Independent and additive effects of atenolol and methionine restriction on lowering rat heart mitochondria oxidative stress

A low rate of mitochondrial ROS production (mitROSp) and a low degree of fatty acid unsaturation are characteristic traits of long-lived animals and can be obtained in a single species by methionine restriction (MetR) or atenolol (AT) treatments. However, simultaneous application of both treatments has never been performed. In the present investigation it is shown that MetR lowers mitROSp and complex I content. Both the MetR and the AT treatments lower protein oxidative modification and oxidative damage to mtDNA and the fatty acid unsaturation degree in rat heart mitochondria. The decrease in fatty acid unsaturation seems to be due, at least in part, to decreases in desaturase and elongase activities or peroxisomal β-oxidation. Furthermore, the phosphorylation of extracellular signal-regulated kinase (ERK) was stimulated by MetR and AT. The decrease in membrane fatty acid unsaturation and protein oxidation, and the changes in fatty acids and p-ERK showed additive effects of both treatments. In addition, the increase in mitROSp induced by AT observed in the present investigation was totally avoided with the combined MetR + AT treatment. It is concluded that the simultaneous treatment with MetR plus atenolol is more beneficial than either single treatment alone to lower oxidative stress in rat heart mitochondria, analogously to what has been reported in long-lived animal species.     

A New Family of Secreted Toxins in Pathogenic Neisseria Species

The genus Neisseria includes both commensal and pathogenic species which are genetically closely related. However, only meningococcus and gonococcus are important human pathogens. Very few toxins are known to be secreted by pathogenic Neisseria species. Recently, toxins secreted via type V secretion system and belonging to the widespread family of contact-dependent inhibition (CDI) toxins have been described in numerous species including meningococcus. In this study, we analyzed loci containing the maf genes in N. meningitidis and N. gonorrhoeae and proposed a novel uniform nomenclature for maf genomic islands (MGIs). We demonstrated that mafB genes encode secreted polymorphic toxins and that genes immediately downstream of mafB encode a specific immunity protein (MafI). We focused on a MafB toxin found in meningococcal strain NEM8013 and characterized its EndoU ribonuclease activity. maf genes represent 2% of the genome of pathogenic Neisseria, and are virtually absent from non-pathogenic species, thus arguing for an important biological role. Indeed, we showed that overexpression of one of the four MafB toxins of strain NEM8013 provides an advantage in competition assays, suggesting a role of maf loci in niche adaptation.     

T Lymphocytes Orient against the Direction of Fluid Flow during LFA-1-Mediated Migration

As they leave the blood stream and travel to lymph nodes or sites of inflammation, T lymphocytes are captured by the endothelium and migrate along the vascular wall to permissive sites of transmigration. These processes take place under the influence of hemodynamic shear stress; therefore, we investigated how migrational speed and directionality are influenced by variations in shear stress. We examined human effector T lymphocytes on intercellular adhesion molecule 1 (ICAM-1)-coated surfaces under the influence of shear stresses from 2 to 60 dyn.cm⁻². T lymphocytes were shown to respond to shear stress application by a rapid (30 s) and fully reversible orientation of their migration against the fluid flow without a change in migration speed. Primary T lymphocytes migrating on ICAM-1 in the presence of uniformly applied SDF-1α were also found to migrate against the direction of shear flow. In sharp contrast, neutrophils migrating in the presence of uniformly applied fMLP and leukemic HSB2 T lymphocytes migrating on ICAM-1 alone oriented their migration downstream, with the direction of fluid flow. Our findings suggest that, in addition to biochemical cues, shear stress is a contributing factor to leukocyte migration directionality.     

The effect of root architecture and root loss through trenching on the anchorage of tropical urban trees (Eugenia grandis Wight)

Eugenia grandis (Wight) is grown in urban environments throughout Malaysia and root systems are often damaged through trenching for the laying down of roads and utilities. We investigated the effect of root cutting through trenching on the biomechanics of mature E. grandis. The force necessary to winch trees 0.2 m from the vertical was measured. Trenches were then dug at different distances (1.5, 1.0 and 0.5 m) from the trunk on the tension side of groups of trees. Each tree was winched sideways again and the uprooting force recorded. No trenches were made in a control group of trees which were winched until failure occurred. Critical turning moment (TM_crit) and tree anchorage rotational stiffness (TARS) before and after trenching were calculated. Root systems were extracted for architectural analysis and relationships between architectural parameters and TM_crit and TARS were investigated. No differences were found between TM_crit and trenching distance. However, in control trees and trees with roots cut at 1.5 m, significant relationships did exist between both TM_crit and TARS with stem dimensions, rooting depth and root plate size. TARS was significantly decreased when roots were cut at 0.5 m only. Surprisingly, no relationships existed between TM_crit and TARS with any root system parameter when trenching was carried out at 0.5 or 1.0 m. Our study showed that in terms of TARS and TM_crit, mechanical stability was not greatly affected by trenching, probably because rooting depth close to the trunk was a major component of anchorage.     

Protein kinase PKC delta and c-Abl are required for mitochondrial apoptosis induction by genotoxic stress in the absence of p53, p73 and Fas receptor

Doxorubicin, cis-diamminedichloroplatinum (II) and 5-fluorouracil used in chemotherapy induce apoptosis in Hep3B cells in the absence of p53, p73, and functional Fas. Since mediators remain unknown, the requirement of PKC delta (PKCdelta) and c-Abl was investigated. Suppression of c-Abl or PKCdelta expression using SiRNAs impaired PARP cleavage, Gleevec and/or rottlerin inhibited the induction of the subG1 phase and the increase of reactive oxygen species level. Co-precipitations and phosphorylations to mitochondria of c-Abl, PKCdelta and Bcl-X(L/s) were induced. A depolarization of the mitochondrial membrane and activations of caspase-2 and -9 were observed. We propose that, in the absence of p53, p73 and Fas, genotoxic drugs could require both PKCdelta and c-Abl to induce apoptosis through the mitochondrial pathway.     

Membrane lateral diffusion and capture of CFTR within transient confinement zones

The cystic fibrosis transmembrane conductance regulator (CFTR) channel interacts with scaffolding and other proteins that are expected to restrict its lateral movement, yet previous studies have reported predominantly free diffusion. We examined the lateral mobility of CFTR channels on live baby hamster kidney cells using three complementary methods. Channels bearing an extracellular biotinylation target sequence were labeled with streptavidin conjugated with fluorescent dyes (Alexa Fluor 488 or 568) or quantum dots (qDot605). Fluorescence recovery after photobleaching and image correlation spectroscopy of the dye-labeled channels revealed a significant immobile population ( approximately 50%), which was confirmed by direct single particle tracking (SPT) of qDot605-labeled CFTR. Adding 10 histidine residues at the C-terminus of CFTR to mask the postsynaptic density 95, Discs large, ZO-1 (PDZ) binding motif abolished its association with EBP50/NHERF1, reduced the immobile fraction, and increased mobility. Other interactions that are not normally detected on this timescale became apparent when binding of PDZ domain proteins was disrupted. SPT revealed that CFTR(His-10) channels diffuse randomly, become immobilized for periods lasting up to 1 min, and in some instances are recaptured at the same location. The impact of transient confinement on the measured diffusion using the three fluorescence techniques were assessed using computer simulations of the biological experiments. Finally, the impact of endosomal CFTR on mobility measurements was assessed by fluorescence correlation spectroscopy. These results reveal unexpected features of CFTR dynamics which may influence its ion channel activity.     

Annexin 2: a novel human immunodeficiency virus type 1 Gag binding protein involved in replication in monocyte-derived macrophages

Human immunodeficiency virus (HIV) replication in the major natural target cells, CD4+ T lymphocytes and macrophages, is parallel in many aspects of the virus life cycle. However, it differs as to viral assembly and budding, which take place on plasma membranes in T cells and on endosomal membranes in macrophages. It has been postulated that cell type-specific host factors may aid in directing viral assembly to distinct destinations. In this study we defined annexin 2 (Anx2) as a novel HIV Gag binding partner in macrophages. Anx2-Gag binding was confined to productively infected macrophages and was not detected in quiescently infected monocyte-derived macrophages (MDM) in which an HIV replication block was mapped to the late stages of the viral life cycle (A. V. Albright, R. M. Vos, and F. Gonzalez-Scarano, Virology 325:328-339, 2004). We demonstrate that the Anx2-Gag interaction likely occurs at the limiting membranes of late endosomes/multivesicular bodies and that Anx2 depletion is associated with a significant decline in the infectivity of released virions; this coincided with incomplete Gag processing and inefficient incorporation of CD63. Cumulatively, our data suggest that Anx2 is essential for the proper assembly of HIV in MDM.     

In vitro assessment of some sperm function following exposure to levonorgestrel in human fallopian tubes

Background  

The mechanism of action of levonorgestrel (LNG) as emergency contraception (EC) remains a subject of debate and its effect on sperm function has been only partially explained. The aim of this study was to assess whether LNG at a similar dose to those found in serum following oral intake for EC could affect spermatozoa when exposed to human fallopian tubes in vitro.