Transport of cholesterol across a BeWo cell monolayer: implications for net transport of sterol from maternal to fetal circulation

The placental transport of various compounds, such as glucose and fatty acids, has been well studied. However, the transport of cholesterol, a sterol essential for proper fetal development, remains undefined in the placenta. Therefore, the purpose of these studies was to examine the transport of cholesterol across a placental monolayer and its uptake by various cholesterol acceptors. BeWo cells, which originated from a human choriocarcinoma, were grown on transwells for 3 days to form a confluent monolayer. The apical side of the cells was radiolabeled with either free cholesterol or LDL cholesteryl ester. After 24 h, the radiolabel was removed and cholesterol acceptors were added to the basolateral chamber. Cholesterol was found to be taken up by the apical surface of the placental monolayer, transported to the basolateral surface of the cell, and effluxed to fetal human serum, fetal HDL, or phospholipid vesicles, but not to apolipoprotein A-I. In addition, increasing the cellular cholesterol concentration further increased the amount of cholesterol transported to the basolateral acceptors. These are the first studies to demonstrate the movement of cholesterol across a placental cell from the maternal circulation (apical side) to the fetal circulation (basolateral side).     

Toll-like receptor 3 mediates a more potent antiviral response than Toll-like receptor 4

We have recently described an IFN regulatory factor 3-mediated antiviral gene program that is induced by both Toll-like receptor (TLR)3 and TLR4 ligands. In our current study, we show that activation of IFN/viral response gene expression in primary macrophage cells is stronger and prolonged with TLR3 stimulation compared with that of TLR4. Our data also reveal that the cytoplasmic tails of both TLR3 and TLR4 can directly interact with myeloid differentiation factor 88 (MyD88). However, although Toll/IL-1 receptor homology domain-containing adaptor protein/MyD88 adaptor-like is able to associate with TLR4, we were unable to detect any interaction between Toll/IL-1 receptor homology domain-containing adaptor protein/MyD88 adaptor-like and TLR3. By using quantitative real-time PCR assays, we found that TLR3 expression is inducible by both TLR3 and TLR4 ligands, while TLR4 expression is not inducible by these same stimuli. Furthermore, using cells derived from mice deficient in the IFN-alphabetaR, we show that both TLR3 and TLR4 require IFN-beta autocrine/paracrine feedback to induce TLR3 expression and activate/enhance genes required for antiviral activity. More specifically, a subset of antiviral genes is initially induced independent of IFN-beta, yet the cytokine further enhances expression at later time points. This was in contrast to a second set of genes (including TLR3) that is induced only after IFN-beta production. Taken together, our data argue that, despite both TLR3 and TLR4 being able to use IFN-beta to activate/enhance antiviral gene expression, TLR3 uses multiple mechanisms to enhance and sustain the antiviral response more strongly than TLR4.     

Adenovirus-transduced dendritic cells injected into skin or lymph node prime potent simian immunodeficiency virus-specific T cell immunity in monkeys

Adenoviral vectors can be used to deliver complex Ag to dendritic cells (DC), and thus may be ideal for stimulating broad T cell responses to viral pathogens and tumors. To test this hypothesis in a relevant primate model, we used recombinant adenovirus serotype 5 vectors expressing SIV Gag Ag to transduce monocyte-derived DC from rhesus macaques, and then immunized donor animals either by intradermal or intranodal injections. T cell responses were evaluated by ELISPOT assay using previously frozen PBMC pulsed with pools of 15-mer peptides representing the Gag sequence. Immunization resulted in rapid and potent induction of T cell responses to multiple regions of Gag, with frequencies approaching 1 Gag-specific T cell per 500 uncultured PBMC. Surprisingly, intradermal and intranodal injections generated a similar intensity and breadth of response, indicating that administration of Ag-expressing DC by either route may be equally effective at inducing immune responses. Detailed analysis of two monkeys revealed CD8(+) T cell responses to several peptide epitopes of Gag not previously described, at least two of which are restricted by MHC class I alleles not currently identified. Repeated vaccination did not induce T cell responses to the adenoviral vector and did not prevent Ag-expressing DC injected under the capsule of the lymph node from migrating to the paracortex and interposing between T cells. However, boost injections of adenovirus-transduced DC were generally limited in efficacy. These findings support the use of adenovirus-transduced DC in the therapy of HIV infection and cancer.     

The Carboxylesterase Gene Family from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Carboxylesterases hydrolyze esters of short-chain fatty acids and have roles in animals ranging from signal transduction to xenobiotic detoxification. In plants, however, little is known of their roles. We have systematically mined the genome from the model plant Arabidopsis thaliana for carboxylesterase genes and studied their distribution in the genome and expression profile across a range of tissues. Twenty carboxylesterase genes (AtCXE) were identified. The AtCXE family shares conserved sequence motifs and secondary structure characteristics with carboxylesterases and other members of the larger / hydrolase fold superfamily of enzymes. Phylogenetic analysis of the AtCXE genes together with other plant carboxylesterases distinguishes seven distinct clades, with an Arabidopsis thaliana gene represented in six of the seven clades. The AtCXE genes are widely distributed across the genome (present in four of five chromosomes), with the exception of three clusters of tandemly duplicated genes. Of the interchromosomal duplication events, two have been mediated through newly identified partial chromosomal duplication events that also include other genes surrounding the AtCXE loci. Eighteen of the 20 AtCXE genes are expressed over a broad range of tissues, while the remaining 2 (unrelated) genes are expressed only in the flowers and siliques. Finally, hypotheses for the functional roles of the AtCXE family members are presented based on the phylogenetic relationships with other plant carboxylesterases of known function, their expression profile, and knowledge of likely esterase substrates found in plants.     

High glucose augments the angiotensin II-induced activation of JAK2 in vascular smooth muscle cells via the polyol pathway

Angiotensin II (Ang II), protein kinase C (PKC), reactive oxygen species (ROS) generated by NADPH oxidase, the activation of Janus kinase 2 (JAK2), and the polyol pathway play important parts in the hyperproliferation of vascular smooth muscle cells (VSMC), a characteristic feature of diabetic macroangiopathy. The precise mechanism, however, remains unclear. This study investigated the relation between the polyol pathway, PKC-beta, ROS, JAK2, and Ang II in the development of diabetic macroangiopathy. VSMC cultured in high glucose (HG; 25 mm) showed significant increases in the tyrosine phosphorylation of JAK2, production of ROS, and proliferation activities when compared with VSMC cultured in normal glucose (5.5 mm (NG)). Both the aldose reductase specific inhibitor (zopolrestat) or transfection with aldose reductase antisense oligonucleotide blocked the phosphorylation of JAK2, the production of ROS, and proliferation of VSMC induced by HG, but it had no effect on the Ang II-induced activation of these parameters in both NG and HG. However, transfection with PKC-beta antisense oligonucleotide, preincubation with a PKC-beta-specific inhibitor (LY379196) or apocynin (NADPH oxidase-specific inhibitor), or electroporation of NADPH oxidase antibodies blocked the Ang II-induced JAK2 phosphorylation, production of ROS, and proliferation of VSMC in both NG and HG. These observations suggest that the polyol pathway hyperactivity induced by HG contributes to the development of diabetic macroangiopathy through a PKC-beta-ROS activation of JAK2.     

Levuglandinyl adducts of proteins are formed via a prostaglandin H2 synthase-dependent pathway after platelet activation

The product of oxygenation of arachidonic acid by the prostaglandin H synthases (PGHS), prostaglandin H(2) (PGH(2)), undergoes rearrangement to the highly reactive gamma-ketoaldehydes, levuglandin (LG) E(2), and LGD(2). We have demonstrated previously that LGE(2) reacts with the epsilon-amine of lysine to form both the levuglandinyl-lysine Schiff base and the pyrrole-derived levuglandinyl-lysine lactam adducts. We also have reported that these levuglandinyl-lysine adducts are formed on purified PGHSs following the oxygenation of arachidonic acid. We now present evidence that the levuglandinyl-lysine lactam adduct is formed in human platelets upon activation with exogenous arachidonic acid or thrombin. After proteolytic digestion of the platelet proteins, and isolation of the adducted amino acid residues, this adduct was identified by liquid chromatography-tandem mass spectrometry. We also demonstrate that formation of these adducts is inhibited by indomethacin, a PGHS inhibitor, and is enhanced by an inhibitor of thromboxane synthase. These data establish that levuglandinyl-lysine adducts are formed via a PGHS-dependent pathway in whole cells, even in the presence of an enzyme that metabolizes PGH(2). They also demonstrate that a physiological stimulus is sufficient to lead to the lipid modification of proteins through the levuglandin pathway in human platelets.     

The spatial organization of apolipoprotein A-I on the edge of discoidal high density lipoprotein particles: a mass specrometry study

The three-dimensional structure of human apoA-I on nascent, discoidal HDL particles has been debated extensively over the past 25 years. Recent evidence has demonstrated that the alpha-helical domains of apoA-I are arranged in a belt-like orientation with the long axis of the helices perpendicular to the phospholipid acyl chains on the disc edge. However, experimental information on the spatial relationships between apoA-I molecules on the disc is lacking. To address this issue, we have taken advantage of recent advances in mass spectrometry technology combined with cleavable cross-linking chemistry to derive a set of distance constraints suitable for testing apoA-I structural models. We generated highly homogeneous, reconstituted HDL particles containing two molecules of apoA-I. These were treated with a thiol-cleavable cross-linking agent, which covalently joined Lys residues in close proximity within or between molecules of apoA-I in the disc. The cross-linked discs were then exhaustively trypsinized to generate a discrete population of peptides. The resulting peptides were analyzed by liquid chromatography/mass spectrometry before and after cleavage of the cross-links, and resulting peaks were identified based on the theoretical tryptic cleavage of apoA-I. We identified at least 8 intramolecular and 7 intermolecular cross-links in the particle. The distance constraints are used to analyze three current models of apoA-I structure. The results strongly support the presence of the salt-bridge interactions that were predicted to occur in the "double belt" model of apoA-I, but a helical hairpin model containing the same salt-bridge docking interface is also consistent with the data.     

Nitric-oxide synthase (NOS) reductase domain models suggest a new control element in endothelial NOS that attenuates calmodulin-dependent activity

Inducible (iNOS) and constitutive (eNOS, nNOS) nitric-oxide synthases differ in their Ca2+-calmodulin (CaM) dependence. iNOS binds CaM irreversibly but eNOS and nNOS, which bind CaM reversibly, have inserts in their reductase domains that regulate electron transfer. These include the 43-45-amino acid autoinhibitory element (AI) that attenuates electron transfer in the absence of CaM, and the C-terminal 20-40-amino acid tail that attenuates electron transfer in a CaM-independent manner. We constructed models of the reductase domains of the three NOS isoforms to predict the structural basis for CaM-dependent regulation. We have identified and characterized a loop (CD2A) within the NOS connecting domain that is highly conserved by isoform and that, like the AI element, is within direct interaction distance of the CaM binding region. The eNOS CD2A loop (eCD2A) has the sequence 834KGSPGGPPPG843, and is truncated to 809ESGSY813 (iCD2A) in iNOS. The eCD2A contributes to the Ca2+ dependence of CaM-bound activity to a level similar to that of the AI element. The eCD2A plays an autoinhibitory role in the control of NO, and CaM-dependent and -independent reductase activity, but this autoinhibitory function is masked by the dominant AI element. Finally, the iCD2A is involved in determining the salt dependence of NO activity at a post-flavin reduction level. Electrostatic interactions between the CD2A loop and the CaM-binding region, and CaM itself, provide a structural means for the CD2A to mediate CaM regulation of intra-subunit electron transfer within the active NOS complex.     

Endoplasmic reticulum stress induces hyaluronan deposition and leukocyte adhesion

There is mounting evidence that perturbations in endoplasmic reticulum (ER) function play a key role in the pathogenesis of a broad range of diseases. We have examined the ability of ER stress to modulate leukocyte binding to colonic and aortic smooth muscle cells. In vitro, control smooth muscle cells bind few leukocytes, but treatment with compounds that induce ER stress, including tunicamycin, A23187, and thapsigargin, promotes leukocyte binding. Likewise, dextran sulfate, another agent capable of inducing ER stress and promoting inflammation in vivo, strongly induces leukocyte adhesion. The bound leukocytes are released by hyaluronidase treatment, indicating a critical role for hyaluronan-containing structures in mediating leukocyte binding. Affinity histochemistry demonstrated that hyaluronan accumulates and is present in cable-like structures in the treated, but not the untreated, cultures and that these structures serve as attachment sites for leukocytes. Hyaluronan-rich regions of both murine and human inflamed colon contain numerous cells that stain intensely for ER-resident chaperones containing the KDEL sequence, demonstrating a relationship between ER stress and hyaluronan deposition in vivo. These results indicate that ER stress may contribute to chronic inflammation by forming a hyaluronan-rich extracellular matrix that is conducive to leukocyte binding.