Apolipoprotein J/Clusterin is a novel structural component of human erythrocytes and a biomarker of cellular stress and senescence

Background

Secretory Apolipoprotein J/Clusterin (sCLU) is a ubiquitously expressed chaperone that has been functionally implicated in several pathological conditions of increased oxidative injury, including aging. Nevertheless, the biological role of sCLU in red blood cells (RBCs) remained largely unknown. In the current study we identified sCLU as a component of human RBCs and we undertook a detailed analysis of its cellular topology. Moreover, we studied the erythrocytic membrane sCLU content during organismal aging, in conditions of increased organismal stress and accelerated RBCs senescence, as well as during physiological in vivo cellular senescence.

Methodology/Principal Findings

By using a combination of molecular, biochemical and high resolution microscopical methods we found that sCLU is a novel structural component of RBCs extra- and intracellular plasma membrane and cytosol. We observed that the RBCs membrane-associated sCLU decreases during organismal aging or exposure to acute stress (e.g. smoking), in patients with congenital hemolytic anemia, as well as during RBCs in vivo senescence. In all cases, sCLU reduction paralleled the expression of typical cellular senescence, redox imbalance and erythrophagocytosis markers which are also indicative of the senescence- and oxidative stress-mediated RBCs membrane vesiculation.

Conclusions/Significance

We propose that sCLU at the mature RBCs is not a silent remnant of the erythroid precursors, but an active component being functionally implicated in the signalling mechanisms of cellular senescence and oxidative stress-responses in both healthy and diseased organism. The reduced sCLU protein levels in the RBCs membrane following cell exposure to various endogenous or exogenous stressors closely correlates to the levels of cellular senescence and redox imbalance markers, suggesting the usefulness of sCLU as a sensitive biomarker of senescence and cellular stress.     

Interactions of phosphorus-containing dendrimers with liposomes

The influence of cationic phosphorus-containing dendrimers generation 3 and 4 on model DMPC or DPPC lipid membranes was studied. Measurements of fluorescence anisotropy and differential scanning calorimetry (DSC) were applied to assess changes in lipid bilayer parameters, including fluidity, anisotropy, and phase-transition temperature. Interaction with both hydrophobic and hydrophilic regions of the bilayer was followed by these methods. Dendrimers of both generations influence lipid bilayers by decreasing membrane fluidity. The results suggest that dendrimers can interact both with the hydrophobic part and the polar head-group region of the phospholipid bilayer. Higher generation dendrimers interact more strongly with model membranes, and the concentration, as well as the generation, is of similar importance.     

Sialyl Lewis-dependent binding of human monocyte-derived dendritic cells to selectins

The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewis^x (sLe^x) in selectin-dependent mo-DC binding. Our data reveal that sLe^x is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLe^x-dependent binding of mo-DC to selectins was further substantiated by using sLe^x free sugar and anti-sLe^x antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLe^x expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.     

Sialofucosylated podocalyxin is a functional E- and L-selectin ligand expressed by metastatic pancreatic cancer cells

Selectin-mediated interactions in the vasculature promote metastatic spread by facilitating circulating tumor cell binding to selectin-expressing host cells. Therefore, identifying the selectin ligand(s) on tumor cells is critical to the prevention of blood-borne metastasis. A current challenge is to distinguish between structures expressed by circulating tumor cells that can bind selectins in vitro from the functional ligands whose depletion suppresses selectin-dependent binding under flow in vivo. Interestingly, podocalyxin (PODXL), which can bind E- and L-selectin, is upregulated in a number of cancers, including those of the breast, colon, and pancreas. In this work, we show that metastatic pancreatic cancer cells overexpress PODXL compared with nonmalignant pancreatic epithelial cells. We further demonstrate via tissue microarray that 69% of pancreatic ductal adenocarcinomas stain positive for PODXL. In cases of focal expression, positive staining is restricted to the invasive front of primary tumors. By combining immunoblot, immunodepletion, short-hairpin RNA-mediated gene silencing, and flow-based adhesion assays, we evaluated the functional role of sialofucosylated PODXL in selectin-mediated adhesion under flow. Our data indicate that sialofucosylated PODXL is a functional E- and L-selectin ligand expressed by metastatic pancreatic cancer cells, as specific depletion of this molecule from the cell surface significantly interferes with selectin-dependent interactions. Cumulatively, these data support a correlation between sialofucosylated PODXL expression and enhanced binding to selectins by metastatic pancreatic cancer cells and offer additional perspective on the upregulation of PODXL in aggressive cancers.     

Novel diamino imidazole and pyrrole-containing polyamides: Synthesis and DNA binding studies of mono- and diamino-phenyl-ImPy*Im polyamides designed to target 5'-ACGCGT-3'

Pyrrole- and imidazole-containing polyamides are widely investigated as DNA sequence selective binding agents that have potential use as gene control agents. The key challenges that must be overcome to realize this goal is the development of polyamides with low molar mass so the molecules can readily diffuse into cells and concentrate in the nucleus. In addition, the molecules must have appreciable water solubility, bind DNA sequence specifically, and with high affinity. It is on this basis that the orthogonally positioned diamino/dicationic polyamide Ph-ImPy*Im 5 was designed to target the sequence 5'-ACGCGT-3'. Py* denotes the pyrrole unit that contains a N-substituted aminopropyl pendant group. The DNA binding properties of diamino polyamide 5 were determined using a number of techniques including CD, ΔT(M), DNase I footprinting, SPR and ITC studies. The effects of the second amino moiety in Py* on DNA binding affinity over its monoamino counterpart Ph-ImPyIm 3 were assessed by conducting DNA binding studies of 3 in parallel with 5. The results confirmed the minor groove binding and selectivity of both polyamides for the cognate sequence 5'-ACGCGT-3'. The diamino/dicationic polyamide 5 showed enhanced binding affinity and higher solubility in aqueous media over its monoamino/monocationic counterpart Ph-ImPyIm 3. The binding constant of 5, determined from SPR studies, was found to be 1.5 × 10(7)M(-1), which is ～3 times higher than that for its monoamino analog 3 (4.8 × 10(6)M(-1)). The affinity of 5 is now approaching that of the parent compound f-ImPyIm 1 and its diamino equivalent 4. The advantages of the design of diamino polyamide 5 over 1 and 4 are its sequence specificity and the ease of synthesis compared to the N-terminus pyrrole analog 2.