Dehydration mediated microRNA response in the African clawed frog Xenopus laevis

Exposure to various environmental stresses induces metabolic rate depression in many animal species, an adaptation that conserves energy until the environment is again conducive to normal life. The African clawed frog, Xenopus laevis, is periodically subjected to arid summers in South Africa, and utilizes entry into the hypometabolic state of estivation as a mechanism of long term survival. During estivation, frogs must typically deal with substantial dehydration as their ponds dry out and X. laevis can endure > 30% loss of its body water. We hypothesize that microRNAs play a vital role in establishing a reversible hypometabolic state and responding to dehydration stress that is associated with amphibian estivation. The present study analyzes the effects of whole body dehydration on microRNA expression in three tissues of X. laevis. Compared to controls, levels of miR-1, miR-125b, and miR-16-1 decreased to 37 ± 6, 64 ± 8, and 80 ± 4% of control levels during dehydration in liver. By contrast, miR-210, miR-34a and miR-21 were significantly elevated by 3.05 ± 0.45, 2.11 ± 0.08, and 1.36 ± 0.05-fold, respectively, in the liver. In kidney tissue, miR-29b, miR-21, and miR-203 were elevated by 1.40 ± 0.09, 1.31 ± 0.05, and 2.17 ± 0.31-fold, respectively, in response to dehydration whereas miR-203 and miR-34a were elevated in ventral skin by 1.35 ± 0.05 and 1.74 ± 0.12-fold, respectively. Bioinformatic analysis of the differentially expressed microRNAs suggests that these are mainly involved in two processes: (1) expression of solute carrier proteins, and (2) regulation of mitogen-activated protein kinase signaling. This study is the first report that shows a tissue specific mode of microRNA expression during amphibian dehydration, providing evidence for microRNAs as crucial regulators of metabolic depression.     

P2X7 receptor activation induces cell death and CD23 shedding in human RPMI 8226 multiple myeloma cells

Background

The extracellular ATP-gated cation channel, P2X7 receptor, has an emerging role in neoplasia, however progress in the field is limited by a lack of malignant cell lines expressing this receptor.

Methods

Immunofluorescence labelling and a fixed-time ATP-induced ethidium⁺ uptake assay were used to screen a panel of human malignant cell lines for the presence of functional P2X7. The presence of P2X7 was confirmed by RT-PCR, immunoblotting and pharmacological approaches. ATP-induced cell death was measured by colourimetric tetrazolium-based and cytofluorometric assays. ATP-induced CD23 shedding was measured by immunofluorescence labelling and ELISA.

Results

RPMI 8226 multiple myeloma cells expressed P2X7 mRNA and protein, as well as P2X1, P2X4 and P2X5 mRNA. ATP induced ethidium⁺ uptake into these cells with an EC₅₀ of ~ 116 μM, and this uptake was reduced in the presence of extracellular Ca²⁺ and Mg²⁺. The P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not UTP, induced ethidium⁺ uptake. ATP-induced ethidium⁺ uptake was impaired by the P2X7 antagonists, KN-62 and A-438079. ATP induced death and CD23 shedding in RPMI 8226 cells, and both processes were impaired by P2X7 antagonists. The metalloprotease antagonists, BB-94 and GM6001, impaired ATP-induced CD23 shedding but not ethidium⁺ uptake.

Conclusions

P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells.

General significance

RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes.     

Increase in CpG DNA-induced inflammatory responses by DNA oxidation in macrophages and mice

Unmethylated CpG dinucleotide (CpG motif) is involved in the exacerbation of DNA-associated autoimmune diseases. We investigated the effect of DNA containing 8-hydroxydeoxyguanosine (oxo-dG), a representative DNA biomarker for oxidative stress in the diseases, on CpG motif-dependent inflammatory responses. ODN1668 and ODN1720 were selected as CpG-DNA and non-CpG DNA, respectively. Deoxyguanosine in the CpG motif (G9) or outside the motif (G15) of ODN1668 was substituted with oxo-dG to obtain oxo(G9)-1668 and oxo(G15)-1668, respectively. Oxo(G15)-1668 induced a significantly higher amount of tumor necrosis factor (TNF)-α from RAW264.7 macrophage-like cells than ODN1668, whereas oxo(G9)-1668, oxo(G8)-1720, or oxo(G15)-1720 hardly did. CpG DNA-induced TNF-α production was significantly increased by addition of oxo(G8)-1720 or oxo(G15)-1720, but not of ODN1720. This oxo-dG-containing DNA-induced increase in TNF-α production was also observed in primary cultured macrophages isolated from wild-type mice, but not observed in those from Toll-like receptor (TLR)-9 knockout mice. In addition, TNF-α production by ligands for TLR3, TLR4, or TLR7 was not affected by oxo-dG-containing DNA. Then, the footpad swelling induced by subcutaneous injection of ODN1668 into mice was increased by coinjection with oxo(G8)-1720, but not with ODN1720. These results indicate that oxo-dG-containing DNA increases the CpG motif-dependent inflammatory responses, which would exacerbate DNA-related autoimmune diseases.     

Requirement of c-Jun NH2-terminal kinase activation in interferon-alpha-induced apoptosis through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma cells

Interferon alpha (IFN-alpha) inhibits growth, at least in part, through induction of apoptosis. However, the molecular mechanisms underlying IFN-alpha-induced apoptosis are not completely understood. In the present study, we found that IFN-alpha induced a sustained activation of c-Jun N-terminal kinase 1 (JNK1), but not extracellular kinases (ERKs), in Daudi B lymphoma cells, as assessed by Western blotting using phospho-specific antibodies. Several lines of evidence support the notion that the IFN-alpha-induced activation of JNK is responsible for IFN-alpha-induced apoptosis, at least in part, through upregulation of TNF-related apoptosis-inducing ligand (TRAIL). First, pretreatment of Daudi cells with a JNK inhibitor reduced IFN-alpha-induced upregulation of TRAIL and loss of mitochondrial membrane potential (DeltaPsim) and annexin-positive cells, which was assessed by flow cytometry. Second, a dominant-negative form of JNK1 (dnJNK1) also reduced these apoptotic events, while a constitutively active form of JNK1, MKK7-JNK1beta, enhanced them. Finally, treatment with IFN-alpha enhanced the promoter activity of the TRAIL gene, which was partially abrogated by either JNK inhibitor or dnJNK1, while it was moderately enhanced by MKK7-JNK1beta. These findings are useful for understanding molecular mechanisms of IFN-alpha-induced apoptosis and also for development of treatment modalities of some tumors with IFN-alpha.     

Lysophosphatidic acid inhibits anti-Fas-mediated apoptosis enhanced by actin depolymerization in epithelial ovarian cancer

Conflicting reports exist on the effect of actin depolymerization in anti-Fas-induced apoptosis. Lysophosphatidic acid (LPA) has been found to inhibit apoptosis in variable cell types. In this study, we evaluated LPA's protective effects on anti-Fas-induced apoptosis enhanced by actin depolymerization and possible mechanisms in epithelial ovarian cancer. OVCAR3 cells were pretreated with vehicle or LPA, then treated with Cytochalasin D (Cyto D), followed with anti-Fas mAb to induce apoptosis. Cells were stained with apoptotic markers and analyzed by flow cytometry. We report that LPA inhibited anti-Fas-induced apoptosis enhanced by actin depolymerization. Immunoprecipition of Fas death-inducing signaling complex (DISC) and Western blot suggested that the actin depolymerization accelerated caspase-8 activation, while LPA inhibited the association and activation of caspase-8 at the DISC. LPA inhibited caspase-3 and 7 activation induced by anti-Fas and/or Cyto D in cytosols. Phosphorylation of ERK and Bad112 by LPA may play a role in preventing caspase-3 activation through mitochondrial pathway induced by Cyto D. Our investigation found that LPA inhibited anti-Fas-induced apoptosis enhanced by actin depolymerization, and LPA may protect epithelial ovarian cancer from immune cell attack and cytoskeleton disrupting reagents induced apoptosis through multiple pathways.     

Detailed analysis of inflammatory and neuromodulatory cytokine secretion from human NT2 astrocytes using multiplex bead array

Astrocytes are a very important cell type in the brain fulfilling roles in both neuroimmunology and neurotransmission. We have conducted the most comprehensive analysis of secreted cytokines conducted to date (astrocytes of any source) to determine whether astrocytes derived from the human Ntera2 (NT2) cell-line are a good model of human primary astrocytes. We have compared the secretion of cytokines from NT2 astrocytes with those produced in astrocyte enriched human brain cultures and additional cytokines implicated in brain injury or known to be expressed in the human brain. The concentration of cytokines was measured in astrocyte conditioned media using multiplex bead array (MBA), where 18 cytokines were measured simultaneously. Resting NT2 astrocytes produced low levels (～1-30 pg/ml) of MIP1α, IL-6 and GM-CSF and higher levels of MCP-1, IP-10 and IL-8 (1-11 ng/ml) under non-inflammatory conditions. All of these in addition to IL-1β, TNFα, and IL-13, were increased by pro-inflammatory activation (TNFα or IL-1β stimulation). In contrast, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, LTα, and IFNγ were not detected in astrocyte conditioned media under any of the culture conditions tested. NT2 astrocytes were unresponsive to IL-2 and the adenyl cyclase agonist, forskolin. Interestingly, IFNγ stimulation selectively increased IP-10 secretion only. As astrocytes stimulated with IL-1β or TNFα produced several chemokines in the ng/ml range, we next assessed the chemoattractant properties of these cells. Conditioned media from TNFα-stimulated astrocytes significantly chemoattracted leukocytes from human blood. This study provides the most comprehensive analysis of cytokine production by human astrocytes thus far, and shows that NT2 astrocytes are highly responsive to pro-inflammatory mediators including TNFα and IL-1β, producing cytokines and chemokines capable of attracting leukocytes from human blood. We conclude that in the absence of adult human primary astrocytes that NT2-astrocytes may provide a valuable alternative to study the immunological behaviour of human astrocytes.     

The significant increase of FcγRIIIA (CD16), a sensitive marker, in patients with coronary heart disease

Our previous studies suggest that Fc receptor III A of immunoglobulin G (FcγRIIIA, also named CD16) is closely correlated to coronary heart disease (CHD). However, whether or not deregulated FcγRIIIA expression is involved in the development of CHD remains largely unclear. Herein, we investigated the FcγRIIIA mRNA expression in the leukocytes, the serum protein level of soluble CD16 (sCD16) and membrane CD16 on monocytes from 100 diagnosed CHD patients and 40 healthy individuals. Our results demonstrated that there was a significant increase of FcγRIIIA at the mRNA level in leukocytes, and at the protein level for both sCD16 in sera and membrane CD16 on monocytes from CHD patients compared to the healthy control. Similarly to the soluble CD14 (sCD14), the level of macrophage colony stimulating factor (M-CSF) in sera was also higher in CHD patients than that in the control individuals. Furthermore, the levels of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), in sera and the mean fluorescent intensity of intercellular adhesion molecule 1 (ICAM-1, CD54) on CD14(+) CD16(+) monocytes were increased in CHD patients. Overall, these data demonstrated that FcγRIIIA (CD16) is involved in the pathogenesis of CHD by activating monocytes and stimulating inflammation. The significant increase of CD14(+) CD16(+) monocytes in CHD patients therefore suggested that the increase of the FcγRIIIA level might be a sensitive marker for the CHD diagnosis.     

An entirely cell-based system to generate single-chain antibodies against cell surface receptors

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.     

Effect of the headgroup variation on the gene transfer properties of cholesterol based cationic lipids possessing ether linkage

Eight cholesterol based cationic lipids differing in the headgroup have been synthesized based on the ether linkage between the cationic headgroup and the cholesterol backbone. All the lipids formed stable suspensions in water. Transfection efficacies were examined in the absence and presence of serum using their optimized liposomal (lipid:DOPE) formulations. Our results showed that the transfection activities depend on the nature of the headgroup. Lipid bearing 4-N,N′-dimethylaminopyridine (DMAP) as headgroup showed the maximum transfection efficacy in the presence of serum. Importantly, the optimized formulation for this cationic lipid does not require DOPE, which is being used by most commercially available formulations. Cytotoxicity studies showed that the introduction of the positive charge decreases the cell viability of the cationic lipid formulations. Gel electrophoresis and Ethidium bromide exclusion assay revealed the different DNA binding abilities of formulations depending upon the headgroup of the cholesteryl lipid.