Cholesteryl ester accumulation and accelerated cholesterol absorption in intestine-specific hormone sensitive lipase-null mice

Hormone sensitive lipase (HSL) regulates the hydrolysis of acylglycerols and cholesteryl esters (CE) in various cells and organs, including enterocytes of the small intestine. The physiological role of this enzyme in enterocytes, however, stayed elusive. In the present study we generated mice lacking HSL exclusively in the small intestine (HSLiKO) to investigate the impact of HSL deficiency on intestinal lipid metabolism and the consequences on whole body lipid homeostasis. Chow diet-fed HSLiKO mice showed unchanged plasma lipid concentrations. In addition, feeding with high fat/high cholesterol (HF/HC) diet led to unaltered triglyceride but increased plasma cholesterol concentrations and CE accumulation in the small intestine. The same effect was observed after an acute cholesterol load. Gavaging of radioactively labeled cholesterol resulted in increased abundance of radioactivity in plasma, liver and small intestine of HSLiKO mice 4h post-gavaging. However, cholesterol absorption determined by the fecal dual-isotope ratio method revealed no significant difference, suggesting that HSLiKO mice take up the same amount of cholesterol but in an accelerated manner. mRNA expression levels of genes involved in intestinal cholesterol transport and esterification were unchanged but we observed downregulation of HMG-CoA reductase and synthase and consequently less intestinal cholesterol biosynthesis. Taken together our study demonstrates that the lack of intestinal HSL leads to CE accumulation in the small intestine, accelerated cholesterol absorption and decreased cholesterol biosynthesis, indicating that HSL plays an important role in intestinal cholesterol homeostasis.

Kinetic assessment of persistent halogenated xenobiotics in cell culture models: comparison of mono- and poly-halogenated compounds

We evaluated the suitability of single and multiple cell type cultures as model systems to characterise cellular kinetics of highly lipophilic compounds with potential ecotoxicological impact. Confluent mono-layers of human skin fibroblasts, rat astrocytoma C6 cells, non-differentiated and differentiated mouse 3T3 cells were kept in culture medium supplemented with 10% foetal calf serum. For competitive uptake experiments up to four different cell types, grown on glass sectors, were exposed for 3h to (14)C-labelled model compounds, dissolved either in organic solvents or incorporated into unilamellar lecithin liposomes. Bromo-, or chloro-benzenes, decabromodiphenylether (DBP), and dichlorodiphenyl ethylene (DDE) were tested in rather high concentration of 20 microM. Cellular toxicity was low. Compound levels were related to protein, DNA, and triglyceride contents. Cellular uptake was fast and dependent on physico-chemical properties of the compounds (lipophilicity, molecular size), formulation, and cell type. Mono-halogenated benzenes showed low and similar uptake levels (=low accumulation compounds). DBP and DDE showed much higher cellular accumulations (=high accumulation compounds) except for DBP in 3T3 cells. Uptake from liposomal formulations was mostly higher than if compounds were dissolved in organic solvents. The extent of uptake correlated with the cellular content of triglycerides, except for DBP. Uptake competition between different cell types was studied in a sectorial multi-cell culture model. For low accumulation compounds negligible differences were found among C6 cells and fibroblasts. Uptake of DDE was slightly and that of DBP highly increased in fibroblasts. Well-defined cell culture systems, especially the sectorial model, are appropriate to screen for bioaccumulation and cytotoxicity of (unknown) chemical entities in vitro.

THERMORESPONSIVE PROPERTIES OF GOLD HYBRID NANOPARTICLES OF POLY(DI(ETHYLENE GLYCOL) METHYL ETHER METHACRYLATE) (PDEGMA) AND ITS BLOCK COPOLYMERS WITH DIFFERENT ANCHORING REGIMES

Thermo-responsive materials have been of interest for many years, and have been studied mostly as thermally stimulated drug delivery vehicles. Recently acrylate and methacrylates with pendant ethylene glycol methyl ethers been studied as thermo responsive materials. This work explores thermo response properties of hybrid nanoparticles of one of these methacrylates (DEGMA) and a block copolymer with one of the acrylates (OEGA), with gold nanoparticle cores of different sizes. We were interested in the effects of gold core size, number and type of end groups that anchored the chains to the gold cores, and location of bonding sites on the thermo-response of the polymer. To control the number and location of anchoring groups we using a type of controlled radical polymerization called Reversible Addition Fragmentation Transfer (RAFT) Polymerization. Smaller gold cores did not show the thermo responsive behavior of the polymer but the gold cores did seem to self-assemble. Polymer anchored to larger gold cores did show thermo responsivity. The anchoring end group did not alter the thermoresponsivity but thiol-modified polymers stabilized gold cores less well than chains anchored by dithioester groups, allowing gold cores to grow larger. Use of multiple bonding groups stabilized the gold core. Using block copolymers we tested the effects of number of thiol groups and the distance between them. We observed that the use of multiple anchoring groups on the block copolymer with a sufficiently large gold core did not prevent thermo responsive behavior of the polymer to be detected which allows a new type of thermo-responsive hybrid nanoparticle to be used and studied for new applications.

Whole body postmortem angiography with a high viscosity contrast agent solution using poly ethylene glycol as contrast agent dissolver

Postmortem minimal invasive angiography has already been implemented to support virtual autopsy examinations. An experimental approach in a porcine model to overcome an initially described artificial tissue edema artifact by using a poly ethylene glycol (PEG) containing contrast agent solution showed promising results. The present publication describes the first application of PEG in a whole corpse angiographic CT examination. A minimal invasive postmortem CT angiography was performed in a human corpse utilizing the high viscosity contrast agent solution containing 65% of PEG. Injection was carried out via the femoral artery into the aortic root in simulated cardiac output conditions. Subsequent CT scanning delivered the 3D volume data of the whole corpse. Visualization of the human arterial anatomy was excellent and the contrast agent distribution was generally limited to the arterial system as intended. As exceptions an enhancement of the brain, the left ventricular myocardium and the renal cortex became obvious. This most likely represented the stage of centralization of the blood circulation at the time of death with dilatation of the precapillary arterioles within these tissues. Especially for the brain this resulted in a distinctively improved visualization of the intracerebral structures by CT. However, the general tissue edema artifact of postmortem minimal invasive angiography examinations could be distinctively reduced.

Bovine primary chondrocyte culture in synthetic matrix metalloproteinase-sensitive poly(ethylene glycol)-based hydrogels as a scaffold for cartilage repair

A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.

Omega-3 poly-unsaturated fatty acids for the prevention of severe neutropenic enterocolitis in patients with acute myeloid leukemia

Neutropenic enterocolitis is a potentially fatal complication of myeloablative chemotherapy in patients with acute myeloid leukemia. Omega-3 polyunsaturated fatty acids (PUFA) are precursors of potent anti-inflammatory prostaglandins. Our aim was to explore the safety and effectiveness of omega-3 PUFA added to parenteral nutrition in protecting leukemia patients from severe enterocolitis. Fourteen patients with acute myeloid leukemia who received omega-3 PUFA in a Phase II trial were compared with 66 consecutive control patients not getting this intervention. We performed crude and adjusted comparisons, using inverse probability of treatment weighting for adjusted analysis, and blind outcome assessment to minimize assessor bias. Primary outcome was severe enterocolitis (≥Grade 3). The crude odds ratio of Grade 3 colitis or higher was 1.36 (95% CI 0.37 to 4.96, P = 0.64), and the adjusted odds ratio was 0.79 (95% CI 0.35 to 1.78, P = 0.57). There was little evidence to suggest differences between groups in serious adverse events and overall mortality. Our results provide little evidence that addition of omega-3 PUFA is beneficial in this condition. Routine treatment with omega-3 PUFA is currently not warranted.

Indocyanine Green Loaded Biocompatible Nanoparticles: Stabilisation of Indocyanine Green (ICG) Using Biocompatible Silica-Poly (&epsilon-Caprolactone) Grafted Nanocomposites

Indocyanine green (ICG) is a chemically labile compound which needs to be stabilized in aqueous media to be used in biomedical applications. In the present study, poly(ε-caprolactone) (PCL), a semi-crystalline polyester, was used to encapsulate and stabilize ICG in a hydrophobic environment. A hydrophobic and biocompatible nanocomposite was obtained by the process of encapsulating inorganic silica. ICG was embedded in the hydrophobic polymer coating by starting from a well-defined silica (Si) core of either 80 nm or 120 nm diameter, which served as a template for a ‘grafting from’ approach using ε-caprolactone. The obtained nanocomposite Si grafted PCL/ICG was based on silica nanoparticles grafted with PCL, in which ICG was adsorbed. The nanoparticles were characterized by IR spectroscopy, thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The change in the surface charge and the colloidal stability of the nanoparticles was followed by zeta potential measurements. This approach of synthesizing nanocomposite-based ICG demonstrates a new route to stabilize ICG. We synthesized biocompatible nanoparticles containing a high ICG concentration and exhibiting excellent stability to aqueous decomposition.

Canine keratinocytes upregulate type I interferons and proinflammatory cytokines in response to poly(dA:dT) but not to canine papillomavirus.

Papillomaviruses (PV) are double stranded (ds) DNA viruses that infect epithelial cells within the skin or mucosa, most often causing benign neoplasms that spontaneously regress. The immune system plays a key role in the defense against PVs. Since these viruses infect keratinocytes, we wanted to investigate the role of the keratinocyte in initiating an immune response to canine papillomavirus-2 (CPV-2) in the dog. Keratinocytes express a variety of pattern recognition receptors (PRR) to distinguish different cutaneous pathogens and initiate an immune response. We examined the mRNA expression patterns for several recently described cytosolic nucleic acid sensing PRRs in canine monolayer keratinocyte cultures using quantitative reverse transcription-polymerase chain reaction. Unstimulated normal cells were found to express mRNA for melanoma differentiation associated gene 5 (MDA5), retinoic acid-inducible gene I (RIG-I), DNA-dependent activation of interferon regulatory factors, leucine rich repeat flightless interacting protein 1, and interferon inducible gene 16 (IFI16), as well as their adaptor molecules myeloid differentiation primary response gene 88, interferon-β promoter stimulator 1, and endoplasmic reticulum-resident transmembrane protein stimulator of interferon genes. When stimulated with synthetic dsDNA [poly(dA:dT)] or dsRNA [poly(I:C)], keratinocytes responded with increased mRNA expression levels for interleukin-6, tumor necrosis factor-α, interferon-β, RIG-I, IFI16, and MDA5. There was no detectable increase in mRNA expression, however, in keratinocytes infected with CPV-2. Furthermore, CPV-2-infected keratinocytes stimulated with poly(dA:dT) and poly(I:C) showed similar mRNA expression levels for these gene products when compared with expression levels in uninfected cells. These results suggest that although canine keratinocytes contain functional PRRs that can recognize and respond to dsDNA and dsRNA ligands, they do not appear to recognize or initiate a similar response to CPV-2.

Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation.

Poly(A)-binding protein (PABP) stimulates translation initiation by binding simultaneously to the mRNA poly(A) tail and eukaryotic translation initiation factor 4G (eIF4G). PABP activity is regulated by PABP-interacting (Paip) proteins. Paip1 binds PABP and stimulates translation by an unknown mechanism. Here, we describe the interaction between Paip1 and eIF3, which is direct, RNA independent, and mediated via the eIF3g (p44) subunit. Stimulation of translation by Paip1 in vivo was decreased upon deletion of the N-terminal sequence containing the eIF3-binding domain and upon silencing of PABP or several eIF3 subunits. We also show the formation of ternary complexes composed of Paip1-PABP-eIF4G and Paip1-eIF3-eIF4G. Taken together, these data demonstrate that the eIF3-Paip1 interaction promotes translation. We propose that eIF3-Paip1 stabilizes the interaction between PABP and eIF4G, which brings about the circularization of the mRNA.

Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation.

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3' poly(A) tail-PABP complex.

Pharmacological characterization of nipecotic acid ethyl ester: A novel cholinergic muscarinic agonist

The aim of this dissertation was to examine the hypothesis that (R)-nipecotic acid ethyl ester ((R)-NAEE) is a cholinergic agonist that is selective for a particular subclass (M$\sb1$ or M$\sb2$) of muscarinic receptors.^ Ligand binding studies indicated that like cholinergic agonists (R)-NAEE selectively interacts with rat heart (M$\sb2$) and brain (M$\sb1$) muscarinic binding sites. Physiological studies revealed that unlike cholinergic agonists (R)-NAEE stimulated only those responses coupled to M$\sb2$ muscarinic receptors (acid secretion, negative inotropic response, smooth muscle contraction). Moreover, in rat brain (R)-NAEE differentiated between M$\sb2$ receptors negatively coupled to adenylate cyclase activity and M$\sb1$ receptors mediating PI turnover, being a weak competitive antagonist at these latter sites. In isolated rat gastric mucosal cells (R)-NAEE also differentiated between two M$\sb2$ coupled responses where it potentiated acid secretion but could not stimulate PI turnover. Atropine, a selective antimuscarinic agent, competitively antagonized all agonist effects of (R)-NAEE.^ Unlike (R)-NAEE, the muscarinic agonist arecoline, which is structurally similar to (R)-NAEE, stimulates both M$\sb1$ and M$\sb2$ receptors. Structure activity studies revealed that saturation of the piperidine ring and the length of the ester side chain of (R)-NAEE are the most important determinants for both M$\sb2$ efficacy and selectivity.^ The results of this dissertation establish that (R)-NAEE is a cholinergic muscarinic receptor agonist that displays greater efficacy at M$\sb2$ than at M$\sb1$ receptors, being a weak antagonist at the M$\sb1$ site. With such selectivity, (R)-NAEE may be regarded as a prototype for a unique class of cholinergic muscarinic M$\sb2$ receptor agonists. Because of these unique properties, (R)-NAEE should be useful in the further characterization of muscarinic receptors, and could lead to the development of a new class of therapeutic agents. ^

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

Osteo-odonto-keratoprosthesis (OOKP) and the testing of three different adhesives for bonding bovine teeth with optical poly-(methyl methacrylate) (PMMA) cylinder.

AIM Preparation of the lamina during osteo-odonto-keratoprosthesis (OOKP) design is complex, and its longevity and watertightness important. To date, only acrylic bone cements have been used for bonding the optical cylinder to the tooth dentine. Our aim was to evaluate different dental adhesives for OOKP preparation. METHODS Specimens of bovine teeth were produced by preparing 1.5-mm thick dentine slices with holes having a diameter of 3.5 mm. Each group (n=10 per group) was luted with either classic poly-(methyl methacrylate) (PMMA) bone cement, universal resin cement or glass ionomer cement. All specimens underwent force measurement using a uniaxial traction machine. RESULTS The highest mean force required to break the bond was measured for PMMA bone cement (128.2 N) followed by universal resin cement (127.9 N), with no statistically significant difference. Glass ionomer cement showed significantly lower force resistance (78.1 N). CONCLUSIONS Excellent bonding strength combined with easy application was found for universal resin cement, and thus, it is a potential alternative to acrylic bone cement in OOKP preparation.