Anti-inflammatory effects of moxifloxacin and human beta-defensin 2 association in human lung epithelial cell line (A549) stimulated with lipopolysaccharide

Epithelia in the human airways, from the nasal aperture to the alveoli, are covered in a protective film of fluid containing a number of antimicrobial proteins. Defensins are single-chain, strongly cationic peptides and are one of the most extensively studied classes of antimicrobial peptides. Moxifloxacin (MXF) is a fluoroquinolone that acts against both Gram positive and Gram negative bacteria. In this study, we evaluated the effects of HBD2, MXF and the association MXF/HBD2 on some cytokines and on the ICAM-1 expression in LPS-stimulated A549 cells. Our results suggest that by lowering the epithelial cell-derived IL-1β, IL-6, IL-8 and ICAM-1 expression, the MXF/HBD2 association interferes with the multifunctional cytokine network evolving during inflammatory processes of the respiratory tract; this anti-inflammatory potential could be of great value in the treatment of inflammatory disorders.     

Smad mediates BMP-2-induced upregulation of FGF-evoked PC12 cell differentiation

To gain insight into presenilin-1 (PS1) structural aspects, we explored the structure–function relationship of its N- and C-terminal (NTF and CTF, respectively) complexes. We demonstrated that both NTF and CTF act as independent but inter-changing binding units capable of binding each other (NTF/CTF) or their homologues (NTF/NTF; CTF/CTF). The Alzheimer’s disease-associated PS1 mutations Y115H and M146L do not affect their ability to hetero- and/or homodimerize, thus conserving their basic integrity and function(s). These results suggest that PS1 associates intra-molecularly to form higher order complexes, which may be needed for endoproteolytic cleavage and/or γ-secretase-associated activity.     

DNA methyltransferase inhibition induces mouse embryonic stem cell differentiation into endothelial cells

Understanding endothelial cell (EC) differentiation is a step forward in tissue engineering, controlling angiogenesis, and endothelial dysfunction. We hypothesized that epigenetic activation of EC lineage specification genes is an important mediator of embryonic stem cell (ESC) differentiation into EC. Mouse ESC was differentiated by removing leukemia inhibitory factor (LIF) from the maintenance media in the presence or absence of the specific DNA methyltransferase (DNMT) inhibitor 5′-aza-2′-deoxycytidine (aza-dC). Expression of EC specification and marker genes was monitored by quantitative PCR, western, immunocytochemistry, and flow cytometry. Functionality of differentiated EC was assessed by angiogenesis assay. The methylation status in the proximal promoter CpGs of the mediators of EC differentiation VEGF-A, BMP4, and EPAS-1 as well as of the mature EC marker VE-cadherin was determined by bisulfite sequencing. ESC differentiation resulted in repression of OCT4 expression in both the absence and presence of aza-dC treatment. However, significant increase in angiogenesis and expression of the mediators of EC differentiation and EC-specific genes was only observed in aza-dC-treated cells. The DNMT inhibition-mediated increase in EC specification and marker gene expression was not associated with demethylation of these genes. These studies suggest that DNMT inhibition is an efficient inducer of EC differentiation from ESC.     

Effects of TFAR19 gene on the growth and biorheological properties of mouse erythroleukemia cell line MEL

Apoptosis is a genetically controlled program of cellular self-destruction, which plays a key role in the development and homeostasis of virtually all animals. In recent years, Liu et al.[1] found a novel apoptosis-related gene TFAR19 (TF-1 cells apoptosis-related gene 19). It is conserved in evolution and distributed extensively in tissues. The related study showed that its coding protein is probably very important for the signal transduction in cell nucleus, regulating the cell proliferat…     

Proteomic-based analysis of nuclear signaling: PLCbeta1 affects the expression of the splicing factor SRp20 in Friend erythroleukemia cells

An extensive body of evidence links inositide-specific phospholipase C (PLC) to the nucleus and the main isoform located in the nucleus is PLCbeta(1). Constitutive overexpression of nuclear PLCbeta(1) has been previously shown to inhibit Friend erythroleukemia cells differentiation and to induce cell cycle progression targeting cyclin D3. The aim of this study was to identify new proteins regulated by PLCbeta(1) overexpression, given the role exerted by its signaling in the nucleus during cell growth and differentiation. To identify novel downstream effectors of nuclear PLCbeta(1)-dependent signaling in Friend erythroleukemia cells, we performed the high-resolution 2-DE-based proteomic analysis. Using a proteomic approach we found that SRp20, a member of the highly conserved SR family of splicing regulators, was down-regulated in cells overexpressing nuclear PLCbeta(1) as compared with wild-type cells. Reduction in SRp20 was confirmed by 2-D Western blotting. Moreover, we have shown that nuclear PLCbeta(1) is bound to the SRp20 splicing factor. Indeed, by immunoprecipitation and subcellular fractioning, we have demonstrated that endogenous PLCbeta(1) and SRp20 physically interact in the nucleus. Here we show the existence of a PLCbeta(1)-specific target, the splicing factor SRp20, whose expression is specifically down-regulated by the nuclear signaling evoked by PLCbeta(1).     

(2'-5')An-dependent endoribonuclease: enzyme levels are regulated by IFN beta, IFN gamma, and cell culture conditions

The levels of a (2'-5')An-dependent endonuclease (RNase L) were determined in extracts prepared from murine L cells and Ehrlich ascites tumor (EAT) cells by measuring specific binding of protein to a labeled derivative of (2'-5')An, (2'-5')A3[32P]pCp. RNase L levels were found to depend both on interferon (IFN) treatment and on cell growth conditions. Treatment of murine L cells and EAT cells with 100-2,000 IRU IFN beta or IFN gamma resulted in a similar 2-4-fold increase in the levels of RNase L when cells were present at low density. The levels of RNase L were also shown to increase 2-3-fold as cells approached saturation density. Serum-starved cells also displayed relatively high levels of RNase L. RNase L levels in cells maintained at high cell density did not change appreciably following treatment with IFN beta or IFN gamma. Regulation of RNase L levels by cell growth conditions as well as by IFN beta or IFN gamma treatment suggests that RNase L may play an important role in regulating the levels of cellular mRNAs as well as acting to degrade viral RNAs.     

The effect of heme on intracellular iron pools during differentiation of friend erythroleukemia cells

The inhibition of cellular iron uptake by hemin described previously in reticulocytes was studied in murine erythroleukemia (Friend) cells that can be induced to differentiate in culture by dimethyl sulfoxide (DMSO). Hemin had no effect on iron uptake into noninduced cells. After the induction by DMSO, hemin inhibited iron uptake into Friend cells and this effect of hemin became more pronounced with the further progress of differentiation. The reduction of cellular iron accumulation was caused mainly by inhibition of iron incorporation into heme, iron uptake into the non-heme pool was influenced by hemin treatment. Inhibition of heme synthesis by isonicotinic acid hydrazide (INH) caused an accumulation of iron in mitochondria in DMSO-induced cells but not in uninduced cells. On the basis of these results, a specific system transporting iron to mitochondria induced by DMSO treatment is suggested as a target for the inhibitory action of hemin. In Friend cells of the Fw line which are deficient in ferrochelatase, heme has no effect on iron uptake. The addition of INH to the Fw cells does not enhance the iron accumulatoni in mitochondria.     

An optimization approach to predicting protein structural class from amino acid composition.

Proteins are generally classified into four structural classes: all-alpha proteins, all-beta proteins, alpha + beta proteins, and alpha/beta proteins. In this article, a protein is expressed as a vector of 20-dimensional space, in which its 20 components are defined by the composition of its 20 amino acids. Based on this, a new method, the so-called maximum component coefficient method, is proposed for predicting the structural class of a protein according to its amino acid composition. In comparison with the existing methods, the new method yields a higher general accuracy of prediction. Especially for the all-alpha proteins, the rate of correct prediction obtained by the new method is much higher than that by any of the existing methods. For instance, for the 19 all-alpha proteins investigated previously by P.Y. Chou, the rate of correct prediction by means of his method was 84.2%, but the correct rate when predicted with the new method would be 100%! Furthermore, the new method is characterized by an explicable physical picture. This is reflected by the process in which the vector representing a protein to be predicted is decomposed into four component vectors, each of which corresponds to one of the norms of the four protein structural classes.     

Close link between development and function of gamma-delta T cells

Murine γδ T cells develop as the first T-cell lineage within the fetal thymus and disproportionately localize in mucosal tissues such as lung, skin, uterus, and intestine of adult mice. These unique developmental features and distribution patterns of γδ T cells enable rapid functioning against various insults from pathogens. γδ T cells are also able to respond to local inflammation and consequently regulate the pathogenesis of autoimmune disorders and development of tumors in mice and humans. Hence, it is clinically important to understand the mechanisms that regulate γδ T cell functions. Recent evidence has shown that generations of effector γδ T cell subsets producing IFN-γ, IL-4, and IL-17 are programmed in the murine thymus before their migration to peripheral tissues. This review outlines our current understanding of the development and function of γδ T cells as they influence both innate and acquired immunity.     

Overexpression of regucalcin suppresses cell response for tumor necrosis factor-alpha or transforming growth factor-beta1 in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The regulatory role of regucalcin on cell responses for tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1) was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2-transfected cells (transfectant) were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture, cells were further cultured for 24-72 h in medium without BS containing either vehicle, TNF-alpha (0.1 or 1.0 ng/ml of medium), or TGF-beta1 (1.0 or 5.0 ng/ml). Culture with TNF-alpha or TGF-beta1 caused a significant decrease in the number of wild-type cells. This decrease was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with TNF-alpha (1.0 ng/ml) or TGF-beta1 (5.0 ng/ml). This DNA fragmentation was significantly suppressed in transfectants. TNF-alpha- or TGF-beta1-induced cell death was significantly prevented in culture with caspase-3 inhibitor (10(-8) M). Nitric oxide (NO) synthase activity in wild-type cells was significantly increased by addition of calcium chloride (10 microM) and calmodulin (5 microg/ml) into the enzyme reaction mixture. This increase was significantly suppressed in transfectants. Culture with TNF-alpha caused a significant increase in NO synthase activity in wild-type cells. The effect of TNF-alpha was not seen in transfectants. Culture with TGF-beta1 did not cause a significant increase in NO synthase activity in wild-type cells and transfectants. Culture with TNF-alpha or TGF-beta1 caused a remarkable increase in alpha-smooth muscle actin in wild-type cells. This increase was significantly prevented in transfectants. The expression of Smad 2 or NF-kappaB mRNAs was significantly increased in transfectants as compared with that of wild-type cells. Smad 3 or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expression was not significantly changed in transfectants. NF-kappaB mRNA expression in wild-type cells was significantly increased with culture of TNF-alpha. Smad 2 mRNA expression was significantly enhanced in wild-type cells cultured with TGF-beta1. These effects of TNF-alpha or TGF-beta1 were not significantly enhanced in transfectants. This study demonstrates that overexpression of regucalcin has suppressive effects on cell responses which are mediated through intracellular signaling pathways of TNF-alpha or TGF-beta1 in kidney NRK52E cells.     

Laminin-5 suppresses chondrogenic differentiation of murine teratocarcinoma cell line ATDC5

Laminin-5 is an important basement membrane protein that regulates cell adhesion and motility. It was previously found that the gamma2 chain of laminin-5 is transiently expressed in embryonic cartilage. This suggests a possible role of laminin-5 in chondrogenesis. Here, we examined this possibility using the murine teratocarcinoma cell line ATDC5. ATDC5 cells transiently and weakly expressed laminin-5 when they were stimulated for differentiation. Exogenous laminin-5 in either insoluble or soluble form strongly inhibited the differentiation phenotypes, i.e. formation of cartilaginous cell aggregates and production of chondrogenic marker proteins through its integrin-binding domain LG3 in the alpha3 chain. Laminin-5 had no effect on cell growth. In addition, we found that the laminin-5 with the 105-kDa, processed gamma2 chain suppressed differentiation more strongly than one with the 150-kDa gamma2 chain. This indicated that the proteolytic processing of gamma2 chain regulated the activity of laminin-5. However, a gamma2 chain short arm fragment had no effect on the chondrogenesis, and it rather suppressed the differentiation at excessive concentrations. These results suggest that laminin-5 and its processing modulate chondrogenic differentiation during development.