Effect of apoA-I Mutations in the Capacity of Reconstituted HDL to Promote ABCG1-Mediated Cholesterol Efflux

ATP binding cassette transporter G1 (ABCG1) mediates the cholesterol transport from cells to high-density lipoprotein (HDL), but the role of apolipoprotein A-I (apoA-I), the main protein constituent of HDL, in this process is not clear. To address this, we measured cholesterol efflux from HEK293 cells or J774 mouse macrophages overexpressing ABCG1 using as acceptors reconstituted HDL (rHDL) containing wild-type or various mutant apoA-I forms. It was found that ABCG1-mediated cholesterol efflux was severely reduced (by 89%) when using rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)]. ABCG1-mediated cholesterol efflux was not affected or moderately decreased by rHDL containing amino-terminal deletion mutants and several mid-region deletion or point apoA-I mutants, and was restored to 69–99% of control by double deletion mutants apoA-I[Δ(1–41)Δ(185–243)] and apoA-I[Δ(1–59)Δ(185–243)]. These findings suggest that the central helices alone of apoA-I associated to rHDL can promote ABCG1-mediated cholesterol efflux. Further analysis showed that rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)] only slightly reduced (by 22%) the ABCG1-mediated efflux of 7-ketocholesterol, indicating that depending on the sterol type, structural changes in rHDL-associated apoA-I affect differently the ABCG1-mediated efflux of cholesterol and 7-ketocholesterol. Overall, our findings demonstrate that rHDL-associated apoA-I structural changes affect the capacity of rHDL to accept cellular cholesterol by an ABCG1-mediated process. The structure-function relationship seen here between rHDL-associated apoA-I mutants and ABCG1-mediated cholesterol efflux closely resembles that seen before in lipid-free apoA-I mutants and ABCA1-dependent cholesterol efflux, suggesting that both processes depend on the same structural determinants of apoA-I.     

Hemodynamic Impact of Absent or Reverse End-Diastolic Flow in the Two Umbilical Arteries in Growth-Restricted Fetuses

Objective

To determine if bilateral absent or reverse end-diastolic (ARED) flow in the two umbilical arteries (UAs) at the perivesical (PVC) segment represents a more severe degree of hemodynamic compromise than unilateral ARED flow at the PVC segment in singleton pregnancies complicated by intrauterine growth restriction (IUGR).

Methods

This was a prospective observational study. One hundred nine fetuses with IUGR underwent a total of 225 ultrasound (US) examinations. We measured the pulsatility index (PI) from the two UAs at the PVC segment, UA in the free floating cord (FFC), middle cerebral artery (MCA), ductus venosus (DV) and the aortic isthmus blood flow index (IFI). Three groups were classified according to bilateral positive end-diastolic (PED) flow, unilateral ARED flow or bilateral ARED flow in the UAs at the PVC segment.

Results

The proportions of US examinations with PED flow, unilateral ARED flow and bilateral ARED flow in the UAs were 54.7%, 20.4%, and 24.9%, respectively. At the last US examination, the IFI z-scores were significantly lower in the bilateral ARED group (-6.28±4.30) compared to the unilateral ARED group (-1.72±3.18, p<0.05) and the bilateral PED group (-0.83±2.36, p<0.05), the DV-PI z-scores were significantly higher in the bilateral ARED group (2.15±3.79) compared to the bilateral PED group (0.64±1.50, p<0.05). Before 32 weeks of gestation, the interval between US examination and delivery was significantly shorter in the bilateral ARED group (8.9 days ±8.2) than the unilateral ARED group (15.9 days ±13.4, p<0.05) and the bilateral PED group (30.3 days±25.7, p<0.05).

Conclusion

There are significant differences in fetal blood fluxes between left and right UA. Doppler examination at the PVC segment significantly improves the comparability of UA-PI between two successive US examinations and allows a longitudinal and independent hemodynamic investigation of each UA. Examination of a single UA in free floating cord may miss a large fraction of unilateral ARED flow. In singleton IUGR fetuses, a bilateral ARED flow in the UAs at the PVC segment indicates more severe hemodynamic compromise and worse fetal conditions than unilateral ARED flow.     

Interaction of copper(II) with the non-steroidal anti-inflammatory drugs naproxen and diclofenac: synthesis, structure, DNA- and albumin-binding

Copper(II) complexes with the non-steroidal anti-inflammatory drugs (NSAIDs) naproxen and diclofenac have been synthesized and characterized in the presence of nitrogen donor heterocyclic ligands (2,2′-bipyridine, 1,10-phenanthroline or pyridine). Naproxen and diclofenac act as deprotonated ligands coordinated to Cu(II) ion through carboxylato oxygens. The crystal structures of (2,2′-bipyridine)bis(naproxenato)copper(II), , (1,10-phenanthroline)bis(naproxenato)copper(II), and bis(pyridine)bis(diclofenac)copper(II), have been determined by X-ray crystallography. The UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that the complexes can bind to CT DNA with (2,2′-bipyridine)bis(naproxenato)copper(II) exhibiting the highest binding constant to CT DNA. Competitive study with ethidium bromide (EB) indicates that the complexes can displace the DNA-bound EB suggesting strong competition with EB. The cyclic voltammograms of the complexes recorded in the presence of CT DNA have shown that the complexes can bind to CT DNA by the intercalative binding mode which has also been verified by DNA solution viscosity measurements. The NSAID ligands and their complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. The biological properties of the previously reported complexes [Cu₂(naproxenato)₄(H₂O)₂], [Cu₂(diclofenac)₄(H₂O)₂] and [Cu(naproxenato)₂(pyridine)₂(H₂O)] have been also evaluated. The dinuclear complexes exhibit similar affinity for CT DNA as the 2,2′-bipyridine or 1,10-phenanthroline containing complexes. The pyridine containing complexes exhibit the lowest affinity for CT DNA and the lowest ability to displace EB from its EB-DNA complex.     

Molecular etiology of a dominant form of type III hyperlipoproteinemia caused by R142C substitution in apoE4

We have used adenovirus-mediated gene transfer in apolipoprotein (apo)E^−/− mice to elucidate the molecular etiology of a dominant form of type III hyperlipoproteinemia (HLP) caused by the R142C substitution in apoE4. It was found that low doses of adenovirus expressing apoE4 cleared cholesterol, whereas comparable doses of apoE4[R142C] greatly increased plasma cholesterol, triglyceride, and apoE levels, caused accumulation of apoE in VLDL/IDL/LDL region, and promoted the formation of discoidal HDL. Co-expression of apoE4[R142C] with lecithin cholesterol acyltransferase (LCAT) or lipoprotein lipase (LPL) in apoE^−/− mice partially corrected the apoE4[R142C]-induced dyslipidemia. High doses of C-terminally truncated apoE4[R142C]-202 partially cleared cholesterol in apoE^−/− mice and promoted formation of discoidal HDL. The findings establish that apoE4[R142C] causes accumulation of apoE in VLDL/IDL/LDL region and affects in vivo the activity of LCAT and LPL, the maturation of HDL, and the clearance of triglyceride-rich lipoproteins. The prevention of apoE4[R142C]-induced dyslipidemia by deletion of the 203-299 residues suggests that, in the full-length protein, the R142C substitution may have altered the conformation of apoE bound to VLDL/IDL/LDL in ways that prevent triglyceride hydrolysis, cholesterol esterification, and receptor-mediated clearance in vivo.     

Expression of the blood-group-related antigens Sialyl Lewis a,Sialyl Lewis x and Lewis y in term placentas of normal,preeclampsia, IUGR- and HELLP-complicated pregnancies

Lewis antigens belong to the blood group of antigens and mediate cellular adhesion through interaction with selectins. Invasive trophoblasts use an array of adhesion molecules to facilitate cell–cell and cell–extracellular matrix interactions. Here, we examined immunohistochemically the expression of Sialyl Lewis a (sLe^a), Sialyl Lewis x (sLe^x) and Lewis y (Le^y) in term placentas obtained from cases of normal, intrauterine growth retardation (IUGR), preeclamptic (PE) and hemolysis, elevated liver enzymes and low platelets syndrome (HELLP) pregnancies. We report the expression of sLe^x in third trimester extravillous trophoblasts (EVT). sLe^x was significantly decreased in IUGR and moderately decreased in PE compared to normal placentas. sLe^x was additionally found in syncytiotrophoblast, without however any significant differences in staining intensity between normal and pathological cases. sLe^a was restricted to amnion epithelium. Finally, Le^y was expressed in cytotrophoblasts and villous endothelial cells. Le^y expression was significantly upregulated in IUGR and HELLP, whereas there was a trend toward increase in PE compared to normal placentas. The present study suggests that downregulation of sLe^x in EVT might be associated with IUGR and PE. Furthermore, Le^y, which was recently described as a potent angiogenic factor, is upregulated in placental villi in conditions associated with placental malperfusion. U. Jeschke and A. Makrigiannakis have contributed equally.     

Activation of FAK/PI3K/Rac1 signaling controls actin reorganization and inhibits cell motility in human cancer cells.

We have recently identified a specific signaling pathway that regulates actin reorganization in malignant human breast and prostate epithelial cells associated with FAK, PI-3K and Rac1 activation. Here we report that this pathway operates in MCF7 cells upon activation of membrane androgen receptors (mAR). Stimulation of mAR by the non-permeable testosterone-BSA conjugate resulted in early actin reorganization documented by quantitative measurements of actin dynamics and morphological analysis of microfilament organization. This effect was regulated by early phosphorylation of FAK and subsequent PI-3K and Rac1 activation. The functional role of this pathway was further shown in A375 melanoma cells. Treatment with the opioid antagonist alpha(s1) casomorphin resulted in rapid and potent actin remodeling in A375 cells, regulated by rapid activation of the FAK/PI-3K/Rac1 signaling. Pretreatment of both cell lines with the specific PI-3K inhibitor wortmannin blocked actin reorganization. Interestingly, wound healing assays revealed that testosterone-BSA and alpha (s1) casomorphin significantly inhibited MCF7 and A375 cell motility respectively. These effects were abrogated through blockade of PI-3K signaling by wortmannin. The results presented here indicate that actin reorganization through FAK/PI3-K/Rac-1 activation operates in various human cancer cell systems supporting a functional role for FAK/PI-3K/Rac1/actin signaling in controlling cell motility.     

In Vitro Digestion of caseinate and Tween 20 Emulsions

Food Biophysics - In vitro digestion experiments are set-up with emulsions stabilized either with sodium caseinate and/or Tween 20, in the presence of mucin in the simulated GI fluids. The progress...     

A patient with partial trisomy 21 and 7q deletion expresses mild Down syndrome phenotype

Backround

Down syndrome (DS) is the most common aneuploidy in live-born individuals and it is well recognized with various phenotypic expressions. Although an extra chromosome 21 is the genetic cause for DS, specific phenotypic features may result from the duplication of smaller regions of the chromosome and more studies need to define genotypic and phenotypic correlations.

Case report

We report on a 26 year old male with partial trisomy 21 presenting mild clinical symptoms relative to DS including borderline intellectual disability. In particular, the face and the presence of hypotonia and keratoconus were suggestive for the DS although the condition remained unnoticed until his adult age array comparative genomic hybridization (aCGH) revealed a 10.1 Mb duplication in 21q22.13q22.3 and a small deletion of 2.2 Mb on chromosomal band 7q36 arising from a paternal translocation t(7;21). The 21q duplication encompasses the gene DYRK1.

Conclusion

Our data support the evidence of specific regions on distal 21q whose duplication results in phenotypes recalling the typical DS face. Although the duplication region contains DYRK1, which has previously been implicated in the causation of DS, our patient has a borderline IQ confirming that their duplication is not sufficient to cause the full DS phenotype.