Regulated expression of mature human insulin in the liver of transgenic mice

AbstractTransgenic mice expressing either human proinsulin cDNA or mutated proinsulin cDNA in the liver were created. The human proinsulin cDNA was mutated to generate a protein cleavable by the ubiquitous prohormone convertase furin, thus leading to mature insulin peptide. All transgenic lines expressed human C-peptide in the blood, whose level varied according to nutritional conditions. High performance liquid chromatography fractionation of mouse serum revealed that mutant proinsulin was effectively processed into mature insulin in vivo. This transgenic mouse model provides a useful tool for further prospects of gene therapy of insulin-dependent diabetes mellitus

pH Dependent Molecular Self-Assembly of Octaphosphonate Porphyrin of Nanoscale Dimensions: Nanosphere and Nanorod Aggregates

Self-assembled nanostructures of zwitterionic octaphosphanatoporphyrin 1, of either nanoparticles or nanorods, depending on small changes in the pH, is demonstrated based on the J-aggregates. Porphyrin 1 self-assembled into nanosphere aggregates with a diameter of about 70–80 nm in the pH range 5–7, and nanorod aggregates were observed at pH 8.5. Hydrogen bonding, π-π stacking and hydrophilic interactions play important roles in the formation of this nanostructure morphology. Nanostructures were characterized by UV/Vis absorbance, fluorescence, atomic force microscopy (AFM) and transmission electron microscopy (TEM). This interesting pH dependent self-assembly phenomenon could provide a basis for development of novel biomaterials

Differential changes in gene expression in human neutrophils following TNF-α stimulation: Up-regulation of anti-apoptotic proteins and down-regulation of proteins involved in death receptor signaling.

Responses of human neutrophils to TNF-α are complex and multifactorial. Exposure of human neutrophils to TNF-α in vitro primes the respiratory burst, delays apoptosis and induces the expression of several genes including chemokines, and TNF-α itself. This study aimed to determine the impact of TNF-α exposure on the expression of neutrophil genes and proteins that regulate apoptosis. Quantitative PCR and RNA-Seq, identified changes in expression of several apoptosis regulating genes in response to TNF-α exposure. Up-regulated genes included TNF-α itself, and several anti-apoptotic genes, including BCL2A1, CFLAR (cFLIP) and TNFAIP3, whose mRNA levels increased above control values by between 4-20 fold (n = 3, P < 0.05). In contrast, the expression of pro-apoptotic genes, including CASP8, FADD and TNFRSF1A and TNFRSF1B, were significantly down-regulated following TNF-α treatment. These changes in mRNA levels were paralleled by decreases in protein levels of caspases 8 and 10, TRADD, FADD, TNFRSF1A and TNFRSF1B, and increased cFLIP protein levels, as detected by western blotting. These data indicate that when neutrophils are triggered by TNF-α exposure, they undergo molecular changes in transcriptional expression to up-regulate expression of specific anti-apoptotic proteins and concomitantly decrease expression of specific proteins involved in death receptor signaling which will alter their function in TNF-α rich environments

Interleukin-18 produced by bone marrow- derived stromal cells supports T-cell acute leukaemia progression

International audienceDevelopment of novel therapies is critical for T-cell acute leukae-mia (T-ALL). Here, we investigated the effect of inhibiting the MAPK/MEK/ERK pathway on T-ALL cell growth. Unexpectedly, MEK inhibitors (MEKi) enhanced growth of 70% of human T-ALL cell samples cultured on stromal cells independently of NOTCH activa-tion and maintained their ability to propagate in vivo. Similar results were obtained when T-ALL cells were cultured with ERK1/ 2-knockdown stromal cells or with conditioned medium from MEKi-treated stromal cells. Microarray analysis identified interleu-kin 18 (IL-18) as transcriptionally up-regulated in MEKi-treated MS5 cells. Recombinant IL-18 promoted T-ALL growth in vitro, whereas the loss of function of IL-18 receptor in T-ALL blast cells decreased blast proliferation in vitro and in NSG mice. The NFKB pathway that is downstream to IL-18R was activated by IL-18 in blast cells. IL-18 circulating levels were increased in T-ALL-xeno-grafted mice and also in T-ALL patients in comparison with controls. This study uncovers a novel role of the pro-inflammatory cytokine IL-18 and outlines the microenvironment involvement in human T-ALL development

The Thrombopoietin Receptor Can Mediate Proliferation Without Activation of the Jak-STAT Pathway

Cytokine receptors of the hematopoietic receptor superfamily lack intrinsic tyrosine kinase domains for the intracellular transmission of their signals. Instead all members of this family associate with Jak family nonreceptor tyrosine kinases. Upon ligand stimulation of the receptors, Jaks are activated to phosphorylate target substrates. These include STAT (signal transducers and activators of transcription) proteins, which after phosphorylation translocate to the nucleus and modulate gene expression. The exact role of the Jak-STAT pathway in conveying growth and differentiation signals remains unclear. Here we describe a deletion mutant of the thrombopoietin receptor (c-mpl) that has completely lost the capacity to activate Jaks and STATs but retains its ability to induce proliferation. This mutant still mediates TPO-induced phosphorylation of Shc, Vav, mitogen-activated protein kinase (MAPK) and Raf-1 as well as induction of c-fos and c-myc, although at somewhat reduced levels. Furthermore, we show that both wild-type and mutant receptors activate phosphatidylinositol (PI) 3-kinase upon thrombopoietin stimulation and that thrombopoietin-induced proliferation is inhibited in the presence of the PI 3-kinase inhibitor wortmannin. These results demonstrate that the Jak-STAT pathway is dispensable for the generation of mitogenic signals by a cytokine receptor

A Senescence-Like Cell-Cycle Arrest Occurs During Megakaryocytic Maturation: Implications for Physiological and Pathological Megakaryocytic Proliferation

During normal megakaryocyte development, in response to thrombopoetin, mature cells enter a senescence-like state in which they shed platelets; this state, characterized by cell cycle arrest, is defective in malignant megakaryocytes

ERK1 Regulates the Hematopoietic Stem Cell Niches

The mitogen-activated protein kinases (MAPK) ERK1 and ERK2 are among the major signal transduction molecules but little is known about their specific functions in vivo. ERK activity is provided by two isoforms, ERK1 and ERK2, which are ubiquitously expressed and share activators and substrates. However, there are not in vivo studies which have reported a role for ERK1 or ERK2 in HSCs and the bone marrow microenvironment. The present study shows that the ERK1-deficient mice present a mild osteopetrosis phenotype. The lodging and the homing abilities of the ERK1−/− HSC are impaired, suggesting that the ERK1−/−-defective environment may affect the engrafment of HSCs. Serial transplantations demonstrate that ERK1 is involved in the maintenance of an appropriate medullar microenvironment, but that the intrinsic properties of HSCs are not altered by the ERK1−/− defective microenvironment. Deletion of ERK1 impaired in vitro and in vivo osteoclastogenesis while osteoblasts were unaffected. As osteoclasts derive from precursors of the monocyte/macrophage lineage, investigation of the monocytic compartment was performed. In vivo analysis of the myeloid lineage progenitors revealed that the frequency of CMPs increased by approximately 1.3-fold, while the frequency of GMPs significantly decreased by almost 2-fold, compared with the respective WT compartments. The overall mononuclear-phagocyte lineage development was compromised in these mice due to a reduced expression of the M-CSF receptor on myeloid progenitors. These results show that the cellular targets of ERK1 are M-CSFR-responsive cells, upstream to osteoclasts. While ERK1 is well known to be activated by M-CSF, the present results are the first to point out an ERK1-dependent M-CSFR regulation on hematopoietic progenitors. This study reinforces the hypothesis of an active cross-talk between HSCs, their progeny and bone cells in the maintenance of the homeostasis of these compartments