Proteomic Profile of Reversible Protein Oxidation Using PROP, Purification of Reversibly Oxidized Proteins

Signal transduction pathways that are modulated by thiol oxidation events are beginning to be uncovered, but these discoveries are limited by the availability of relatively few analytical methods to examine protein oxidation compared to other signaling events such as protein phosphorylation. We report here the coupling of PROP, a method to purify reversibly oxidized proteins, with the proteomic identification of the purified mixture using mass spectrometry. A gene ontology (GO), KEGG enrichment and Wikipathways analysis of the identified proteins indicated a significant enrichment in proteins associated with both translation and mRNA splicing. This methodology also enabled the identification of some of the specific cysteine residue targets within identified proteins that are reversibly oxidized by hydrogen peroxide treatment of intact cells. From these identifications, we determined a potential consensus sequence motif associated with oxidized cysteine residues. Furthermore, because we identified proteins and specific sites of oxidation from both abundant proteins and from far less abundant signaling proteins (e.g. hepatoma derived growth factor, prostaglandin E synthase 3), the results suggest that the PROP procedure was efficient. Thus, this PROP-proteomics methodology offers a sensitive means to identify biologically relevant redox signaling events that occur within intact cells

Nucleotide receptor signalling and the generation of reactive oxygen species

Elevated levels of extracellular nucleotides are present at sites of inflammation, platelet degranulation and cellular damage or lysis. These extracellular nucleotides can lead to the activation of purinergic (nucleotide) receptors on various leukocytes, including monocytes, macrophages, eosinophils, and neutrophils. In turn, nucleotide receptor activation has been linked to increased cellular production and release of multiple inflammatory mediators, including superoxide anion, nitric oxide and other reactive oxygen species (ROS). In the present review, we will summarize the evidence that extracellular nucleotides can facilitate the generation of multiple ROS by leukocytes. In addition, we will discuss several potential mechanisms by which nucleotide-enhanced ROS production may occur. Delineation of these mechanisms is important for understanding the processes associated with nucleotide-induced antimicrobial activities, cell signalling, apoptosis, and pathology

Efficacy of single-injection unilateral thoracic paravertebral block for post open cholecystectomy pain relief: a prospective randomized study at Gondar University Hospital

Demeke Yilkal Fentie, Endale Gebreegziabher Gebremedhn, Zewditu Abdissa Denu, Amare Hailekiros Gebreegzi Department of Anesthesia, School of Medicine, Gondar College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia Background: Cholecystectomy can be associated with considerable postoperative pain. While the benefits of paravertebral block (PVB) on pain after thoracotomy and mastectomy have been demonstrated, not enough investigations on the effects of PVB on pain after open cholecystectomy have been conducted. We tested the hypothesis that a single-injection thoracic PVB reduces pain scores, decreases opioid consumption, and prolongs analgesic request time after cholecystectomy.Methods: Of 52 patients recruited, 50 completed the study. They were randomly allocated into two groups: the paravertebral group and the control group. The outcome measures were the severity of pain measured on numeric pain rating scale, total opioid consumption, and first analgesic request time during the first postoperative 24 hours.Result: The main outcomes recorded during 24 hours after surgery were Numerical Rating Scale (NRS) pain scores (NRS, 0–10), cumulative opioid consumption, and the first analgesic request time. Twenty four hours after surgery, NRS at rest was 4 (3–6) vs 5 (5–7) and at movement 4 (4–7) vs 6 (5–7.5) for the PVB and control groups, respectively. The difference between the groups over the whole observation period was statistically significant (P<0.05). Twenty-four hours after surgery, median (25th–75th percentile) cumulative morphine consumption was 0 (0–2) vs 2.5 (2–4) mg (P<0.0001) and cumulative tramadol consumption was 200 (150–250) mg vs 300 (200–350) mg in the paravertebral and in the control group, respectively (P=0.003). After surgery, the median (25th–75th percentile) first analgesic requirement time was prolonged in the PVB group in statistically significant fashion (P<0.0001).Conclusion and recommendations: Single-shot thoracic PVB as a component of multimodal analgesic regimen provided superior analgesia when compared with the control group up to 24 postoperative hours after cholecystectomy, and we recommend this block for post cholecystectomy pain relief. Keywords: open cholecystectomy, postoperative pain, paravertebral block, landmark technique&nbsp

Cell signaling via the P2X7 nucleotide receptor: linkage to ROS production, gene transcription, and receptor trafficking

Extracellular nucleotides can act as important intercellular signals in diverse biological processes, including the enhanced production of factors that are key to immune response regulation. One receptor that binds extracellular adenosine triphosphate released at sites of infection and injury is P2X7, which is an ionotrophic receptor that can also lead to the formation of a non-specific pore, activate multiple mitogen-activated protein kinases (MAPKs), and stimulate the production of immune mediators including interleukin family members and reactive oxygen species (ROS). In the present report, we have investigated the signaling mechanisms by which P2X7 promotes monocytic cell mediator production and induces transcription factor expression/phosphorylation, as well as how receptor-associated pore activity is regulated by intracellular trafficking. We report that P2X7 stimulates ROS production in macrophages through the MAPKs ERK1/2 and the nicotinamide adenine dinucleotide phosphate oxidase complex, activates several transcription factors including cyclic-AMP response element-binding protein and components of the activating protein-1 complex, and contains specific sequences within its intracellular C-terminus that appear critical for its activity. Altogether, these data further implicate P2X7 activation and signaling as a fundamental modulator of macrophage immune responses