Cellular FLICE-inhibitory protein (c-FLIP) signalling: A key regulator of receptor-mediated apoptosis in physiologic context and in cancer

Cellular FLICE-inhibitory protein (c-FLIP) is a catalytically inactive procaspase-8/10 homologue that associates with the signalling complex downstream of death-receptors negatively interfering with apoptotic signalling. Three c-FLIP splice variants have been identified: c-FLIP_L, c-FLIP_S and c-FLIP_R, with all three functioning as apoptosis inhibitors involved in modulation of caspase-8/10 activity in both physiologic and pathologic contexts. Furthermore, a cell-type specific pro-apoptotic role, depending on caspase-8 to c-FLIP_L ratio, has also been described for the long isoform. The present review summarizes recent findings concerning c-FLIP proteins’ function and regulation, with a main focus on the c-FLIP_L deregulated expression in cancer. The role of c-FLIP_L as anti-apoptotic pro-survival factor in tumors and the potential utility of this molecule as a possible alternative therapeutic target are discussed.     

The activation of liver X receptors inhibits toll‐like receptor‐9‐induced foam cell formation

Toll‐like receptors (TLRs) are related to foam cell formation (FCF), key event in the establishment/progression of atherosclerosis. The activation of TLR2 and TLR4 can increase FCF. The aim of this study was to evaluate the role of TLR9 in FCF. Murine macrophages were treated with CpG‐ODN, TLR9 agonist, and oxidized particles of LDL (Paz‐PC) and FCF was analyzed by means of Oil Red O staining. The administration of CpG‐ODN plus Paz‐PC onto macrophages increased the amount of lipid droplets, correlated to increased levels of tumor necrosis factor (TNF)‐α, IFNβ, and IP‐10. The underlying mechanism by which TLR9 ligation influenced Paz‐PC in the FCF was NF‐κB‐ and IRF7‐dependent, as observed by higher levels of phosphorylated IκBα, increased nuclear translocation of the p65 subunit, lower levels of the total IKKα protein and higher release of interferon‐dependent cytokines, such as IP‐10. Liver X receptors (LXRs) regulate lipid cellular transport and negatively modulate TLR‐dependent signaling pathways. Indeed, the addition of GW3965, synthetic LXRs agonist, significantly reduced FCF after CpG‐ODN plus Paz‐PC stimulation. In this condition, we observed decreased levels of the nuclear translocation of the p65 subunit, related to the higher presence of LXRα into the nucleus. TNF‐α, IP‐10, and IFNβ levels were reduced by the administration of GW3965 following CpG‐ODN and Paz‐PC treatment. In conclusion, the activation of TLR9 facilitates the formation of foam cells in an NF‐κB‐ and IRF7‐dependent manner, countered by the activation of LXRs. This study further support LXRs as potential anti‐atherosclerotic target. J. Cell. Physiol. 223: 158–167, 2010. © 2009 Wiley‐Liss, Inc.     

Discovery and Characterization of Three New Escherichia coli Septal Ring Proteins That Contain a SPOR Domain: DamX,DedD, and RlpA

SPOR domains are ∼70 amino acids long and occur in >1,500 proteins identified by sequencing of bacterial genomes. The SPOR domains in the FtsN cell division proteins from Escherichia coli and Caulobacter crescentus have been shown to bind peptidoglycan. Besides FtsN, E. coli has three additional SPOR domain proteins—DamX, DedD, and RlpA. We show here that all three of these proteins localize to the septal ring in E. coli. The loss of DamX or DedD either alone or in combination with mutations in genes encoding other division proteins resulted in a variety of division phenotypes, demonstrating that DamX and DedD participate in cytokinesis. In contrast, RlpA mutants divided normally. Follow-up studies revealed that the SPOR domains themselves localize to the septal ring in vivo and bind peptidoglycan in vitro. Even SPOR domains from heterologous organisms, including Aquifex aeolicus, localized to septal rings when produced in E. coli and bound to purified E. coli peptidoglycan sacculi. We speculate that SPOR domains localize to the division site by binding preferentially to septal peptidoglycan. We further suggest that SPOR domain proteins are a common feature of the division apparatus in bacteria. DamX was characterized further and found to interact with multiple division proteins in a bacterial two-hybrid assay. One interaction partner is FtsQ, and several synthetic phenotypes suggest that DamX is a negative regulator of FtsQ function.Cell division in Escherichia coli is mediated by a collection of approximately 20 proteins, all of which localize to the midcell, where they form a structure called the septal ring, or divisome. About half of these proteins are essential for cell division. The corresponding temperature-sensitive mutants or depletion strains become filamentous and die under nonpermissive conditions. The remaining proteins are not essential under most laboratory conditions. In some cases null mutations reveal modest division defects, but in other cases division defects become apparent only under certain growth conditions or in combination with mutations in genes for other division proteins. For reviews of this topic, see references 18, 22, 29, and 67.One of the essential cell division proteins is a bitopic membrane protein named FtsN (see Fig. ​Fig.1A)1A) (13, 14). How FtsN facilitates cell division is not clear. Because overproduction of FtsN rescues a variety of mutants with lesions in genes for other cell division proteins [ftsA(Ts), ftsI(Ts) ftsQ(Ts), ftsEX null, ftsK null, and ftsP (sufI) null strains], it seems likely that one function of FtsN is to improve the assembly and/or stability of the septal ring (13, 20, 24, 30, 58, 63). Very recent evidence indicates that FtsN plays an important role in triggering constriction, probably by allosteric activation of some other component of the septal ring (26).Open in a separate windowFIG. 1.SPOR domain proteins included in this study. (A) Membrane topology and number of amino acids in each domain as retrieved from UniProt release 15.7 (http://www.uniprot.org) or the GTOP update of 15 December 2008 (http://spock.genes.nig.ac.jp/∼genome/gtop.html). N, amino terminus; CM, cytoplasmic membrane; OM, outer membrane. RlpA and VPA1294 have a covalently attached lipid at their amino termini. (B) Multiple-sequence alignment of SPOR domains shown in the present study to localize to the septal ring of E. coli. Sequences were aligned manually to the position-specific scoring matrix (PSSM) from http://www.ncbi.nlm.nih.gov/Class/Structure/pssm/pssm_viewer.cgi with the SPOR domain (Pfam accession no. 05036) as the PSSM identifier (PSSM ID). Residues with identity to those in the consensus sequence from the PSSM alignment are shaded gray. Numbers to the left refer to the first positions of the SPOR domains in the indicated proteins.A notable feature of FtsN is that it contains at its C terminus a peptidoglycan (PG) binding domain known as a SPOR domain (Pfam accession no. 05036) (23, 65, 72). SPOR domains are both common and widespread in bacteria. At the time of this writing (August 2009), over 1,500 proteins that contain a SPOR domain are listed in the Pfam database (23). These proteins come from over 500 bacterial species. The domain is named after the founding member of the protein family, a Bacillus subtilis protein named CwlC that is produced relatively late in the process of sporulation (41). CwlC, which comprises an N-terminal amidase domain and a C-terminal SPOR domain, facilitates release of the mature spore by degrading PG in the mother cell (48, 61).Our interest in SPOR domain proteins was piqued during a study of Vibrio parahaemolyticus (in collaboration with Linda McCarter) when we observed that a gene of unknown function, designated vpa1294, is highly induced in V. parahaemolyticus swarmer cells. The VPA1294 protein was annotated as a “putative DamX-related protein” (44; http://genome.gen-info.osaka-u.ac.jp/bacteria/vpara/). To learn about DamX, we turned to the EcoGene website (http://ecogene.org/) (57), which noted that (i) DamX from E. coli has an essentially unknown function, (ii) overproduction of DamX inhibits cell division (43), and (iii) DamX is one of four E. coli proteins that contain a SPOR domain, the others being the cell division protein FtsN and two proteins of unknown function, DedD and RlpA. Based on this information, we decided to investigate whether DamX, DedD, and RlpA are involved in cell division in E. coli. While this work was in progress, the Thanbichler laboratory demonstrated that Caulobacter crescentus has an FtsN-like protein that is needed for cell division (49) and the de Boer laboratory published a report on DamX, DedD, and RlpA from E. coli (26). We also learned that J. Maddock''s laboratory has been investigating DamX, DedD, and RlpA from E. coli (personal communication). Importantly, the major findings from all four laboratories are in general agreement: SPOR domain proteins are widespread in bacteria, many of these proteins are involved in cell division, and SPOR domains are sufficient for septal localization, probably because SPOR domains bind to septal PG.     

Synthesis and characterization of a C6 nucleoside analogue for the in vivo imaging of the gene expression of herpes simplex virus type-1 thymidine kinase (HSV1 TK)

The synthesis and biological evaluation of '6-(1,3-dihydroxyisobutyl)thymine' (DHBT; 1), which corresponds to 6-[3-hydroxy-2-(hydroxymethyl)propyl]-5-methylpyrimidine-2,4(1H,3H)-dione, is reported. DHBT (1) was designed as a new substrate for herpes simplex virus type-1 thymidine kinase (HSV1 TK). The compound was found to be exclusively phosphorylated by HSV1 TK, and to exhibit good binding affinity (Ki = 35.3+/-1.3 microM). Cell-proliferation assays with HSV1-TK-transduced human osteosarcoma cells (143B-TK+-HSV1-WT) and with both human-thymidine-kinase-1-negative (143B-TK-) and non-transduced parental (MG-63) cells indicate that 1 is less cytotoxic than the standard drug Ganciclovir. Thus, DHBT (1) represents a promising precursor of a nontoxic reporter probe for the monitoring of HSV1 TK gene expression by means of positron-emission tomography (PET).     

Optimization of enantioselective resolution of racemic ibuprofen by native lipase from Aspergillus niger

Resolution of (R,S)-ibuprofen (2-(4-isobutylphenyl)propionic acid) enantiomers by esterification reaction with 1-propanol in different organic solvents was studied using native Aspergillus niger lipase. The main variables controlling the process (enzyme concentration and 1-propanol:ibuprofen molar ratio) have been optimized using response surface methodology based on a five-level, two-variable central composite rotatable design, in which the selected objective function was enantioselectivity. This enzyme preparation showed preferentially catalyzes the esterification of R(−)-ibuprofen, and under optimum conditions (7% w/v of enzyme and molar ratio of 2.41:1) the enantiomeric excess of active S(+)-ibuprofen and total conversion values were 79.1 and 48.0%, respectively, and the E-value was 32, after 168 h of reaction in isooctane.     

Phytoremediation of two types of sediment contaminated with Zn by Schoenoplectus americanus

The effect of different sediments on growth, Zn uptake, Zn plant distribution, and morphometric variables of Schoenoplectus americanus were investigated under controlled conditions. Two types of sediments were assayed: from a large natural levee (LS) and alluvial sediments (AS), the former with lower organic matter (OM) and nutrients content than AS, without and with added Zn (2500 microg Zn/g air-dry sediment). Zinc partition in sediment was determined. Increases in water conductivity and Zn concentrations in water and sediments were observed in artificially contaminated treatments. Plants showed a lower above ground growth rate, height, and width of shoots, and a higher Zn concentration in shoots and rhizomes. In the contaminated treatments, AS treatment showed lower Zn concentration in water and higher Zn concentration in sediments (total, exchangeable, and OM fractions) than LS treatment, due to Zn displacement from floodwater to sediments. The presence of a high level of OM and nutrients also increased aboveground biomass growth, whereas it decreased Zn concentration in shoots. Although the translocation factor increased with Zn addition, it was lower in AS treatment Sediments of AS treatments are a suitable environment for growth of S. americanus, which partially compensates the toxic effects of Zn. Our results provide an encouraging basis for planning larger scale experiments to test the role of OM and nutrients in improving phytoremediation.     

Immunoconjugate generation between the ribosome inactivating protein restrictocin and an anti-human breast carcinoma MAB

Summary Treatment of Wistar/AG rats with a single i.p. injection of 1 mg/kg of synthetic double-stranded polynucleotides, either polyadenylic-polyuridylic acid (rA_n.rU_n), or a mismatched analogue of polyinosinic-polycytidylic acid (rI_n.r(C₁₂U)_n), enhanced the cytotoxicity of natural killer (NK) cells among peripheral blood leukocytes and lung intracapillary leukocytes (LICL). The enhancement reached a peak 24 h after treatment and returned to control values after 4 days. In rats given repeated injections of double-stranded polynucleotides (2 per week), the NK cytotoxicity expressed by LICL reached more than ten times (in lytic units) the control levels between day 8, after 3 injections, and day 360, after 100 injections. No hypore-sponsiveness was observed. Moreover, NK activity was frequently and significantly higher in rats given multiple injections than in those given a single injection. In rats with experimentally induced P77 lung fibrohistiocytoma colonies, repeated injections of rI_n.r(C₁₂U)_n stimulated NK activity and reduced the number of metastatic nodules from 172 to 19. The same significant reduction (from 172 to 27) was also observed in animals given repeated injections of rA_n.rU_n. However, with two models of spontaneous metastases, significant reduction in lung metastases (M37 bronchioloalveolar carcinoma) or lack of effect (S₄T₁₉ rhabdomyosarcoma) were observed.     

Comparison Between Two Human Endogenous Retrovirus (HERV)-Rich Regions Within the Major Histocompatibility Complex

Sixteen human endogenous retrovirus (HERV) sequences were detected within 656 kb of genomic sequence obtained from the alpha- and beta-block of the class I region of the major histocompatibility complex (MHC). The HERVs were identified and characterized as family members of HERV-16 (11 copies), HERV-L (1 copy), HERV-I (2 copies), HERV-K91 (1 copy), and HARLEQUIN (1 copy) by sequence comparison using CENSOR or Repeat Masker, BLAST searches, and dot plots. The 11 copies of HERV-16 arose as products of duplication of genomic segments containing HLA class I (HLAcI) and PERB11 (MIC) genes inter alia, whereas the other five HERVs arose after duplication probably as a consequence of single insertion events or translocations. HERV-L and HERV-I are located between the duplicated genes PERB11.2 (MICB) and PERB11.1 (MICA), and HLA-B and HLA-C, respectively, whereas HERV-K91 and HARLEQUIN are located telomeric of HLA-C. A highly fragmented copy of HERV-I was also found telomeric of PERB11.4. Structural analysis of open reading frames (ORFs) revealed the absence of intact coding sequence within the putative gag, pol, and env gene regions of all the HERVs with the exception of HERV-K91, which had two large ORFs within the region of the putative protease and pol genes. In addition, the 5′-LTR of HERV-L contained a 2.5-kb element that was AT-rich and large ORFs with putative amino acid sequences rich in tyrosines and isoleucines. HERV-I, HARLEQUIN, and at least four copies of HERV-16 appear to have been receptors for the insertion of other retrotransposons including Alu elements and fragments of L1 and THE1. Examination of flanking sequences suggests that HERV-I and HERV-L had occurred by insertion into ancient L1 fragments. This study has revealed that the alpha- and beta-block region within the MHC is rich in HERV sequences occurring at a much higher ratio (10 to 1) than normally observed in the human genome. These HERV sequences will therefore enhance further studies on disease associations and differences between human haplotypes and primates and their role in the evolution of class I genes in the MHC. Received: 17 September 1998 / Accepted: 8 January 1999