Involvement of the P2X7 Purinergic Receptor in Colonic Motor Dysfunction Associated with Bowel Inflammation in Rats

Background and Purpose

Recent evidence indicates an involvement of P2X7 purinergic receptor (P2X7R) in the fine tuning of immune functions, as well as in driving enteric neuron apoptosis under intestinal inflammation. However, the participation of this receptor in the regulation of enteric neuromuscular functions remains undetermined. This study was aimed at investigating the role of P2X7Rs in the control of colonic motility in experimental colitis.

Experimental Approach

Colitis was induced in rats by 2,4-dinitrobenzenesulfonic acid. P2X7R distribution was examined by immunofluorescence analysis. The effects of A804598 (selective P2X7R antagonist) and BzATP (P2X7R agonist) were tested on contractions of longitudinal smooth muscle evoked by electrical stimulation or by carbachol in the presence of tetrodotoxin.

Key Results

P2X7Rs were predominantly located in myenteric neurons, but, in the presence of colitis, their expression increased in the neuromuscular layer. In normal preparations, A804598 elicited a negligible increase in electrically induced contractions, while a significant enhancement was recorded in inflamed tissues. In the presence of N^ω-propyl-L-arginine (NPA, neuronal nitric oxide synthase inhibitor) the A804598 effects were lost. P2X7R stimulation with BzATP did not significantly affect electrical-induced contractions in normal colon, while a marked reduction was recorded under inflammation. The inhibitory effect of BzATP was antagonized by A804598, and it was also markedly blunted by NPA. Both P2X7R ligands did not affect carbachol-induced contractions.

Conclusions and Implications

The purinergic system contributes to functional neuromuscular changes associated with bowel inflammation via P2X7Rs, which modulate the activity of excitatory cholinergic nerves through a facilitatory control on inhibitory nitrergic pathways.     

The fine-tuning of TRAF2–GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex

We provide the first biochemical evidence of a direct interaction between the glutathione transferase P1-1 (GSTP1-1) and the TRAF domain of TNF receptor-associated factor 2 (TRAF2), and describe how ligand binding modulates such an equilibrium. The dissociation constant of the heterocomplex is K_d=0.3 μM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (K_d≥2.6 μM) or is inactivated by oxidation (K_d=1.7 μM). This indicates that GSTP1-1''s TRAF2-binding region involves the GSH-binding site. The GSTP1-1 inhibitor NBDHEX further decreases the complex''s binding affinity, as compared with when GSH is the only ligand; this suggests that the hydrophobic portion of the GSTP1-1 active site also contributes to the interaction. We therefore hypothesize that TRAF2 binding inactivates GSTP1-1; however, analysis of the data, using a model taking into account the dimeric nature of GSTP1-1, suggests that GSTP1-1 engages only one subunit in the complex, whereas the second subunit maintains the catalytic activity or binds to other proteins. We also analyzed GSTP1-1''s association with TRAF2 at the cellular level. The TRAF2–GSTP1-1 complex was constitutively present in U-2OS cells, but strongly decreased in S, G2 and M phases. Thus the interaction appears regulated in a cell cycle-dependent manner. The variations in the levels of individual proteins seem too limited to explain the complex''s drastic decline observed in cells progressing from the G0/G1 to the S–G2–M phases. Moreover, GSH''s intracellular content was so high that it always saturated GSTP1-1. Interestingly, the addition of NBDHEX maintains the TRAF2–GSTP1-1 complex at low levels, thus causing a prolonged cell cycle arrest in the G2/M phase. Overall, these findings suggest that a reversible sequestration of TRAF2 into the complex may be crucial for cell cycle progression and that multiple factors are involved in the fine-tuning of this interaction.     

Changing the heme ligation in flavocytochrome b 2: substitution of histidine-66 by cysteine

Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate. Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.     

Protein degradation sets the fraction of active ribosomes at vanishing growth

Growing cells adopt common basic strategies to achieve optimal resource allocation under limited resource availability. Our current understanding of such “growth laws” neglects degradation, assuming that it occurs slowly compared to the cell cycle duration. Here we argue that this assumption cannot hold at slow growth, leading to important consequences. We propose a simple framework showing that at slow growth protein degradation is balanced by a fraction of “maintenance” ribosomes. Consequently, active ribosomes do not drop to zero at vanishing growth, but as growth rate diminishes, an increasing fraction of active ribosomes performs maintenance. Through a detailed analysis of compiled data, we show that the predictions of this model agree with data from E. coli and S. cerevisiae. Intriguingly, we also find that protein degradation increases at slow growth, which we interpret as a consequence of active waste management and/or recycling. Our results highlight protein turnover as an underrated factor for our understanding of growth laws across kingdoms.     

Controlled X‐chromosome dynamics defines meiotic potential of female mouse in vitro germ cells

The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X‐chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X‐inactivation and reactivation dynamics using a tailor‐made in vitro system of primordial germ cell‐like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X‐inactivation in PGCLCs in vitro and in germ cell‐competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X‐inactivation is followed by step‐wise X‐reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X‐inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine‐tuned X‐chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis.     

The cryo-EM structure of the S-layer deinoxanthin-binding complex of Deinococcus radiodurans informs properties of its environmental interactions

The radiation-resistant bacterium Deinococcus radiodurans is known as the world’s toughest bacterium. The S-layer of D. radiodurans, consisting of several proteins on the surface of the cellular envelope and intimately associated with the outer membrane, has therefore been useful as a model for structural and functional studies. Its main proteinaceous unit, the S-layer deinoxanthin-binding complex (SDBC), is a hetero-oligomeric assembly known to contribute to the resistance against environmental stress and have porin functional features; however, its precise structure is unknown. Here, we resolved the structure of the SDBC at ∼2.5 Å resolution by cryo-EM and assigned the sequence of its main subunit, the protein DR_2577. This structure is characterized by a pore region, a massive β-barrel organization, a stalk region consisting of a trimeric coiled coil, and a collar region at the base of the stalk. We show that each monomer binds three Cu ions and one Fe ion and retains one deinoxanthin molecule and two phosphoglycolipids, all exclusive to D. radiodurans. Finally, electrophysiological characterization of the SDBC shows that it exhibits transport properties with several amino acids. Taken together, these results highlight the SDBC as a robust structure displaying both protection and sieving functions that facilitates exchanges with the environment.     

T cell stimulation remodels the latently HIV-1 infected cell population by differential activation of proviral chromatin

The reservoir of latently HIV-1 infected cells is heterogeneous. To achieve an HIV-1 cure, the reservoir of activatable proviruses must be eliminated while permanently silenced proviruses may be tolerated. We have developed a method to assess the proviral nuclear microenvironment in single cells. In latently HIV-1 infected cells, a zinc finger protein tethered to the HIV-1 promoter produced a fluorescent signal as a protein of interest came in its proximity, such as the viral transactivator Tat when recruited to the nascent RNA. Tat is essential for viral replication. In these cells we assessed the proviral activation and chromatin composition. By linking Tat recruitment to proviral activity, we dissected the mechanisms of HIV-1 latency reversal and the consequences of HIV-1 production. A pulse of promoter-associated Tat was identified that contrasted to the continuous production of viral proteins. As expected, promoter H3K4me3 led to substantial expression of the provirus following T cell stimulation. However, the activation-induced cell cycle arrest and death led to a surviving cell fraction with proviruses encapsulated in repressive chromatin. Further, this cellular model was used to reveal mechanisms of action of small molecules. In a proof-of-concept study we determined the effect of modifying enhancer chromatin on HIV-1 latency reversal. Only proviruses resembling active enhancers, associated with H3K4me1 and H3K27ac and subsequentially recognized by BRD4, efficiently recruited Tat upon cell stimulation. Tat-independent HIV-1 latency reversal of unknown significance still occurred. We present a method for single cell assessment of the microenvironment of the latent HIV-1 proviruses, used here to reveal how T cell stimulation modulates the proviral activity and how the subsequent fate of the infected cell depends on the chromatin context.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.