Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells

Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells. The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions.     

The interaction between dimethylsulfoxide and nitrate reducing pathways in Rhodobacter capsulatus

Abstract The interaction between nitrate- and dimethyl-sulphoxide (DMSO)-reducing pathways was demonstrated in intact cells of Rhodobacter capsulatus AD2 removed from cultures grown under different conditions. The results provide evidence of competition between the DMSO and nitrate reductases for a common electron pool. Furthermore, strong inhibition was observed of the anaerobic dark DMSO-dependent growth of R. capsulatus by nitrate in the growth medium. This phenomenon is also discussed.     

EGF-Induced VEGF Exerts a PI3K-Dependent Positive Feedback on ERK and AKT through VEGFR2 in Hematological In Vitro Models

EGFR and VEGFR pathways play major roles in solid tumor growth and progression, however, little is known about these pathways in haematological tumors. This study investigated the crosstalk between EGFR and VEGFR2 signaling in two hematological in vitro models: THP1, a human monocytic leukemia, and Raji, a Burkitt’s lymphoma, cell lines. Results showed that both cell lines express EGFR and VEGFR2 and responded to EGF stimulation by activating EGFR, triggering VEGF production and phosphorylating ERK, AKT, and p38 very early, with a peak of expression at 10–20min. Blocking EGFR using Tyrphostin resulted in inhibiting EGFR induced activation of ERK, AKT, and p38. In addition, EGF stimulation caused a significant and immediate increase, within 1min, in pVEGFR2 in both cell lines, which peaked at ~5–10 min after treatment. Selective inhibition of VEGFR2 by DMH4, anti-VEGFR2 antibody or siRNA diminished EGF-induced pAKT and pERK, indicating a positive feedback exerted by EGFR-induced VEGF. Similarly, the specific PI3K inhibitor LY294002, suppressed AKT and ERK phosphorylation showing that VEGF feedback is PI3K-dependent. On the other hand, phosphorylation of p38, initiated by EGFR and independent of VEGF feedback, was diminished using PLC inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. Moreover, measurement of intracellular [Ca²⁺] and ROS following VEGFR2 inhibition and EGF treatment proved that VEGFR2 is not implicated in EGF-induced Ca²⁺ release whereas it boosts EGF-induced ROS production. Furthermore, a significant decrease in pAKT, pERK and p-p38 was shown following the addition of the ROS inhibitor NAC. These results contribute to the understanding of the crosstalk between EGFR and VEGFR in haematological malignancies and their possible combined blockade in therapy.     

Regulatory aspects of inorganic nitrogen metabolism in the Rhodospirillaceae

Regulatory aspects of the assimilation of inorganic nitrogen compounds (ammonia, nitrate, nitrogen) were studied in 12 strains belonging to the Rhodospirillaceae. All strains possessed an ammonium transport system, as demonstrated by ¹⁴C-methylammonium uptake. This uptake showed saturation kinetics (K _m between 50–150 M), and was competitively inhibited by ammonium (K _i between 5–18 M). The ammonium transport systems were repressed by ammonium in the growth medium. The nitrogenase activity of all strains was reversibly inhibited by ammonium (switch-off). This effect was not shown under nitrogen starvation conditions with the exception of some strains of Rhodopseudomonas capsulata, the nitrogenase of which was always susceptible to switch-off by ammonium. Assimilation of nitrate was confined to some strains of Rhodopseudomonas capsulata.Abbreviations ATCC American Type culture Collection, Rockville, MD, USA - DSM Deutsche Sammlung von Mikroorganismen, Göttingen, FRG     

dlk1/FA1 regulates the function of human bone marrow mesenchymal stem cells by modulating gene expression of pro-inflammatory cytokines and immune response-related factors

dlk1/FA1 (delta-like 1/fetal antigen-1) is a member of the epidermal growth factor-like homeotic protein family whose expression is known to modulate the differentiation signals of mesenchymal and hematopoietic stem cells in bone marrow. We have demonstrated previously that Dlk1 can maintain the human bone marrow mesenchymal stem cells (hMSC) in an undifferentiated state. To identify the molecular mechanisms underlying these effects, we compared the basal gene expression pattern in Dlk1-overexpressing hMSC cells (hMSC-dlk1) versus control hMSC (negative for Dlk1 expression) by using Affymetrix HG-U133A microarrays. In response to Dlk1 expression, 128 genes were significantly up-regulated (with >2-fold; p < 0.001), and 24% of these genes were annotated as immune response-related factors, including pro-inflammatory cytokines, in addition to factors involved in the complement system, apoptosis, and cell adhesion. Also, addition of purified FA1 to hMSC up-regulated the same factors in a dose-dependent manner. As biological consequences of up-regulating these immune response-related factors, we showed that the inhibitory effects of dlk1 on osteoblast and adipocyte differentiation of hMSC are associated with Dlk1-induced cytokine expression. Furthermore, Dlk1 promoted B cell proliferation, synergized the immune response effects of the bacterial endotoxin lipopolysaccharide on hMSC, and led to marked transactivation of the NF-kappaB. Our data suggest a new role for Dlk1 in regulating the multiple biological functions of hMSC by influencing the composition of their microenvironment "niche." Our findings also demonstrate a role for Dlk1 in mediating the immune response.     

Up-regulation of nerve growth factor and interleukin-10 in inflamed and non-inflamed intestinal segments in rats with experimental colitis

Inflammatory bowel diseases are characterized by dysregulated immune response to the normal microflora and structural and functional changes of the enteric nervous system which occur in inflamed as well as non-inflamed areas of the bowel. This study describes the changes in the expression of nerve growth factor (NGF) and interleukin-10 (IL-10) in the colon and in various segments of the small intestine in two rat models of experimental colitis induced by iodoacetamide or 2,4,6-trinitrobenzene sulfonic acid (TNBS). Levels of NGF and IL-10 were measured by ELISA in tissue homogenate sampled from duodenum, jejunum, ileum and colon at different time intervals. NGF and IL-10 increased significantly in homogenates of strips isolated from all small intestinal segments, 3-6h after iodoacetamide or TNBS administration and remained elevated until the colonic inflammation subsided. Similar but more pronounced increase occurred in areas of the colon adjacent to the ulcer. Histologic examinations revealed inflammatory changes in the colon; however, examination of sections from the small intestines did not reveal significant differences between controls and rats with colitis. The marked up-regulation of nerve growth factor and interleukin-10 in colitis suggests that they play a role in limiting or resolving inflammation and in preventing it from becoming uncontrolled. It also suggests that experimental colitis may be associated with latent inflammation in the small bowel.