IFN-ε Is Constitutively Expressed by Cells of the Reproductive Tract and Is Inefficiently Secreted by Fibroblasts and Cell Lines

Type-I interferons (IFNs) form a large family of cytokines that primarily act to control the early development of viral infections. Typical type-I IFN genes, such as those encoding IFN-α or IFN-β are upregulated by viral infection in many cell types. In contrast, the gene encoding IFN-ε was reported to be constitutively expressed by cells of the female reproductive tract and to contribute to the protection against vaginal infections with herpes simplex virus 2 and Chlamydia muridarum. Our data confirm the lack of induction of IFN-ε expression after viral infection and the constitutive expression of IFN-ε by cells of the female but also of the male reproductive organs. Interestingly, when expressed from transfected expression plasmids in 293T, HeLa or Neuro2A cells, the mouse and human IFN-ε precursors were inefficiently processed and secretion of IFN-ε was minimal. Analysis of chimeric constructs produced between IFN-ε and limitin (IFN-ζ) showed that both the signal peptide and the mature moiety of IFN-ε contribute to poor processing of the precursor. Immunofluorescent detection of FLAG-tagged IFN-ε in transfected cells suggested that IFN-ε and chimeric proteins were defective for progression through the secretory pathway. IFN-ε did not, however, act intracellularly and impart an antiviral state to producing cells. Given the constitutive expression of IFN-ε in specialized cells and the poor processing of IFN-ε precursor in fibroblasts and cell lines, we hypothesize that IFN-ε secretion may require a co-factor specifically expressed in cells of the reproductive organs, that might secure the system against aberrant release of this IFN.     

MicroRNA-153 Inhibits Osteosarcoma Cells Proliferation and Invasion by Targeting TGF-β2

Increasing evidence indicates that microRNAs (miRNAs), a class of small noncoding RNAs, participate in almost every step of cellular processes. MiRNAs are aberrantly expressed in human cancers and contribute to cancer development and progression. Study of miRNAs may provide a new clue for understanding the mechanism of carcinogenesis and a new tool for cancer treatment. In the present study, miR-153 was downregulated in human osteosarcoma tissues and cell lines. Introduction of miR-153 mimics into the MG-63 cells inhibited cell proliferation and invasion. Our results further revealed that transforming growth factor beta 2 (TGF-β2) was negatively regulated by miR-153. Furthermore, overexpression of miR-153 decreased p-SMAD2, p-SMAD3, epidermal growth factor receptor (EGFR) and insulin-like growth factor binding protein-3 (IGFBP-3) expressions, which were the downstream signaling molecules of TGF-β. Furthermore, miRNA-153 suppressed TGF-β-mediated MG-63 proliferation and migration. Therefore, our results suggest that miR-153 may act as a tumor suppressor in osteosarcoma through targeting TGF-β2.     

Inactivation of Nocardiophages φC and φEC by Extracts of Bacteriophage-Attachable Cells

Cultures of several species of Nocardia, including N. erythropolis Mat-Ce and Mat-cE mating strains, were extracted with solvents in an attempt to isolate an inactivating complex for nocardiophages phiC and phiEC. Ethanol was the only solvent found effective in solubilizing an inhibitory substance. Inactivating extracts were obtained from the cells of all species to which the phage were able to attach. After extraction of whole cells or cell wall preparations, the phage could not effectively attach to them. Both phages phiC and phiEC were inactivated by the same complex. However, phage phiEC inactivation was 10-fold greater than phiC inactivation. The velocity of inactivation was about 4.1 x 10(2) plaque-forming units per microgram per minute for phiC and 1.1 x 10(3) plaque-forming units per microgram per minute for phage phiEC. The cell extracts required divalent cations for phage inactivation. The inhibitory capacity of the cell extracts was reduced or lost by the activity of proteolytic enzymes, Tween 80, 2-mercaptoethanol, thymol, and sodium lauryl sulfate. Boiling the extract for 10 min did not alter its activity. The inactivating substance was postulated to be a lipoprotein of considerable complexity, unique in the ease with which it is solubilized from host cells by ethanol.     

Inhibition by α-Amanitin of Adenovirus 12 Replication in Human Embryo Kidney Cells and of Adenovirus Transformation of Hamster Cells

ADENOVIRUS infection of human embryonic kidney (HEK) cultures seems to induce cellular RNA synthesis, which is preceded by a transient increase in the activities of the Mg²⁺-activated and Mn²⁺-(NH₄)₂SO₄-activated DNA dependent RNA polymerases and in the rate of histone acetylation¹. The two polymerase reactions, assayed in isolated cell nuclei, apparently reflect the activities of distinct nucleolar and nucleo-plasmic RNA polymerases^2,3. We were therefore prompted to test the effect of a specific inhibitor of the mammalian DNA-dependent RNA polymerase function, α-amanitin, on the multiplication of adenovirus. α-Amanitin is a bicyclic octapeptide isolated from the poisonous mushroom Amanita phalloides⁴ and which blocks RNA synthesis in intact animals^5,6. Nuclei isolated from the livers of such animals show a reduced activity of the RNA polymerases associated with nucleoplasm^5,6 and the nucleolus⁶.     

Intracellular Calcium Changes in Neuronal Cells Induced by Alzheimer's ß-Amyloid Protein Are Blocked by Estradiol and Cholesterol

1. The elevation of intracellular Ca²⁺ levels ([Ca²⁺]_i) in immortalized hypothalamic neurons (GT1–7 cells) after exposure to Alzheimer's ß-amyloid protein (AßP[25–35]) was investigated using a multisite fluorometry system.2. The marked rise in [Ca²⁺]_i appeared afterexposure to 5–20-M AßP[25–35]. Analysis of the spatiotemporal patterns of [Ca²⁺]_i changes revealed that the magnitude and the latency of the response to AßP in each cell werehighly heterogeneous.3. The preadministration of 17ß-estradiol, 17-estradiol, phloretin and cholesterol, which influence the properties of membranes, such as membrane fluidity or membrane potential, significantly decreased the rise in [Ca²⁺]_i.4. These findings support the idea that disruption of calcium homeostasis by AßP channels may be the molecular basis of the neurotoxicity of AßP and of the pathogenesis of Alzheimer's disease. It is also suggested that membrane properties may play key roles in the expression of neurotoxicity.     

5′-Phosphodiesterase Formation by Cultured Plant Cells

5′-Phosphodiesterase (5′-PDase) which degrades RNA to nucleoside-5′-monophosphates was investigated in various kinds of plant calli, and the calli of Vinca rosea and Phytolacca americana were found to have the high activity. The liquid culture conditions of the cells of V. rosea were examined. Three mg of kinetin and 0.5 mg of 2,4-dichlorophenoxyacetic acid per liter in the Murashige and Skoog medium were optimal for the growth and the 5′-PDase formation. Under the optimal conditions, time courses of the cell growth and the enzyme formation were measured.

The 5′-PDase of the cultured cells of V. rosea in suspension showed the maximal activity at pH 8.0 and 60°C. A comparison of 5′-PDase of the cultured cells and of the mother plant of V. rosea was carried out and it was found that the cultured cells had more than 30 times as much 5′-PDase activity as the mother plant on dry cell weight basis.     

Enhancement of Regeneration Potential and Variability by γ-Irradiation in Cultured Cells of Scilla Indica

Induced mutagenesis in callus tissues was studied in the medicinal plant Scilla indica irradiated with different doses of -radiation ranging from 2.5 to 20 Gy. Low doses accelerated the cell division and growth rate of the tissues whereas high doses repressed growth rate and resulted in lethality of tissues. Various cytological and chromosomal abnormalities were observed in the irradiated calli, the degree of which depended upon the dosage. Low doses of irradiation also promoted the regenerating capacity of the calli tissues and plants regenerating from them exhibited better growth and vigour compared to normal plants. High doses led to loss of regenerating capacity and promoted formation of malformed and stunted plants. Cytological study of regenerants revealed both diploid and mixoploid plants but no tetraploids were obtained.     

Stem Cells and β Cells: The Same,but Different?

Researchers have long debated whether new pancreatic β cells derive from stem cells or from pre-existing β cells. A new study in this issue of Cell Stem Cell (Smukler et al., 2011) suggests that both sides may be right.     

γδ T Cells Are Reduced and Rendered Unresponsive by Hyperglycemia and Chronic TNFα in Mouse Models of Obesity and Metabolic Disease

Epithelial cells provide an initial line of defense against damage and pathogens in barrier tissues such as the skin; however this balance is disrupted in obesity and metabolic disease. Skin γδ T cells recognize epithelial damage, and release cytokines and growth factors that facilitate wound repair. We report here that hyperglycemia results in impaired skin γδ T cell proliferation due to altered STAT5 signaling, ultimately resulting in half the number of γδ T cells populating the epidermis. Skin γδ T cells that overcome this hyperglycemic state are unresponsive to epithelial cell damage due to chronic inflammatory mediators, including TNFα. Cytokine and growth factor production at the site of tissue damage was partially restored by administering neutralizing TNFα antibodies in vivo. Thus, metabolic disease negatively impacts homeostasis and functionality of skin γδ T cells, rendering host defense mechanisms vulnerable to injury and infection.     

Dual Control by Divalent Cations and Mitogenic Cytokines of α4β1 and α5β1 Integrin Avidity Expressed by Human Hemopoietic Cells

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function α4β1 and α5β1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by “inside-out” mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn²⁺, Mg²⁺ and Ca²⁺ for α4β1 and α5β1 activation by “inside-out” mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-β₁ integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of β₁ integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, α4β1 and α5β1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca²⁺, Mg²⁺ or Mn²⁺ were able to restore adhesive functions of α4β1 and α5β1 when activated by 8A2, only Mg²⁺ and Mn²⁺ were able to support activation of α5β1 and α5β1 by cytokines. Furthermore, high concentrations of Ca²⁺ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn²⁺ and cytokines but not by 8A2. On the contrary, in the presence of both Ca²⁺ and Mg²⁺, Mn²⁺ had an additive effect on the activation of α5β1 and α5β1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca²⁺, Mg²⁺ and Mn²⁺ may modulate in vivo α4β1 and α5β1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.     

Control of Murine Cytomegalovirus Infection by γδ T Cells

Infections with cytomegalovirus (CMV) can cause severe disease in immunosuppressed patients and infected newborns. Innate as well as cellular and humoral adaptive immune effector functions contribute to the control of CMV in immunocompetent individuals. None of the innate or adaptive immune functions are essential for virus control, however. Expansion of γδ T cells has been observed during human CMV (HCMV) infection in the fetus and in transplant patients with HCMV reactivation but the protective function of γδ T cells under these conditions remains unclear. Here we show for murine CMV (MCMV) infections that mice that lack CD8 and CD4 αβ-T cells as well as B lymphocytes can control a MCMV infection that is lethal in RAG-1^-/- mice lacking any T- and B-cells. γδ T cells, isolated from infected mice can kill MCMV infected target cells in vitro and, importantly, provide long-term protection in infected RAG-1^-/- mice after adoptive transfer. γδ T cells in MCMV infected hosts undergo a prominent and long-lasting phenotypic change most compatible with the view that the majority of the γδ T cell population persists in an effector/memory state even after resolution of the acute phase of the infection. A clonotypically focused Vγ1 and Vγ2 repertoire was observed at later stages of the infection in the organs where MCMV persists. These findings add γδ T cells as yet another protective component to the anti-CMV immune response. Our data provide clear evidence that γδ T cells can provide an effective control mechanism of acute CMV infections, particularly when conventional adaptive immune mechanisms are insufficient or absent, like in transplant patient or in the developing immune system in utero. The findings have implications in the stem cell transplant setting, as antigen recognition by γδ T cells is not MHC-restricted and dual reactivity against CMV and tumors has been described.