Deactivation of the lipopolysaccharide antagonist eritoran (E5564) by high-density lipoprotein-associated apolipoproteins

Lipid A, the active moiety of LPS, exerts its effects through interaction with TLR4, triggering a signalling cascade that results in the release of pro-inflammatory cytokines. Eritoran is a lipid A analogue that competes with LPS for binding to TLR4; however, after intravenous administration, it undergoes a time-dependent deactivation as a consequence of binding to high-density lipoproteins (HDLs). The site of eritoran association with HDL remains unknown. Therefore the aim of this study was to determine if HDL-associated apolipoproteins A1, A2, serum amyloid A (SAA) and C1, inhibit the ability of eritoran to block LPS-induced TNF-α release from whole blood. Eritoran activity after LPS stimulation in human whole blood was assessed in the presence of reconstituted HDL (rHDL) containing different apos. In rHDL, the major apolipoproteins in both the healthy and septic state, A1 and SAA, caused a significant reduction in eritoran antagonistic activity and had a greater effect than minor apolipoproteins A2 and C1. Apolipoproteins associated with HDL are likely to facilitate eritoran deactivation. Apolipoproteins A1 and SAA should be of particular focus as they are the major apos found on HDL in both the healthy and septic state. Further evaluation of the physical association between apolipoproteins and eritoran should be explored.     

Unusual role of a cysteine residue in substrate binding and activity of human AP-endonuclease 1

The mammalian AP-endonuclease (APE1) repairs apurinic/apyrimidinic (AP) sites and strand breaks with 3′ blocks in the genome that are formed both endogenously and as intermediates during base excision repair. APE1 has an unrelated activity as a redox activator (and named Ref-1) for several trans-acting factors. In order to identify whether any of the seven cysteine residues in human APE1 affects its enzymatic function, we substituted these singly or multiply with serine. The repair activity is not affected in any of the mutants except those with C99S mutation. The Ser99-containing mutant lost affinity for DNA and its activity was inhibited by 10 mM Mg²⁺. However, the Ser99 mutant has normal activity in 2 mM Mg²⁺. Using crystallographic data and molecular dynamics simulation, we have provided a mechanistic basis for the altered properties of the C99S mutant. We earlier predicted that Mg²⁺, with potential binding sites A and B, binds at the B site of wild-type APE1-substrate complex and moves to the A site after cleavage occurs, as observed in the crystal structure. The APE1-substrate complex is stabilized by a H bond between His309 and the AP site. We now show that this bond is broken to destabilize the complex in the absence of the Mg²⁺. This effect due to the mutation of Cys99, ∼ 16 Å from the active site, on the DNA binding and activity is surprising. Mg²⁺ at the B site promotes stabilization of the C99S mutant complex. At higher Mg²⁺ concentration the A site is also filled, causing the B-site Mg²⁺ to shift together with the AP site. At the same time, the H bond between His309 and the AP site shifts toward the 5′ site of DNA. These shifts could explain the lower activity of the C99S mutant at higher [Mg²⁺]. The unexpected involvement of Cys99 in APE1's substrate binding and catalysis provides an example of involvement of a residue far from the active site.     

Submembraneous microtubule cytoskeleton: biochemical and functional interplay of TRP channels with the cytoskeleton

Much work has focused on the electrophysiological properties of transient receptor potential channels. Recently, a novel aspect of importance emerged: the interplay of transient receptor potential channels with the cytoskeleton. Recent data suggest a direct interaction and functional repercussion for both binding partners. The bi-directionality of physical and functional interaction renders therefore, the cytoskeleton a potent integration point of complex biological signalling events, from both the cytoplasm and the extracellular space. In this minireview, we focus mostly on the interaction of the cytoskeleton with transient receptor potential vanilloid channels. Thereby, we point out the functional importance of cytoskeleton components both as modulator and as modulated downstream effector. The resulting implications for patho-biological situations are discussed.     

New functionalized 1,2,4-trioxepanes: synthesis and antimalarial activity against multi-drug resistant P. yoelii in mice

A series of new amino functionalized 1,2,4-trioxepanes 8-16 and ester functionalized 1,2,4-trioxepanes 17-19 have been synthesized and evaluated against multi-drug resistant Plasmodium yoelii in Swiss mice. Amino functionalized trioxepanes 14, the most active compound of the series, showed 100% clearance of parasitaemia by oral route on day 4 and 75% protection to the treated mice beyond day 28.     

Specificity of age-related carbonylation of plasma proteins in the mouse and rat.

The purpose of this study was to determine (1) whether oxidative damage to plasma proteins in mice and rats, accrued during aging and manifested as carbonyl modifications, was selective or random, and (2) whether the putative carbonylated proteins could be used as markers of oxidative stress and aging. The total protein carbonyl content of the plasma significantly increased with age in mice but not in rats. Immunostaining of mouse plasma proteins, resolved by SDS-PAGE to localize carbonyls, revealed that only two specific proteins exhibited an age-associated increase in carbonylation. These proteins with molecular weights of 68 and 75 kDa, were identified as albumin and transferrin, respectively. In the rat, albumin and a 167-kDa protein, alpha1-macroglobulin (alpha-1M), showed significant age-dependent accrual of carbonylation. In the plasma of middle age Rhesus monkeys, in addition to albumin, a 54-kDa protein showed carbonylation. However, neither transferrin nor alpha-1M were carbonylated in the plasma of Rhesus monkey. Albumin was the only protein that showed carbonylation in all the three species examined. Results of this study indicate that age-associated increase in protein carbonylation is a selective and not a random phenomenon. However, the set of proteins that become carbonylated differs in different species.     

Activities of hydrolytic enzymes in callus cultures of tobacco during organogenesis

Starch, total sugars, reducing sugars and protein contents and the specific activities of hydrolytic enzymes such as amylase, Phosphorylase, soluble acid invertase, wall-bound acid invertase, sucrose synthetase, acid and alkaline phosphatases and ribonuclease were determined in root forming, shoot forming and non-organ-forming callus cultures of tobacco. Organ-forming cultures not only showed higher amounts of the above metabolites but also higher enzyme activities compared to non-organ-forming cultures. The activities of these enzymes in relation to organogenesis is discussed.     

Mastication and enamel microstructure in <Emphasis Type="Italic">Cambaytherium</Emphasis>, a perissodactyl-like ungulate from the early Eocene of India

The dentition of Cambaytherium was investigated in terms of dental wear, tooth replacement and enamel microstructure. The postcanine tooth row shows a significant wear gradient, with flattened premolars and anterior molars at a time when the last molars are only little worn. This wear gradient, which is more intensive in Cambaytherium thewissi than in Cambaytherium gracilis, and the resulting flattened occlusal surfaces, may indicate a preference for a durophagous diet. The tooth replacement (known only in C. thewissi) shows an early eruption of the permanent premolars. They are in function before the third molars are fully erupted. During the dominant phase I of the chewing cycle the jaw movement is very steep, almost orthal, with a slight mesiolingual direction and changes into a horizontal movement during phase II. The enamel microstructure shows Hunter-Schreger-bands (HSB) in the inner zone of the enamel. In some teeth the transverse orientation of the HSB is modified into a zig-zag pattern, possibly an additional indicator of a durophagous diet.     

Conformation and Trimer Association of the Transmembrane Domain of the Parainfluenza Virus Fusion Protein in Lipid Bilayers from Solid-State NMR: Insights into the Sequence Determinants of Trimer Structure and Fusion Activity

Enveloped viruses enter cells by using their fusion proteins to merge the virus lipid envelope and the cell membrane. While crystal structures of the water-soluble ectodomains of many viral fusion proteins have been determined, the structure and assembly of the C-terminal transmembrane domain (TMD) remains poorly understood. Here we use solid-state NMR to determine the backbone conformation and oligomeric structure of the TMD of the parainfluenza virus 5 fusion protein. ¹³C chemical shifts indicate that the central leucine-rich segment of the TMD is α-helical in POPC/cholesterol membranes and POPE membranes, while the Ile- and Val-rich termini shift to the β-strand conformation in the POPE membrane. Importantly, lipid mixing assays indicate that the TMD is more fusogenic in the POPE membrane than in the POPC/cholesterol membrane, indicating that the β-strand conformation is important for fusion by inducing membrane curvature. Incorporation of para-fluorinated Phe at three positions of the α-helical core allowed us to measure interhelical distances using ¹⁹F spin diffusion NMR. The data indicate that, at peptide:lipid molar ratios of ~ 1:15, the TMD forms a trimeric helical bundle with inter-helical distances of 8.2–8.4 Å for L493F and L504F and 10.5 Å for L500F. These data provide high-resolution evidence of trimer formation of a viral fusion protein TMD in phospholipid bilayers, and indicate that the parainfluenza virus 5 fusion protein TMD harbors two functions: the central α-helical core is the trimerization unit of the protein, while the two termini are responsible for inducing membrane curvature by transitioning to a β-sheet conformation.     

Theoretical investigation on the kinetics and branching ratio of the gas phase reaction of sevoflurane with Cl atom

The present work deals with the theoretical investigation on the Cl initiated H-atom abstraction reaction of sevoflurane, (CF₃)₂CHOCH₂F. A dual-level procedure has been adopted for studying the kinetics of the reaction. Geometrical optimization and frequency calculation were performed at DFT(BHandHLYP)/6-311G(d,p) while single-point energy calculation was made at CCSD(T)/6-311G(d,p) level of theory. The intrinsic reaction coordinate (IRC) calculation has also been performed to confirm the smooth transition from the reactant to product through the respective transition state. The rate constants were calculated using conventional transition state theory (TST). It has been found that 99 % of the reaction proceeded via the H-atom abstraction from the –CH₂F end of the sevoflurane. The rate constant of the dominant path is found to be 1.13 × 10^?13 cm³ molecule^?1 s^?1. This is in excellent agreement with the reported experimental rate constant of 1.10 × 10^?13 cm³ molecule^?1 s^?1 obtained by relative rate method using FTIR/Smog chamber and LP/LIF techniques.