REST mediates resolution of HIF-dependent gene expression in prolonged hypoxia

The hypoxia-inducible factor (HIF) is a key regulator of the cellular response to hypoxia which promotes oxygen delivery and metabolic adaptation to oxygen deprivation. However, the degree and duration of HIF-1α expression in hypoxia must be carefully balanced within cells in order to avoid unwanted side effects associated with excessive activity. The expression of HIF-1α mRNA is suppressed in prolonged hypoxia, suggesting that the control of HIF1A gene transcription is tightly regulated by negative feedback mechanisms. Little is known about the resolution of the HIF-1α protein response and the suppression of HIF-1α mRNA in prolonged hypoxia. Here, we demonstrate that the Repressor Element 1-Silencing Transcription factor (REST) binds to the HIF-1α promoter in a hypoxia-dependent manner. Knockdown of REST using RNAi increases the expression of HIF-1α mRNA, protein and transcriptional activity. Furthermore REST knockdown increases glucose consumption and lactate production in a HIF-1α- (but not HIF-2α-) dependent manner. Finally, REST promotes the resolution of HIF-1α protein expression in prolonged hypoxia. In conclusion, we hypothesize that REST represses transcription of HIF-1α in prolonged hypoxia, thus contributing to the resolution of the HIF-1α response

Mast Cell Survival and Mediator Secretion in Response to Hypoxia

Tissue hypoxia is a consequence of decreased oxygen levels in different inflammatory conditions, many associated with mast cell activation. However, the effect of hypoxia on mast cell functions is not well established. Here, we have investigated the effect of hypoxia per se on human mast cell survival, mediator secretion, and reactivity. Human cord blood derived mast cells were subjected to three different culturing conditions: culture and stimulation in normoxia (21% O2); culture and stimulation in hypoxia (1% O2); or 24 hour culture in hypoxia followed by stimulation in normoxia. Hypoxia, per se, did not induce mast cell degranulation, but we observed an increased secretion of IL-6, where autocrine produced IL-6 promoted mast cell survival. Hypoxia did not have any effect on A23187 induced degranulation or secretion of cytokines. In contrast, cytokine secretion after LPS or CD30 treatment was attenuated, but not inhibited, in hypoxia compared to normoxia. Our data suggests that mast cell survival, degranulation and cytokine release are sustained under hypoxia. This may be of importance for host defence where mast cells in a hypoxic tissue can react to intruders, but also in chronic inflammations where mast cell reactivity is not inhibited by the inflammatory associated hypoxia

Involvement of Hypoxia-Inducible Factor-1 in the Inflammatory Responses of Human LAD2 Mast Cells and Basophils

We recently showed that hypoxia-inducible factor 1 (HIF-1) plays a crucial role in the pro-allergic functions of human basophils by transcriptional control of energy metabolism via glycolysis as well as directly triggering expression of the angiogenic cytokine vascular endothelium growth factor (VEGF). Here, we investigated HIF-1 involvement in controlling the synthesis of angiogenic and inflammatory cytokines from various human effector cells stimulated by IgE-dependent or innate immune triggers. Purified primary human basophils, LAD2 human mast cells and THP-1 human myeloid cells were used for investigations of FcεRI and Toll-like receptor (TLR) ligand-induced responses. In contrast to basophils, LAD2 mast cells expressed background levels of HIF-1α, which was largely independent of the effects of stem cell factor (SCF). Both mast cells and basophils expressed TLR2 and 4, albeit weakly compared to THP-1 cells. Cytokine production in mast cells following TLR ligand stimulation was markedly reduced by HIF-1α knockdown in LAD2 mast cells. In contrast, although HIF-1 is involved in IgE-mediated IL-4 secretion from basophils, it is not clearly induced by peptidoglycan (PGN). HIF-1α accumulation is critical for sustaining human allergic effector cell survival and function. This transcription complex facilitates generation of both pro-angiogenic and inflammatory cytokines in mast cells but has a differential role in basophil stimulation comparing IgE-dependent triggering with innate immune stimuli

Preoperative quantification of aortic valve stenosis: comparison of 64-slice computed tomography with transesophageal and transthoracic echocardiography and size of implanted prosthesis

Precise measurements of aortic complex diameters are essential for preoperative examinations of patients with aortic stenosis (AS) scheduled for aortic valve (AV) replacement. We aimed to prospectively compare the accuracy of transthoracic echocardiography (TTE), transoesophageal echocardiography (TEE) and multi-slice computed tomography (MSCT) measurements of the AV complex and to analyze the role of the multi-modality aortic annulus diameter (AAd) assessment in the selection of the optimal prosthesis to be implanted in patients surgically treated for degenerative AS. 20 patients (F/M: 3/17; age: 69 ± 6.5 years) with severe degenerative AS were enrolled into the study. TTE, TEE and MSCT including AV calcium score (AVCS) assessment were performed in all patients. The values of AAd obtained in the long AV complex axis (TTE, TEE, MSCT) and in multiplanar perpendicular imaging (MSCT) were compared to the size of implanted prosthesis. The mean AAd was 24 ± 3.6 mm using TTE, 26 ± 4.2 mm using TEE, and 26.9 ± 3.2 in MSCT (P = 0.04 vs. TTE). The mean diameter of the left ventricle out-flow tract in TTE (19.9 ± 2.7 mm) and TEE (19.5 ± 2.7 mm) were smaller than in MSCT (24.9 ± 3.3 mm, P < 0.001 for both). The mean size of implanted prosthesis (22.2 ± 2.3 mm) was significantly smaller than the mean AAd measured by TTE (P = 0.0039), TEE (P = 0.0004), and MSCT (P < 0.0001). The implanted prosthesis size correlated significantly to the AAd: r = 0.603, P = 0.005 for TTE, r = 0.592, P = 0.006 for TEE, and r = 0.791, P < 0.001 for MSCT. Obesity and extensive valve calcification (AV calcium score ≥ 3177Ag.U.) were identified as potent factors that caused a deterioration of both TTE and MSCT performance. The accuracy of AAd measurements in TEE was only limited by AV calcification. In multivariate regression analysis the mean value of the minimum and maximum AAd obtained in MSCT-multiplanar perpendicular imaging was an independent factor (r = 0.802, P < 0.0001) predicting the size of implanted prosthesis. In patients with AS echocardiography remains the main diagnostics tool in clinical practice. MSCT as a 3-dimentional modality allows for accurate measurement of entire AV complex and facilitates optimal matching of prosthesis size

Cell cycle-dependent phosphorylation of pRb-like protein in root meristem cells of Vicia faba

The retinoblastoma tumor suppressor protein (pRb) regulates cell cycle progression by controlling the G1-to-S phase transition. As evidenced in mammals, pRb has three functionally distinct binding domains and interacts with a number of proteins including the E2F family of transcription factors, proteins with a conserved LxCxE motif (D-type cyclin), and c-Abl tyrosine kinase. CDK-mediated phosphorylation of pRb inhibits its ability to bind target proteins, thus enabling further progression of the cell cycle. As yet, the roles of pRb and pRb-binding factors have not been well characterized in plants. By using antibody which specifically recognizes phosphorylated serines (S807/811) in the c-Abl tyrosine kinase binding C-domain of human pRb, we provide evidence for the cell cycle-dependent changes in pRb-like proteins in root meristems cells of Vicia faba. An increased phosphorylation of this protein has been found correlated with the G1-to-S phase transition

Free Radicals, Salicylic Acid and Mycotoxins in Asparagus After Inoculation with Fusarium proliferatum and F. oxysporum

Electron paramagnetic resonance was used to monitor free radicals and paramagnetic species like Fe, Mn, Cu generation, stability and status in Asparagus officinalis infected by common pathogens Fusarium proliferatum and F. oxysporum. Occurrence of F. proliferatum and F. oxysporum, level of free radicals and other paramagnetic species, as well as salicylic acid and mycotoxins content in roots and stems of seedlings were estimated on the second and fourth week after inoculation. In the first term free and total salicylic acid contents were related to free radicals level in stem (P = 0.010 and P = 0.033, respectively). Concentration of Fe3+ ions in porphyrin complexes (g = 2.3, g = 2.9) was related to the species of pathogen. There was no significant difference between Mn2+ concentrations in stem samples; however, the level of free radicals in samples inoculated with F. proliferatum was significantly higher when compared to F. oxysporum