Selective Uncoupling of Individual Mitochondria within a Cell Using a Mitochondria-Targeted Photoactivated Protonophore

Depolarization of an individual mitochondrion or small clusters of mitochondria within cells has been achieved using a photoactivatable probe. The probe is targeted to the matrix of the mitochondrion by an alkyltriphenylphosphonium lipophilic cation and releases the protonophore 2,4-dinitrophenol locally in predetermined regions in response to directed irradiation with UV light via a local photolysis system. This also provides a proof of principle for the general temporally and spatially controlled release of bioactive molecules, pharmacophores, or toxins to mitochondria with tissue, cell, or mitochondrion specificity

Neurosteroid Dehydroepiandrosterone Interacts with Nerve Growth Factor (NGF) Receptors, Preventing Neuronal Apoptosis

The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75NTR membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [3H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75NTR receptors (KD: 7.4±1.75 nM and 5.6±0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75NTR receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75NTR receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor

Acute effects of intracranial hypertension and ARDS on pulmonary and neuronal damage: a randomized experimental study in pigs

Abstract PURPOSE: To determine reciprocal and synergistic effects of acute intracranial hypertension and ARDS on neuronal and pulmonary damage and to define possible mechanisms. METHODS: Twenty-eight mechanically ventilated pigs were randomized to four groups of seven each: control; acute intracranial hypertension (AICH); acute respiratory distress syndrome (ARDS); acute respiratory distress syndrome in combination with acute intracranial hypertension (ARDS + AICH). AICH was induced with an intracranial balloon catheter and the inflation volume was adjusted to keep intracranial pressure (ICP) at 30-40 cmH2O. ARDS was induced by oleic acid infusion. Respiratory function, hemodynamics, extravascular lung water index (ELWI), lung and brain computed tomography (CT) scans, as well as inflammatory mediators, S100B, and neuronal serum enolase (NSE) were measured over a 4-h period. Lung and brain tissue were collected and examined at the end of the experiment. RESULTS: In both healthy and injured lungs, AICH caused increases in NSE and TNF-alpha plasma concentrations, extravascular lung water, and lung density in CT, the extent of poorly aerated (dystelectatic) and atelectatic lung regions, and an increase in the brain tissue water content. ARDS and AICH in combination induced damage in the hippocampus and decreased density in brain CT. CONCLUSIONS: AICH induces lung injury and also exacerbates pre-existing damage. Increased extravascular lung water is an early marker. ARDS has a detrimental effect on the brain and acts synergistically with intracranial hypertension to cause histological hippocampal damage

Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures

DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling we used biotinylated-trimethylpsoralen as a DNA structure probe to show the genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF binding sites. Under-wound domains are transcriptionally active, enriched in topoisomerase I, “open” chromatin fibers and DNaseI sites, but are depleted of topoisomerase II. Furthermore DNA supercoiling impacts on additional levels of chromatin compaction as under-wound domains are cytologically decondensed, topologically constrained, and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation providing an evolutionary purpose for clustering genes along chromosomes

Super-silent FRET Sensor Enables Live Cell Imaging and Flow Cytometric Stratification of Intracellular Serine Protease Activity in Neutrophils

Abstract Serine proteases are released by neutrophils to act primarily as antimicrobial proteins but excessive and unbalanced serine protease activity results in serious host tissue damage. Here the synthesis of a novel chemical sensor based on a multi-branched fluorescence quencher is reported. It is super-silent, exhibiting no fluorescence until de-quenched by the exemplar serine protease human neutrophil elastase, rapidly enters human neutrophils, and is inhibited by serine protease inhibitors. This sensor allows live imaging of intracellular serine protease activity within human neutrophils and demonstrates that the unique combination of a multivalent scaffold combined with a FRET peptide represents a novel and efficient strategy to generate super-silent sensors that permit the visualisation of intracellular proteases and may enable point of care whole blood profiling of neutrophils