Molecular dynamic simulations of Co(III) and Ru(II) polypyridyl complexes and docking studies with dsDNA

The binding studies of Co(III) and Ru(II) polypyridyl complexes with dsDNA were carried out by molecular modeling studies to identify the binding interactions. The 3D structures of the metal complexes [Ru(phen)₂ippip]²⁺ (RP-ippip), [Co(phen)₂ippip]³⁺ (CP-ippip), [Ru(bpy)₂ippip]²⁺ (RB-ippip), and [Co(bpy)₂ippip]³⁺ (CB-ippip), where ippip = 4-(isopropylbenzaldehyde)imidazo[4,5-f][1,10] phenanthroline, phen = 1,10-phenanthroline, and bpy = bypyridine, were simulated using molecular dynamic simulations for stable conformers. The energy-minimized 3D structures of metal complexes were docked to the double-stranded dodecamer 5′-D(*AP * CP * CP * GP * AP * CP * GP * TP * CP * GP * GP * T)-3′. The aromatic ligand, ippip, facilitates the binding of the metal complex with DNA through intercalation. The effect of ancillary ligands, phen and bpy, was investigated. The ancillary ligands were found to be involved in bond formation with the phosphate backbone of nucleotide base pairs in metal complex–DNA docked complex. The significant interactions of metal complexes in the major groove of DNA are the prerequisite features of the metal complexes to be considered as DNA-intercalator. The molecular docking data are well substantiated by the available experimental data. The modeling results should extend knowledge about the nature of binding of these complexes with DNA.     

Red blood cells induce hypoxic lung inflammation

Hypoxia, which commonly associates with respiratory and cardiovascular diseases, provokes an acute inflammatory response. However, underlying mechanisms are not well understood. Here we report that red blood cells (RBCs) induce hypoxic inflammation by producing reactive oxygen species (ROS) that diffuse to endothelial cells of adjoining blood vessels. Real-time fluorescence imaging of rat and mouse lungs revealed that in the presence of RBC-containing vascular perfusion, hypoxia increased microvascular ROS, and cytosolic Ca²⁺, leading to P-selectin–dependent leukocyte recruitment. However, in the presence of RBC-free perfusion, all hypoxia-induced responses were completely inhibited. Because hemoglobin (Hb) autoxidation causes RBC superoxide formation that readily dismutates to H₂O₂, hypoxia-induced responses were lost when we inhibited Hb autoxidation with CO or nitrite, or when the H₂O₂ inhibitor, catalase was added to the infusion to neutralize the RBC-derived ROS. By contrast, perfusion with RBCs from BERK-trait mice that are more susceptible to Hb autoxidation and to hypoxia-induced superoxide production enhanced the hypoxia-induced responses. We conclude that in hypoxia, increased Hb autoxidation augments superoxide production in RBCs. Consequently, RBCs release H₂O₂ that diffuses to the lung microvascular endothelium, thereby initiating Ca²⁺-dependent leukocyte recruitment. These findings are the first evidence that RBCs contribute to hypoxia-induced inflammation.      

Red cell interactions with amyloid-beta(1-40) fibrils in a murine model

Vascular amyloidosis in Alzheimer's disease (AD) results in the exposure of red blood cells to beta-amyloid fibrils (A beta). The potential in vivo ramifications of this exposure have been investigated by injecting A beta(1-40) alone or A beta-bound mouse red blood cells into the circulation of C57BL/6 mice. Results indicate that when A beta(1-40) is injected alone, a transient uptake of the fibrils by red blood cells occurs in vivo. When A beta-bound red blood cells were injected, beta-amyloid is rapidly removed from these cells in vivo. Double-labeling experiments indicate that while some of the red blood cells bound to A beta(1-40) are removed from circulation, a major fraction of these cells remain in circulation even after A beta is removed. Immunohistochemistry of murine tissue samples obtained after sacrificing the animals suggests that within 1 h after injection of A beta(1-40) or A beta-bound red blood cells, A beta is found in spleen phagocytes and liver Kupffer cells. Heme staining further indicates that the binding of A beta(1-40) to red blood cells enhances red cell phagocytosis by the spleen.     

Inhibitory effect of eugenol on non-enzymatic lipid peroxidation in rat liver mitochondria.

The anti-peroxidative activity of eugenol on Fe(2+)-ascorbate- and Fe(2+)-H2O2-induced lipid peroxidation was studied using rat liver mitochondria. Eugenol inhibited thiobarbituric acid reactive substance (TBARS) formation induced by both the systems in addition to oxygen uptake and mitochondrial swelling induced by Fe(2+)-ascorbate. Time course studies on TBARS formation indicated the ability of eugenol to inhibit initiation and propagation reactions. There was no measurable chemical modification of eugenol during the course of mitochondrial peroxidation by both the systems. Mitochondrial peroxidation by Fe(2+)-H2O2 was inhibited by hydroxyl radical (OH) scavengers like mannitol, benzoate, formate and dimethyl sulfoxide apart from eugenol. The OH scavenging ability of eugenol was evident from its inhibitory effect on OH-mediated deoxyribose degradation. The second-order rate constant for the reaction of OH with eugenol was about 4.8 x 10(10) M-1 sec-1. Eugenol reduced Fe3+ ions and Fe3+ chelated to citrate or ADP but it did not exhibit pro-oxidant activity in OH-mediated deoxyribose degradation. Incubation of mitochondria with eugenol resulted in the uptake of small but significant quantities of eugenol which inhibited subsequent lipid peroxidation by acting as a chain breaking antioxidant.     

Redox reactions of hemoglobin

Redox reactions of hemoglobin have gained importance because of the general interest of the role of oxidative stress in diseases and the possible role of red blood cells in oxidative stress. Although electron paramagnetic resonance (EPR) is extremely valuable in studying hemoglobin redox reactions it has not been adequately used. We have focused in this review on the important contributions of EPR to our understanding of hemoglobin redox reactions. We have limited our discussion to the redox reactions thought to occur under physiological conditions. This includes autoxidation as well as the reactions of hydrogen peroxide generated by superoxide dismutation. We have also discussed redox reactions associated with nitric oxide produced in the circulation. We have pinpointed the value of using EPR to detect and study the paramagnetic species and free radicals formed during these reactions. We have shown how EPR not only identifies the paramagnetic species formed but can also be used to provide insights into the mechanism involved in the redox reactions.     

SlmA forms a higher-order structure on DNA that inhibits cytokinetic Z-ring formation over the nucleoid

The spatial and temporal control of Filamenting temperature sensitive mutant Z (FtsZ) Z-ring formation is crucial for proper cell division in bacteria. In Escherichia coli, the synthetic lethal with a defective Min system (SlmA) protein helps mediate nucleoid occlusion, which prevents chromosome fragmentation by binding FtsZ and inhibiting Z-ring formation over the nucleoid. However, to perform its function, SlmA must be bound to the nucleoid. To deduce the basis for this chromosomal requirement, we performed biochemical, cellular, and structural studies. Strikingly, structures show that SlmA dramatically distorts DNA, allowing it to bind as an orientated dimer-of-dimers. Biochemical data indicate that SlmA dimer-of-dimers can spread along the DNA. Combined structural and biochemical data suggest that this DNA-activated SlmA oligomerization would prevent FtsZ protofilament propagation and bundling. Bioinformatic analyses localize SlmA DNA sites near membrane-tethered chromosomal regions, and cellular studies show that SlmA inhibits FtsZ reservoirs from forming membrane-tethered Z rings. Thus, our combined data indicate that SlmA DNA helps block Z-ring formation over chromosomal DNA by forming higher-order protein-nucleic acid complexes that disable FtsZ filaments from coalescing into proper structures needed for Z-ring creation.Accurate cell division demands a tight synchronization of chromosome replication, segregation, and septum formation. In bacteria, cell division is mediated by the tubulin-like protein, FtsZ (1–5). FtsZ self-assembles into linear protofilaments (pfs) in a GTP-dependent manner by the interaction of the plus end of one subunit with the minus end of another subunit. These pfs combine to form a septal ring-like structure called the Z ring, which drives cell division (1). The precise organization of FtsZ filaments in the Z ring has not been resolved. However, recent cryo-electron microscopy (EM) tomography and superresolution imaging data suggest that lateral contacts between FtsZ molecules in the Z ring are loosely arranged (6–9). Notably, the intracellular levels of FtsZ remain unchanged during the cell cycle and exceed the critical concentration required for Z-ring formation (3). Hence, Z-ring assembly is affected by a diverse repertoire of FtsZ binding proteins that ensure that the ring forms at the correct time and place during cell division (2, 4, 10). Two key regulatory systems, the Min and nucleoid occlusion (NO) systems, ensure that Z rings do not form at cell poles and over chromosomal DNA (11–13). In Escherichia coli, the Min system creates a gradient of FtsZ polymer inhibition at the cell poles (14). In contrast to the pole proximal effect of the Min system, NO prevents Z rings from forming over chromosomal DNA (15).Although the effects of NO are well known, only in recent years have effectors of this process been identified in both gram-negative and gram-positive model organisms. Surprisingly, the gram-positive model organism, Bacillus subtilis, uses a ParB-like protein called Noc, whereas in E. coli the TetR-family member, SlmA, has been shown to be involved in NO (13, 16–18). Recent studies showed that SlmA binds directly to FtsZ and that this interaction antagonizes Z-ring formation when SlmA is bound to specific DNA sites (16, 17). These DNA sequences, called SlmA binding sites (SBSs), are evenly distributed on the chromosome, with the notable exception of the Ter chromosomal region, which is the last to partition. Similar to SlmA, the unrelated Noc protein also binds specific DNA sites that are distributed in all chromosomal regions but the Ter containing domain (18, 19). Thus, NO factors coordinate DNA segregation and cell division.How SlmA prevents Z-ring formation by FtsZ is unclear. However, a key finding was that this inhibition necessitates that SlmA be bound to its specific DNA sites (16, 17). In fact, SlmA alone does not affect FtsZ filament formation, making SlmA the only known FtsZ assembly/disassembly factor that requires DNA for its function. To determine the molecular basis for the specific chromosomal DNA requirement for SlmA’s NO function, we performed structural, bioinformatic, cellular, and biochemical studies. Strikingly, these data show that SlmA interacts with specific SBS sites to form a higher-order structure that can spread on the DNA. Combined with biochemical and bioinformatics analyses, these findings suggest a molecular model for how SlmA participates in NO in gram-negative bacteria.     

Influence of beta-amyloid fibrils on the interactions between red blood cells and endothelial cells

Alzheimer's disease is associated with vascular amyloidosis. As blood flows through the microcirculation, red blood cells (RBCs) come in contact with the vasculature. RBCs as well as endothelial cells (ECs) are known to bind beta amyloid fibrils. This suggests that a potential effect of amyloidosis may involve the interactions of RBCs with ECs lining the wall of the blood vessels mediated by amyloid fibrils. We have studied the effect of beta-amyloid peptide[1-40] (Abeta1-40) fibrils on the interactions of murine RBCs with ECs derived from bovine lung microvascular endothelium (BLMVEC) as well as bovine pulmonary arterial endothelium (BPAEC) in culture. We show that the initial incorporation of Abeta fibrils onto either RBCs or ECs cause RBCs to adhere to the ECs with greater affinity for the microvascular cells than the arterial cells. In addition, there is a transfer of Abeta fibrils between the RBCs and the ECs. Both the transfer and adhesion occurs when the amyloid fibrils are on the ECs or on the RBCs. However, with the amyloid fibrils on the RBCs, the adhesion and the transfer are greater than with the fibrils on the ECs. These results suggest that amyloidosis may affect the flow of RBCs through the microcirculation and that RBCs may play a role in propagating amyloidosis through the vasculature.