The Warburg Effect Is Genetically Determined in Inherited Pheochromocytomas

The Warburg effect describes how cancer cells down-regulate their aerobic respiration and preferentially use glycolysis to generate energy. To evaluate the link between hypoxia and Warburg effect, we studied mitochondrial electron transport, angiogenesis and glycolysis in pheochromocytomas induced by germ-line mutations in VHL, RET, NF1 and SDH genes. SDH and VHL gene mutations have been shown to lead to the activation of hypoxic response, even in normoxic conditions, a process now referred to as pseudohypoxia. We observed a decrease in electron transport protein expression and activity, associated with increased angiogenesis in SDH- and VHL-related, pseudohypoxic tumors, while stimulation of glycolysis was solely observed in VHL tumors. Moreover, microarray analyses revealed that expression of genes involved in these metabolic pathways is an efficient tool for classification of pheochromocytomas in accordance with the predisposition gene mutated. Our data suggest an unexpected association between pseudohypoxia and loss of p53, which leads to a distinct Warburg effect in VHL-related pheochromocytomas.     

A novel primary immunodeficiency with specific natural-killer cell deficiency maps to the centromeric region of chromosome 8

We describe four children with a novel primary immunodeficiency consisting of specific natural-killer (NK) cell deficiency and susceptibility to viral diseases. One child developed an Epstein-Barr virus-driven lymphoproliferative disorder; two others developed severe respiratory illnesses of probable viral etiology. The four patients are related and belong to a large inbred kindred of Irish nomadic descent, which suggests autosomal recessive inheritance of this defect. A genomewide scan identified a single 12-Mb region on chromosome 8p11.23-q11.21 that was linked to this immunodeficiency (maximum LOD score 4.51). The mapping of the disease-causing genomic region paves the way for the identification of a novel pathway governing NK cell differentiation in humans.     

The shedding activity of ADAM17 is sequestered in lipid rafts

The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease-disintegrin responsible for the cleavage of several biologically active transmembrane proteins. However, the substrate specificity of ADAM17 and the regulation of its shedding activity are still poorly understood. Here, we report that during its transport through the Golgi apparatus, ADAM17 is included in cholesterol-rich membrane microdomains (lipid rafts) where its prodomain is cleaved by furin. Consequently, ADAM17 shedding activity is sequestered in lipid rafts, which is confirmed by the fact that metalloproteinase inhibition increases the proportion of ADAM17 substrates (TNF and its receptors TNFR1 and TNFR2) in lipid rafts. Membrane cholesterol depletion increases the ADAM17-dependent shedding of these substrates demonstrating the importance of lipid rafts in the control of this process. Furthermore, ADAM17 substrates are present in different proportions in lipid rafts, suggesting that the entry of each of these substrates in these particular membrane microdomains is specifically regulated. Our data support the idea that one of the mechanisms regulating ADAM17 substrate cleavage involves protein partitioning in lipid rafts.     

Low molecular weight squash trypsin inhibitors from Sechium edule seeds

Nine chromatographic components containing trypsin inhibitor activity were isolated from Sechium edule seeds by acetone fractionation, gel filtration, affinity chromatography and RP-HPLC in an overall yield of 46% of activity and 0.05% of protein. The components obtained with highest yield of total activity and highest specific activity were sequenced by Edman degradation and their molecular masses determined by mass spectrometry. The inhibitors contained 31, 32 and 27 residues per molecule and their sequences were: SETI-IIa, EDRKCPKILMRCKRDSDCLAKCTCQESGYCG; SETI-IIb, EEDRKCPKILMRCKRDSDCLAKCTCQESGYCG and SETI-V, CPRILMKCKLDTDCFPTCTCRPSGFCG. SETI-IIa and SETI-IIb, which differed by an amino-terminal E in the IIb form, were not separable under the conditions employed. The sequences are consistent with consensus sequences obtained from 37 other inhibitors: CPriI1meCk_DSDCla_C_C_G_CG, where capital letters are invariant amino acid residues and lower case letters are the most preserved in this position. SETI-II and SETI-V form complexes with trypsin with a 1:1 stoichiometry and have dissociation constants of 5.4x10(-11)M and 1.1x10(-9)M, respectively.     

Functional regulatory T cells are collected in stem cell autografts by mobilization with high-dose cyclophosphamide and granulocyte colony-stimulating factor

High-dose cyclophosphamide (Cy) and G-CSF are widely used to mobilize hemopoietic stem cells for treating patients with high-dose chemotherapy and autologous stem cell transplantation (ASCT). Because lymphocyte count in the graft collected after Cy-G-CSF treatment is an independent survival factor after ASCT for patients with multiple myeloma, our purpose was to study how Cy-G-CSF treatment affects the phenotype and function of T cells in patients with multiple myeloma. Cy induced a 3-fold decrease of T cell counts with a slow and partial T cell recovery of one-third at the time of hemopoietic stem cell collection. Cy-G-CSF treatment did not affect the relative ratios of central memory, effector memory, and late effector CD4+ or CD8+ T cells, but a decrease in the percentage of naive CD4+ cells was observed. The percentages of CD25+ cells increased 2- to 3-fold in CD4+ and CD8+ T cells, the former including both activated CD25low and CD25high cells. CD4+CD25high cells were regulatory T cells (Treg) that expressed high levels of FOXP3, CTLA-4, and GITR and displayed in vitro suppressive properties. The recovery of Treg absolute counts after Cy-G-CSF treatment was higher than the recovery of other lymphocyte subpopulations. In conclusion, Cy-G-CSF treatment induces a severe T cell count decrease without deleting Treg, which are potent inhibitors of antitumor response. The present data encourage novel therapeutic strategies to improve T cell recovery following ASCT while limiting Treg expansion.     

The apelin receptor is coupled to Gi1 or Gi2 protein and is differentially desensitized by apelin fragments

The apelin receptor is a G protein-coupled receptor to which two ligand fragments, apelin-(65-77) and apelin-(42-77), can bind. To address the physiological significance of the existence of dual ligands for a single receptor, we first compared the ability of the apelin fragments to regulate intracellular effectors, to promote G protein coupling, and to desensitize the response in Chinese hamster ovary cells expressing the murine apelin receptor. We found that both apelin fragments inhibited adenylyl cyclase and increased the phosphorylation of ERK or Akt. Using stably transfected cells expressing a pertussis toxin-insensitive alpha(i) subunit, we demonstrated that each apelin fragment promoted coupling of the apelin receptor to either Galpha(i1) or Galpha(i2) but not to Galpha(i3). Although preincubation with each apelin fragment induced a desensitization at the level of the three effectors, preincubation with apelin-(42-77) also increased basal effector activity. In addition, a C-terminal deletion of the apelin receptor decreased the desensitization induced by apelin-(65-77) but did not alter the desensitization pattern induced by apelin-(42-77). Finally, in umbilical endothelial cells, which we have recently shown to express the apelin receptor, the Galpha(i1) and Galpha(i2) subunits are also expressed, ERK and Akt phosphorylation is desensitized after preincubation with apelin-(65-77), and basal levels of Akt phosphorylation are increased after preincubation with apelin-(42-77). In summary, apelin fragments regulate the same effectors, via the preferential coupling of the apelin receptor to G(i1) or G(i2), but they promote a differential desensitization pattern that may be central to their respective physiological roles.     

2,4-D impact on bacterial communities, and the activity and genetic potential of 2,4-D degrading communities in soil

The key role of telluric microorganisms in pesticide degradation is well recognized but the possible relationships between the biodiversity of soil microbial communities and their functions still remain poorly documented. If microorganisms influence the fate of pesticides, pesticide application may reciprocally affect soil microorganisms. The objective of our work was to estimate the impact of 2,4-D application on the genetic structure of bacterial communities and the 2,4-D-degrading genetic potential in relation to 2,4-D mineralization. Experiments combined isotope measurements with molecular analyses. The impact of 2,4-D on soil bacterial populations was followed with ribosomal intergenic spacer analysis. The 2,4-D degrading genetic potential was estimated by real-time PCR targeted on tfdA sequences coding an enzyme specifically involved in 2,4-D mineralization. The genetic structure of bacterial communities was significantly modified in response to 2,4-D application, but only during the intense phase of 2,4-D biodegradation. This effect disappeared 7 days after the treatment. The 2,4-D degrading genetic potential increased rapidly following 2,4-D application. There was a concomitant increase between the tfdA copy number and the 14C microbial biomass. The maximum of tfdA sequences corresponded to the maximum rate of 2,4-D mineralization. In this soil, 2,4-D degrading microbial communities seem preferentially to use the tfd pathway to degrade 2,4-D.     

Plant, soil microbial and soil inorganic nitrogen responses to elevated CO2: a study in microcosms of Holcus lanatus

The impact of elevated atmospheric CO₂ concentrations on the nitrogen cycle was evaluated in a 2-month experiment in monospecific grassland microcosms (Holcus lanatus L.) grown on reconstituted grassland soil. The responses of the N pools in the plants, soil, and soil microbes were studied. The impact of high CO₂ on key stages of the N cycle, especially nitrification and denitrification processes, were also measured. Our study showed a strong plant response to high CO₂: total biomass increased by 76% (P < 0.001) and root length density increased by 77% (P = 0.010). However, total plant N was not significantly modified by high CO₂, because the percent N in the plant decreased by 40% (P < 0.001). We observed a large decrease in soil NO₃^– concentration under elevated CO₂ (–50%; P = 0.002). Soil ammonium concentrations were much less affected by CO₂ enrichment, and only in resin bags (–8%, P = 0.019). Soil nitrifying enzyme activity (NEA) had a tendency to increase (+17%; P = 0.061) and denitrifying enzyme activity (DEA) decreased (-12%; P = 0.013). We found evidence of increased microbial N sink (microbial N increased by 17%, P = 0.004). This and other studies suggest that rising CO₂ often reduces soil nitrate concentrations, which may lead to decreased nitrate leaching. Elevated CO₂ led to environmental conditions that were less favourable for denitrification in our study.     

A regulatory network comprising let-7 miRNA and SMUG1 is associated with good prognosis in ER+ breast tumours

Single-strand selective uracil–DNA glycosylase 1 (SMUG1) initiates base excision repair (BER) of uracil and oxidized pyrimidines. SMUG1 status has been associated with cancer risk and therapeutic response in breast carcinomas and other cancer types. However, SMUG1 is a multifunctional protein involved, not only, in BER but also in RNA quality control, and its function in cancer cells is unclear. Here we identify several novel SMUG1 interaction partners that functions in many biological processes relevant for cancer development and treatment response. Based on this, we hypothesized that the dominating function of SMUG1 in cancer might be ascribed to functions other than BER. We define a bad prognosis signature for SMUG1 by mapping out the SMUG1 interaction network and found that high expression of genes in the bad prognosis network correlated with lower survival probability in ER⁺ breast cancer. Interestingly, we identified hsa-let-7b-5p microRNA as an upstream regulator of the SMUG1 interactome. Expression of SMUG1 and hsa-let-7b-5p were negatively correlated in breast cancer and we found an inhibitory auto-regulatory loop between SMUG1 and hsa-let-7b-5p in the MCF7 breast cancer cells. We conclude that SMUG1 functions in a gene regulatory network that influence the survival and treatment response in several cancers.     

Pannexin 1 activity in astroglia sets hippocampal neuronal network patterns

Astroglial release of molecules is thought to actively modulate neuronal activity, but the nature, release pathway, and cellular targets of these neuroactive molecules are still unclear. Pannexin 1, expressed by neurons and astrocytes, form nonselective large pore channels that mediate extracellular exchange of molecules. The functional relevance of these channels has been mostly studied in brain tissues, without considering their specific role in different cell types, or in neurons. Thus, our knowledge of astroglial pannexin 1 regulation and its control of neuronal activity remains very limited, largely due to the lack of tools targeting these channels in a cell-specific way. We here show that astroglial pannexin 1 expression in mice is developmentally regulated and that its activation is activity-dependent. Using astrocyte-specific molecular tools, we found that astroglial-specific pannexin 1 channel activation, in contrast to pannexin 1 activation in all cell types, selectively and negatively regulates hippocampal networks, with their disruption inducing a drastic switch from bursts to paroxysmal activity. This decrease in neuronal excitability occurs via an unconventional astroglial mechanism whereby pannexin 1 channel activity drives purinergic signaling-mediated regulation of hyperpolarisation-activated cyclic nucleotide (HCN)-gated channels. Our findings suggest that astroglial pannexin 1 channel activation serves as a negative feedback mechanism crucial for the inhibition of hippocampal neuronal networks.

Astrocytes have mostly been shown to boost neuronal activity. This study reveals that activity-dependent activation of astroglial pannexin 1 channels inhibits hippocampal neuronal networks by decreasing neuronal excitability via purinergic signaling, uncovering a novel astroglial negative feedback loop mechanism.