Endocannabinoid-related compounds in gastrointestinal diseases

The endocannabinoid system (ECS) is an endogenous signalling pathway involved in the control of several gastrointestinal (GI) functions at both peripheral and central levels. In recent years, it has become apparent that the ECS is pivotal in the regulation of GI motility, secretion and sensitivity, but endocannabinoids (ECs) are also involved in the regulation of intestinal inflammation and mucosal barrier permeability, suggesting their role in the pathophysiology of both functional and organic GI disorders. Genetic studies in patients with irritable bowel syndrome (IBS) or inflammatory bowel disease have indeed shown significant associations with polymorphisms or mutation in genes encoding for cannabinoid receptor or enzyme responsible for their catabolism, respectively. Furthermore, ongoing clinical trials are testing EC agonists/antagonists in the achievement of symptomatic relief from a number of GI symptoms. Despite this evidence, there is a lack of supportive RCTs and relevant data in human beings, and hence, the possible therapeutic application of these compounds is raising ethical, political and economic concerns. More recently, the identification of several EC-like compounds able to modulate ECS function without the typical central side effects of cannabinomimetics has paved the way for emerging peripherally acting drugs. This review summarizes the possible mechanisms linking the ECS to GI disorders and describes the most recent advances in the manipulation of the ECS in the treatment of GI diseases

Ferrous Campylobacter jejuni truncated hemoglobin P displays an extremely high reactivity for cyanide - A comparative study

Campylobacter jejuni hosts two hemoglobins (Hbs). The Camplylobacter jejuni single-domain Hb (called Cgb) is homologous to the globin domain of flavohemoglobin, and it has been proposed to protect the bacterium against nitrosative stress. The second Hb is called Ctb (hereafter Cj-trHbP), belongs to truncated Hb group III, and has been hypothesized to be involved in O 2 chemistry. Here, the kinetics and thermodynamics of cyanide binding to ferric and ferrous Cj-trHbP [Cj-trHbP(III) and Cj-trHbP(II), respectively] are reported and analyzed in parallel with those of related heme proteins, with particular reference to those from Mycobacterium tuberculosis. The affinity of cyanide for Cj-trHbP(II) is higher than that reported for any known (in)vertebrate globin by more than three orders of magnitude (K = 1.2 × 10-6 m). This can be fully attributed to the highest (ever observed for a ferrous Hb) cyanide-binding association rate constant (kon = 3.3 × 103 m-1·s-1), even though the binding process displays a rate-limiting step (kmax = 9.1 s -1). Cj-trHbP(III) shows a very high affinity for cyanide (L = 5.8 × 10-9 m); however, cyanide association kinetics are independent of cyanide concentration, displaying a rate-limiting step (l max = 2.0 × 10-3 s-1). Values of the first-order rate constant for cyanide dissociation from Cj-trHbP(II)-cyanide and Cj-trHbP(III)-cyanide (koff =5.0 × 10-3 s -1 and loff ≥ 1 × 10-4 s-1, respectively) are similar to those reported for (in)vertebrate globins. The very high affinity of cyanide for Cj-trHbP(II), reminiscent of that of horseradish peroxidase(II), suggests that this globin may participate in cyanide detoxification. © 2008 The Authors

Rifaximin improves Clostridium difficile toxin A-induced toxicity in Caco-2 cells by the PXR-dependent TLR4/MyD88 /NF-?B pathway

Background: Clostridium difficile infections (CDIs) caused by Clostridium difficile toxin A (TcdA) lead to severe ulceration, inflammation and bleeding of the colon, and are difficult to treat. Aim: The study aimed to evaluate the effect of rifaximin on TcdA-induced apoptosis in intestinal epithelial cells and investigate the role of PXR in its mechanism of action. Methods: Caco‐2 cells were incubated with TcdA and treated with rifaximin (0.1−10 μM) with or without ketoconazole (10 μM). The transepithelial electrical resistance (TEER) and viability of the treated cells was determined. Also, the expression of zona occludens‐1 (ZO‐1), toll‐like receptor 4 (TLR4), Bcl‐2‐associated X protein (Bax), transforming growth factor‐β‐activated kinase‐1 (TAK1), myeloid differentiation factor 88 (MyD88) and nuclear factor‐kappaB (NF‐κB) was determined. Results Rifaximin treatment (0.1, 1.0 and 10 μM) caused a significant and concentration-dependent increase in the TEER of Caco-2 cells (360%, 480% and 680% vs TcdA treatment) 24 hours after the treatment and improved their viability (61%, 79% and 105%). Treatment also concentration-dependently decreased the expression of Bax protein (–29%, –65% and –77%) and increased the expression of ZO-1 (25%, 54% and 87%) and occludin (71%, 114% and 262%) versus TcdA treatment. The expression of TLR4 (–33%, –50% and –75%), MyD88 (–29%, –60% and –81%) and TAK1 (–37%, –63% and –79%) were also reduced with rifaximin versus TcdA treatment. Ketoconazole treatment inhibited these effects. Conclusions: Rifaximin improved TcdA‐induced toxicity in Caco‐2 cells by the PXR‐dependent TLR4/MyD88/NF‐κB pathway mechanism, and may be useful in the treatment of CDIs

Enteric glia: A new player in inflammatory bowel diseases

In addition to the well-known involvement of macrophages and neutrophils, other cell types have been recently reported to substantially contribute to the onset and progression of inflammatory bowel diseases (IBD). Enteric glial cells (EGC) are the equivalent cell type of astrocyte in the central nervous system (CNS) and share with them many neurotrophic and neuro-immunomodulatory properties. This short review highlights the role of EGC in IBD, describing the role played by these cells in the maintenance of gut homeostasis, and their modulation of enteric neuronal activities. In pathological conditions, EGC have been reported to trigger and support bowel inflammation through the specific over-secretion of S100B protein, a pivotal neurotrophic factor able to induce chronic inflammatory changes in gut mucosa. New pharmacological tools that may improve the current therapeutic strategies for inflammatory bowel diseases (IBD), lowering side effects (i.e. corticosteroids) and costs (i.e. anti-TNFα monoclonal antibodies) represent a very important challenge for gastroenterologists and pharmacologists. Novel drugs capable to modulate enteric glia reactivity, limiting the pro-inflammatory release of S100B, may thus represent a significant innovation in the field of pharmacological interventions for inflammatory bowel diseases

SPECIFIC DYSPEPTIC SYMPTOMS ARE ASSOCIATED WITH POOR RESPONSE TO THERAPY IN PATIENTS WITH GASTROESOPHAGEAL REFLUX DISEASE

Background: In gastroesophageal reflux disease (GORD) patients, coexistence of functional dyspepsia (FD) is known to be associated with poor response to proton pump inhibitors (PPIs), but the contribution of specific dyspepsia symptoms has not been systematically investigated yet. Objective: To characterize the impact of dyspepsia symptoms on PPIs response in GORD patients. Methods:. The enrolled subjects were 100 patients with diagnosis of GORD. All patients underwent a 24 hour pH-impedance test, while on PPIs-therapy. Patients were divided into two groups, refractory and responders, according to the persistence of GORD symptoms. A standardized questionnaire for FD was also administered to assess presence of dyspepsia symptoms. Results: In the subgroup of refractory patients FD was more prevalent than in responder ones, with postprandial fullness, nausea, vomiting, early satiation and epigastric pain being significantly prevalent in refractory GORD-patients. In the multivariate analysis only early satiation and vomiting were significantly associated with poor response to PPIs Conclusion: Coexistence of FD is associated with refractory-GORD. We showed that only early satiation and vomiting are risk factors for poor response to PPIs therapy. Our findings suggest that symptoms of early satiation and vomiting would help to identify the subset of PPIs-refractory GORD patients

S100B‐p53 disengagement by pentamidine promotes apoptosis and inhibits cellular migration via aquaporin‐4 and metalloproteinase‐2 inhibition in C6 glioma cells

S100 calcium‐binding protein B (S100B) is highly expressed in glioma cells and promotes cancer cell survival via inhibition of the p53 protein. In melanoma cells, this S100B‐p53 interaction is known to be inhibited by pentamidine isethionate, an antiprotozoal agent. Thus, the aim of the present study was to evaluate the effect of pentamidine on rat C6 glioma cell proliferation, migration and apoptosis in vitro. The change in C6 cell proliferation following treatment with pentamidine was determined by performing a 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyltetrazolium bromide‐formazan assay. Significant dose‐dependent decreases in proliferation were observed at pentamidine concentrations of 0.05 μM (58.5±5%; P<0.05), 0.5 μM (40.6±7%; P<0.01) and 5 μM (13±4%; P<0.001) compared with the control (100% viability). Furthermore, treatment with 0.05, 0.5 and 5 μM pentamidine was associated with a significant increase in apoptosis versus the untreated cells, as determined by DNA fragmentation assays, immunofluorescence analysis of C6 chromatin using Hoechst staining, and immunoblot analysis of B‐cell lymphoma‐2 (Bcl‐2)‐associated X protein (100%, P<0.05; 453%, P<0.01; and 1000%, P<0.001, respectively) and Bcl‐2 (‐60%, P<0.001; ‐80.13%, P<0.001; ‐95%, P<0.001, respectively). In addition, the administration of 0.05, 0.5 and 5 μM pentamidine significantly upregulated the protein expression levels of p53 (681±87.5%, P<0.05; 1244±94.3%, P<0.01; and 2244±111%, P<0.001, respectively), and significantly downregulated the expression levels of matrix metalloproteinase‐2 (42±2.3%, P<0.05; 71±2.5%, P<0.01; and 95.8±3.3%, P<0.001, respectively) and aquaporin 4 (38±2.5%, P<0.05; 69±2.6%, P<0.01; and 88±3.0%, P<0.001, respectively), compared with the untreated cells. The wound healing assay demonstrated that cell migration was significantly impaired by treatment with 0.05, 0.5 and 5 μM pentamidine compared with untreated cells (88±4.2%, P<0.05; 64±2%, P<0.01; and 42±3.1%, P<0.001, respectively). Although additional in vivo studies are required to clarify the current in vitro data, the present study indicates that pentamidine and S100B‐p53 inhibitors may represent a novel approach for the treatment of glioma

Dissipationless Formation and Evolution of the Milky Way Nuclear Star Cluster

Abridged: In one widely discussed model for the formation of nuclear star clusters (NSCs), massive globular clusters spiral into the center of a galaxy and merge to form the nucleus. It is now known that at least some NSCs coexist with supermassive black holes (SBHs); this is the case, for instance, in the Milky Way (MW). In this paper, we investigate how the presence of a SMBH at the center of the MW impacts the merger hypothesis for the formation of its NSC. Starting from a model consisting of a low-density nuclear stellar disk and the SMBH, we use N-body simulations to follow the successive inspiral and merger of globular clusters. The clusters are started on circular orbits of radius 20 pc, and their initial masses and radii are set up in such a way as to be consistent with the galactic tidal field at that radius. The total accumulated mass by ~10 clusters is about 1.5x10^7 Solar masses. Each cluster is disrupted by the SMBH at a distance of roughly one parsec. The density profile that results after the final inspiral event is characterized by a core of roughly this radius, and an envelope with density that falls off as 1/r^2. These properties are similar to those of the MW NSC, with the exception of the core size, which in the MW is a little smaller. But by continuing the evolution of the model after the final inspiral event, we find that the core shrinks substantially via gravitational encounters in a time (when scaled to the MW) of 10 Gyr as the stellar distribution evolves toward a Bahcall-Wolf cusp. We also show that the luminosity function of the MW NSC is consistent with the hypothesis that a large fraction of the mass comes from (~10Gyr) old stars, brought in by globular clusters. We conclude that a model in which a large fraction of the mass of the MW NSC arose from infalling globular clusters is consistent with existing observational constraints.Comment: 15 pages, 13 figures. ApJ accepte

HIV-1 Tat-induced diarrhea is improved by the PPARalpha agonist, palmitoylethanolamide, by suppressing the activation of enteric glia

Diarrhea is a severe complication in HIV-1-infected patients with Trans-activator of transcription (HIV-1 Tat) protein being recognized as a major underlying cause. Beside its direct enterotoxic effects, Tat protein has been recently shown to affect enteric glial cell (EGC) activity. EGCs regulate intestinal inflammatory responses by secreting pro-inflammatory molecules; nonetheless, they might also release immune-regulatory factors, as palmytoilethanolamide (PEA), which exerts anti-inflammatory effects by activating PPARα receptors. We aimed at clarifying whether EGCs are involved in HIV-1 Tat-induced diarrhea and if PEA exerts antidiarrheal activity

S100B‑p53 disengagement by pentamidine promotes apoptosis and inhibits cellular migration via aquaporin‑4 and metalloproteinase‑2 inhibition in C6 glioma cells

S100 calcium‑binding protein B (S100B) is highly expressed in glioma cells and promotes cancer cell survival via inhibition of the p53 protein. In melanoma cells, this S100B‑p53 interaction is known to be inhibited by pentamidine isethionate, an antiprotozoal agent. Thus, the aim of the present study was to evaluate the effect of pentamidine on rat C6 glioma cell proliferation, migration and apoptosis in vitro. The change in C6 cell proliferation following treatment with pentamidine was determined by performing a 3‑[4,5‑dimethylthiazol‑2‑yl]‑2,5 diphenyltetrazolium bromide‑formazan assay. Significant dose‑dependent decreases in proliferation were observed at pentamidine concentrations of 0.05 μM (58.5±5%; P<0.05), 0.5 μM (40.6±7%; P<0.01) and 5 μM (13±4%; P<0.001) compared with the control (100% viability). Furthermore, treatment with 0.05, 0.5 and 5 μM pentamidine was associated with a significant increase in apoptosis versus the untreated cells, as determined by DNA fragmentation assays, immunofluorescence analysis of C6 chromatin using Hoechst staining, and immunoblot analysis of B‑cell lymphoma‑2 (Bcl‑2)‑associated X protein (100%, P<0.05; 453%, P<0.01; and 1000%, P<0.001, respectively) and Bcl‑2 (‑60%, P<0.001; ‑80.13%, P<0.001; ‑95%, P<0.001, respectively). In addition, the administration of 0.05, 0.5 and 5 μM pentamidine significantly upregulated the protein expression levels of p53 (681±87.5%, P<0.05; 1244±94.3%, P<0.01; and 2244±111%, P<0.001, respectively), and significantly downregulated the expression levels of matrix metalloproteinase‑2 (42±2.3%, P<0.05; 71±2.5%, P<0.01; and 95.8±3.3%, P<0.001, respectively) and aquaporin 4 (38±2.5%, P<0.05; 69±2.6%, P<0.01; and 88±3.0%, P<0.001, respectively), compared with the untreated cells. The wound healing assay demonstrated that cell migration was significantly impaired by treatment with 0.05, 0.5 and 5 μM pentamidine compared with untreated cells (88±4.2%, P<0.05; 64±2%, P<0.01; and 42±3.1%, P<0.001, respectively). Although additional in vivo studies are required to clarify the current in vitro data, the present study indicates that pentamidine and S100B‑p53 inhibitors may represent a novel approach for the treatment of glioma

HisE11 and HisF8 Provide Bis-histidyl Heme Hexa-coordination in the Globin Domain of Geobacter sulfurreducens Globin-coupled Sensor

Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS162) is reported. A combination of X-ray crystallography (crystal structure at 1.5 Å resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS162 as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS162 is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS162 is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS162 is a penta-coordinated species, where the proximal HisF8-Fe bond is severed. © 2008 Elsevier Ltd. All rights reserved