 556 +/- 51 pmol 14C-cholesteryl oleate.mg cell protein-1.20 h-1 for low-density lipoproteins isolated from the Type 2 diabetic patients and control subjects, respectively, when they were incubated with human macrophages. A group of approximately one-third of the patients was selected for separate analyses because very low-density lipoproteins isolated from these patients did stimulate more cholesteryl ester synthesis when incubated with macrophages. There were no significant differences in the lipid composition of the lipoproteins isolated from the three groups of subjects. The relative proportion of apoprotein C to apoprotein E was significantly decreased (p less than 0.002) in the very low-density lipoproteins from diabetic patients and was further decreased in samples from these selected diabetic patients. The apoprotein C-I content of very low-density lipoproteins isolated from diabetic patients was increased compared to control subjects and was further increased in samples from the selected diabetic patients (p less than 0.02). There were no significant differences in the proportions of apoproteins C-III-0, C-III-1, or C-III-2 among the three groups. These studies suggest that in normolipidaemic Type 2 diabetic patients, the apoprotein composition of VLDL is abnormal and this may alter VLDL macrophage interactions and thus contribute to the increased prevalence of atherosclerosis in diabetic patients.

Interaction of very-low-density lipoprotein isolated from type I (insulin-dependent) diabetic subjects with human monocyte-derived macrophages

Very-low-density lipoproteins (VLDL) (density less than 1.006 g/mL) were isolated from type I (insulin-dependent) diabetic patients in good to fair glycemic control and from age-, sex-, and race-matched, nondiabetic, control subjects. VLDL were incubated with human, monocyte-derived macrophages obtained from nondiabetic donors, and the rates of cellular cholesteryl ester synthesis and cholesterol accumulation were determined. VLDL isolated from diabetic patients stimulated significantly more cholesteryl ester synthesis than did VLDL isolated from control subjects (4.04 +/- 1.01 v 1.99 +/- 0.39 nmol 14C-cholesteryl oleate synthesized/mg cell protein/20 h; mean +/- SEM, P less than .05). The stimulation of cholesteryl ester synthesis in macrophages incubated with VLDL isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (P less than .05). The increase in cholesteryl ester synthesis and accumulation in macrophages were mediated by a significant increase in the receptor mediated, high affinity degradation (2.55 +/- 0.23 v 2.12 +/- 0.20 micrograms degraded/mg cell protein/20 h) and accumulation (283 +/- 35 v 242 +/- 33 ng/mg cell protein/20 h) of 125I-VLDL isolated from diabetic patients compared with VLDL from control subjects. To determine if changes in VLDL apoprotein composition were responsible for the observed changes in cellular rates of cholesteryl ester synthesis and accumulation, we also examined the apoprotein composition of the VLDL from both groups. There were no significant differences between the apoproteins B, E, and C content of VLDL from both groups. We also determined the chemical composition of VLDL isolated from both groups of subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycosylation of low-density lipoprotein enhances cholesteryl ester synthesis in human monocyte-derived macrophages

Glucose can react with the lysine residues of low-density lipoproteins (LDLs) and convert the lipoprotein to a form with a receptor-mediated uptake by cultured cells that is impaired. However, in contrast to other modified lipoproteins taken up by both murine and human macrophages via the scavenger-receptor pathway that may induce the formation of foam cells, glycosylated LDL is not recognized by murine macrophages, and thus far, it has not been shown to lead to marked intracellular accumulation of cholesterol in human macrophages. This study illustrates that glycosylated LDL incubated with human monocyte-derived macrophages, at a concentration of 100 micrograms LDL/ml medium, stimulates significantly more cholesteryl ester (CE) synthesis than does control LDL (10.65 +/- 1.5 vs. 4.8 +/- 0.13 nmol.mg-1 cell protein.20 h-1; P less than .05). At LDL concentrations similar to those of plasma, the rate of CE synthesis in macrophages incubated with glycosylated LDL is more markedly enhanced than that observed in cells incubated with control LDL (3-fold increase). The marked stimulation of CE synthesis in human macrophages exposed to glycosylated LDL is paralleled by a significant increase in CE accumulation in these cells (P less than .001). The increase in CE synthesis and accumulation seem to be mediated by an increase in the degradation of glycosylated LDL by human macrophages. Glycosylated LDL enters the macrophages and is degraded by the classic LDL-receptor pathway in slightly smaller amounts than control LDL, but its degradation by pathways other than the classic LDL receptor or scavenger receptor is markedly enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of cholesteryl ester synthesis in human monocyte-derived macrophages by low-density lipoproteins from type 1 (insulin-dependent) diabetic patients:the influence of non-enzymatic glycosylation of low-density lipoproteins

Diabetes mellitus is an independent risk factor in the development of atherosclerosis. In this study we aimed to demonstrate whether there is an abnormal interaction between low-density lipoproteins from diabetic patients and human macrophages. We measured cholesteryl ester synthesis and cholesteryl ester accumulation in human monocyte-derived macrophages (obtained from non-diabetic donors) incubated with low density lipoproteins from Type 1 (insulin-dependent) diabetic patients in good or fair glycaemic control. Low density lipoproteins from the diabetic patients stimulated more cholesteryl ester synthesis than low density lipoproteins from non-diabetic control subjects (7.19 +/- 1.19 vs 6.11 +/- 0.94 nmol/mg cell protein/20 h, mean +/- SEM, p less than 0.05). The stimulation of cholesteryl ester synthesis by low density lipoproteins isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (p less than 0.02). There were no significant differences in the lipid composition of low density lipoproteins between the diabetic and control groups. Non-enzymatic glycosylation of low density lipoproteins was higher in the diabetic group (p less than 0.01) and correlated significantly with cholesteryl ester synthesis (r = 0.58). Similarly, low-density lipoproteins obtained from non-diabetic subjects and glycosylated in vitro stimulated more cholesteryl ester synthesis in macrophages than control low density lipoproteins. The increase in cholesteryl ester synthesis and accumulation by cells exposed to low density lipoproteins from diabetic patients seems to be mediated by an increased uptake of these lipoproteins by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Transmission of two novel mutations in a pedigree with familial lecithin:cholesterol acyltransferase deficiency:structure-function relationships and studies in a compound heterozygous proband

Two novel mutations were identified in a compound heterozygous male with lecithin:cholesterol acyltransferase (LCAT) deficiency. Exon sequence determination of the LCAT gene of the proband revealed two novel heterozygous mutations in exons one (C110T) and six (C991T) that predict non-conservative amino acid substitutions (Thr13Met and Pro307Ser, respectively). To assess the distinct functional impact of the separate mutant alleles, studies were conducted in the proband's 3-generation pedigree. The compound heterozygous proband had negligible HDL and severely reduced apolipoprotein A-I, LCAT mass, LCAT activity, and cholesterol esterification rate (CER). The proband's mother and two sisters were heterozygous for the Pro307Ser mutation and had low HDL, markedly reduced LCAT activity and CER, and the propensity for significant reductions in LCAT protein mass. The proband's father and two daughters were heterozygous for the Thr13Met mutation and also displayed low HDL, reduced LCAT activity and CER, and more modest decrements in LCAT mass. Mean LCAT specific activity was severely impaired in the compound heterozygous proband and was reduced by 50% in individuals heterozygous for either mutation, compared to wild type family members. It is also shown that the two mutations impair both catalytic activity and expression of the circulating protein.

Design of an intravaginal ring for the controlled delivery of 17b-estradiol as its 3-acetate ester

Suitable ester prodrugs of 17b-estradiol are identified, thus permitting effective sustained and controlled estrogen replacement therapy (ERT) from an elastomeric, silicone intravaginal ring (IVR). IVR devices of reservoir design were prepared by blending silicone elastomer base with n-propylorthosilicate (cross-linker) and 10% w/w of 17b-estradiol or an ester prodrug, the mix being activated with 0.5% w/w stannous octoate and cured at 808C for 2 min. A rate-controlling membrane was similarly prepared, without the active agent. IVR devices were of cross-sectional diameter 9 mm, outer diameter 54 mm, with core cross-sectional diameter of 2 mm and core length varied as required. Sink conditions were evident for the 17b-estradiol esters in 1.0% aqueous benzalkonium chloride solution. The low release rates into 0.9% w/v saline of the lipophilic valerate and benzoate esters were due to their intrinsically low aqueous solubilities. In vivo, these esters failed to raise plasma estradiol above baseline levels in postmenopausal human volunteers, despite good in vitro release characteristics under sink conditions. The best release rates under sink conditions, in combination with substantial aqueous solubilities as indicated by the release rates into saline, were observed for the acetate and propionate esters. A
combination of drug release characteristics, short plasma half-life and a toxicologically acceptable hydrolysis product indicated that 17b-estradiol-3-acetate was the prodrug of choice for IVR delivery of ERT. In vivo, an IVR device releasing
100 mg/day of estradiol as its 3-acetate ester maintained over 84 days a circulating plasma concentration in the region of 300 pmol l , within the clinically desirable range for ERT.

Cytosolic phospholipase A2 gene expression in rat mesangial cells is regulated post-transcriptionally

Cytosolic phospholipase A2 (cPLA2) is thought to be the rate-limiting enzyme in the arachidonic acid/eicosanoid cascade. The ability of various agonists to increase steady-state cPLA2 mRNA levels has previously been reported. The current study delineates the contributions of transcriptional and post-transcriptional processes to the regulation of cPLA2 gene expression in response to a variety of agonists in cultured rat glomerular mesangial cells. Epidermal growth factor, platelet-derived growth factor, serum and phorbol myristate acetate all increase the half-life of cPLA2 mRNA transcripts, indicating a role for post-transcriptional modulation of gene expression. The presence of three ATTTA motifs in the 3' untranslated region (3'UTR) of the rat cPLA2 cDNA is ascertained. Heterologous expression of chimeric constructs with different 3'UTRs ligated into the 3' end of the luciferase coding region reveals that the presence of the cPLA2 3'UTR results in reduced luciferase activity compared with constructs without the cPLA2 3'UTR. Furthermore, the luciferase activity in the constructs with the cPLA2 3'UTR is increased in response to the same agonists which stabilize endogenous cPLA2 mRNA. A negligible effect of these agonists on transcriptional control of cPLA2 is evident using promoter-reporter constructs expressed in transient and stable transfectants. Taken together, these results indicate predominant post-transcriptional regulation of cPLA2 mRNA levels.

An Update of the Rules for Pyranoside Sulfonate Displacement

The original 1967 Richardson–Hough rules for predicting SN2 displacement viability in carbohydrate sulfonate derivatives with external nucleophiles have now been updated. Not only do the original rules still hold, but the newly updated rules rationalize why O-triflates (trifluoromethanesulfonate esters) frequently allow many seemingly “disallowed” pyranosidic nucleophilic substitutions to proceed. The new guidelines, which are based on three decades of experimental evidence, allow the feasibility of many pyranosidic O-triflate SN2 displacements to be gauged beforehand.