TTDA/hTFB5 gene. One of these mutations completely inactivates the protein, whereas other TFIIH genes only tolerate point mutations that do not compromise the essential role in transcription. Nevertheless, the severe NER-deficiency in TTD-A suggests that the TTDA protein is critical for repair. Using a fluorescently tagged and biologically active version of TTDA, we have investigated the involvement of TTDA in repair and transcription in living cells. Under non-challenging conditions, TTDA is present in two distinct kinetic pools: one bound to TFIIH, and a free fraction that shuttles between the cytoplasm and nucleus. After induction of NER-specific DNA lesions, the equilibrium between these two pools dramatically shifts towards a more stable association of TTDA to TFIIH. Modulating transcriptional activity in cells did not induce a similar shift in this equilibrium. Surprisingly, DNA conformations that only provoke an abortive-type of NER reaction do not result into a more stable incorporation of TTDA into TFIIH. These findings identify TTDA as the first TFIIH subunit with a primarily NER-dedicated role in vivo and indicate that its interaction with TFIIH reflects productive NER.Transcription/repair factor IIH (TFIIH) is essential for RNA polymerase II transcription and nucleotide excision repair (NER). This multi-subunit complex consists of ten polypeptides, including the recently identified small 8-kDa trichothiodystrophy group A (TTDA)/ hTFB5 protein. Patients belonging to the rare neurodevelopmental repair syndrome TTD-A carry inactivating mutations in the Transcription/repair factor IIH (TFIIH) is a multi-subunit protein complex essential for RNA polymerase II transcription and nucleotide excision repair (NER). The authors show that the TTDA subunit is associated with TFIIH specifically during NER. DNA-damaging agents constantly challenge the integrity of DNA. A network of distinct DNA-repair systems collectively removes most injuries and safeguards the stability of the genome . NucleotBesides GG-NER and TC-NER, TFIIH is also engaged in RNA polymerase II transcription initiation, RNA polymerase I transcription, activated transcription, and cell cycle regulation –20. TFITTDA gene [TTDA gene of three non-related patients with TTD-A, lead either to the complete absence of the protein (mutation on the first ATG), or to non-functional truncated peptides, making TTDA the first TFIIH subunit for which a complete absence is compatible with life [Cells from patients with TTD-A have reduced steady-state levels of TFIIH, due to mutatedDA gene , suggestth life . DespiteIn order to investigate the participation of TTDA in DNA repair and transcription, we measured the differences in mobility of TTDA during repair and transcription in living cells by confocal imaging of a functional green fluorescent protein (GFP)-tagged TTDA (TTDA-GFP) expressed in TTDA-deficient transformed fibroblasts. To compare the mobility of TTDA with others TFIIH subunits, we also measured kinetic parameters of XPB-GFP and XPD-GFP in each of these processes.To investigate the localization, behavior and dynamic properties of TTDA in the most relevant biologically context—the living cell, and under different conditions, such as transcriptional interference and introduction of DNA lesions, we have generated a GFP-tagged TTD A . To compImmunoprecipitation assays showed that the TFIIH core factor, XPB, co-precipitates with both fusion proteins, using anti-GFP cross-linked sepharose beads and extracts of TTDA-GFP D. In addHigh-resolution confocal imaging  of TTDA-The cytoplasmic localization of both XPD-GFP and TTDA-GFP argues for a possible assembly of TFIIH-related subcomplexes within the cytoplasm. In order to investigate this, we measured the mobility of TTDA-GFP, XPB-GFP, XPD-GFP, and GFP in both the cytoplasm and the nucleus by photo-bleaching experiments. We applied a tailor-made variant of fluorescence recovery after photo-bleaching (FRAP)  in whichWithin the cytoplasm A, the moIn order to eliminate the fluorescence of this non-incorporated fraction, we modified the FRAP procedure by pre-bleaching the cytoplasmic fraction see. AssuminTo investigate whether the presence of DNA damage influences the equilibrium between the various TTDA-GFP pools, we compared the dynamic behavior of TTDA-GFP before and after UV irradiation (measured between 5 to 30 min after exposure) . FRAP_abWe further explored the participation of the three TFIIH components in NER by determining the binding time of each of the components when actively engaged in NER. To that aim, we applied FRAP on locally UV-damaged cells C, as wasThis tighter association of TTDA-GFP to TFIIH during repair can be explained in two ways: TTDA is more tightly anchored to TFIIH when  bound to DNA or TTDA is indeed a NER-specific subunit of TFIIH docking with higher affinity to the complex when actively repairing. A standard not NER-related DNA interaction of TFIIH is of course its transient interaction with promoter sequences during transcription initiation. Varying the transcriptional competence of the cells would thus also affect the equilibrium of TFIIH-bound versus free TTDA. To verify this hypothesis, we measure the mobility of TTDA-GFP during inhibition of transcription by DRB , known to increase overall mobility of XPB-GFP by the reduced number of interactions with transcription initiation complexes . As showNext we tested the effect of actinomycin D (ActD), classically used as a transcription elongation inhibitor but also known to interfere with other DNA transactions –35. In TFIIH is implicated in a multitude of DNA-transactions, ranging from basal and activated RNA polymerase II transcription, to ribosomal RNA gene expression, participation in GG-NER and TC-NER, and possibly cell-cycle regulation. The complexity of this factor is also reflected by the dynamic composition of distinct subcomplexes, as well as its multiple enzymatic activities: DNA-dependent ATPases and helicases, kinase, and ubiquitin ligase. Participation in multiple reactions complicates the analysis of specific functions when studied in standard in vitro assays. Dynamic regulation of the activity in either one or another process and the existence of different kinetic pools usually escape scrutiny by test tube analysis. Moreover, thermodynamic parameters within mammalian cell nuclei, critical for regulating activity , cannot be fully mimicked in vitro. Recent developments in cell biology have opened new opportunities for studying complex processes in the most relevant context, i.e. the living cell.Prior to in depth live cell analysis of protein dynamics, the biological activity should be carefully checked. In this study we thoroughly analyzed the function of GFP-tagged TTDA. The most pertinent cellular phenotype of TTDA-insufficiency is the NER defect. Remarkably, despite the 4-fold larger size of the tag compared to TTDA itself and the fact that it has to fit in a complex with nine other subunits, that should have numerous interaction partners, TTDA-GFP appeared normally incorporated into TFIIH C, able tIn our live cell studies we have shown that TTDA-GFP and XPD-GFP reside both in the cytoplasm and in the nucleus, contrasting to the strictly nuclear localization of XPB. Photo-bleaching experiments indicated that both proteins do not interact in the cytoplasm. However, application of a newly developed variant of FRAP, FRAP_abc, revealed that TTDA and XPD do interact with TFIIH in the nucleus. In striking contrast to XPB, the association of TTDA to TFIIH  is much more dynamic. Previously we found by subcellular fractionation (unpublished data) and others found, by GFP-tagging , that enBoth TTDA-GFP and XPD-GFP accumulate in nucleolar foci (previously observed in fixed TTDA-HA-expressing fibroblasts ). NucleoReduced steady-state levels of TFIIH in TTD-A cultured fibroblasts appeared to be a critical determinant in NER efficiency , but thiThe in vitro NER studies  showed tThe complete absence of TTDA in patients, as deduced from the mutation abolishing the start codon, shows that cell viability is not critically depending on TTDA and on high amounts of TFIIH. This is in striking contrast with deletion mutants of each of the previously known TFIIH subunits, which are not viable in yeast and mammals. Incompatibility with live associated with deletions of TFIIH encoding genes was explained by the vital transcriptional role of TFIIH . Extrapo6 normal nucleotides) cannot be accomplished in one step [TTDA can be considered as integral component of TFIIH in biochemical terms  with 2-d intervals. Next, from these mass populations, stably expressing GFP-fusion protein clones were isolated after single cell sorting using the FACSVantage . Single-cell sorted clones were further analyzed for GFP expression by microscopic evaluation and immunoblot analysis.Stably expressing cells were isolated after subsequent rounds of selection; first Neomycin-dominant marker selection using G418, followed by selection on UV-resistance by exposing them three times to a dose of 6 J/m2 and 5% CO2.Cell strains used were SV40-immortalized human fibroblasts: TTD1BR-Sv (TTD-A), with and without stably expressing TTDA-GFP; XPCS2B-Sv (XP-B) stably expressing XPB-GFP ; XP6BE-S2 was used for total irradiation, and 40 J/m2 for local irradiation, through a micro-porous filter [Treatment with UV-C was performed using a Philips germicidal lamp. A dose of 8 J/m filter . Transcr3H-thymidine, as described previously [For UV-survival experiments, cells were exposed to different UV-C doses, 2 d after plating. Survival was determined 3 d after UV irradiation by incubation at 37 °C withviously .We prepared whole-cell extracts by isolating cells from six petri dishes (14 cm) per cell line. Cells were washed with phosphate-buffered saline (PBS) before lysing them by douncing  in 2 ml of buffer A , supplemented with anti-proteases. Cellular extracts were incubated overnight at 4 °C in buffer A with Rabbit polyclonal antibodies to GFP  and mouse monoclonal p44 (1H5), cross-linked to protein A-sepharose beads . Before immunoprecipitations, cross-linked beads were washed three times with buffer A. After incubation with the extracts, the cross-linked beads were washed extensively with buffer A. Subsequently, beads were boiled in Laemli SDS-PAGE sample buffer, separated on 11% SDS-PAGE, blotted onto nitrocellulose and analyzed using the following antibodies: anti-XPB (1B3) and monoclonal anti-GFP (Roche).To analyze expression levels of the fusion proteins we prepared whole-cell extracts by sonication. These were separated on an 11% SDS-PAGE and transferred to 0.45 μm nitrocellulose membranes (Millipore). Expression of the fusion proteins was analyzed by hybridizing the membranes with a monoclonal anti-GFP (Roche), followed by a secondary antibody (rabbit anti-mouse) conjugated with horseradish peroxidase  and detected using enhanced chemo-luminescence (ECL+ Detection Kit) (Amersham).+ (PBS containing 0.15 % glycine and 0.5 % BSA). Cells were incubated at room temperature with primary antibodies  for 2 h in a moist chamber. Subsequently, coverslips were washed three times with PBS/TritonX-100 and PBS+, incubated 1 h with secondary antibodies,  at room temperature and again washed three times in PBS/TritonX-100. Samples were embedded in Vectashield mounting medium  containing 0.1 mg/ml DAPI (4′-6-diamino-2-phenylindole). Images were obtained by confocal laser scanning microscopy imaging, carried out with a LSM 510 microscope .Cells were grown on glass coverslips and fixed with 2% paraformaldehyde at 37 °C. Coverslips were washed with PBS containing 0.1% Triton X-100, three times for 5 min, and subsequently washed with PBS3 d prior to microscopy experiments, cells were seeded onto 24-mm-diameter coverslips. Imaging and FRAP were performed on a Zeiss LSM 510 meta confocal laser scanning microscope .FRAP analysis was performed at high time resolution . Briefly. After local irradiation of TTDA-GFP, XPB-GFP, and XPD-GFP fibroblasts with UV-C light, locally damaged areas were photo-bleached during 3 s with 100% laser intensity. Monitoring of the fluorescence recovery was followed by imaging the cell every 5 s for 200 s. A single image was taken prior to the photo-bleaching. Results are expressed by the ratio between damaged area and undamaged area. The first data point after photo-bleaching is set to 0, while final recovery is normalized to 1. Error bars, in the inset of the curves, represent the standard error of the mean. Statistical significance was checked, whenever two distinct curves were not easily dissociable, by using Student'st-test  within an appropriate time window: right after the photo-bleaching when evaluating mobility differences or after complete recovery when immobile fractions are being compared.To determine residence times of the different TFIIH components at locally damaged areas, we applied FRAP on local damageFigure S1Mobility of TTDA-GFP in the cytoplasm after bleaching the nuclear pool (red line) compared to the mobility of TTDA-GFP in the cytoplasm without bleaching the nuclear pool (green line). The relative fluorescence (fluorescence post-bleach divided by fluorescence pre-bleach) is plotted against time in seconds.(193 KB PPT)Click here for additional data file.Figure S2(A) Scheme of the plasmid used to transfect TTD-A-GFP expressing cells. The construct expresses small-interference RNA against XPC (sequence available upon request), under the control of an H1 (RNA polymerase III) promoter. The plasmid also contains a Mito DsRED_IRES_Hygro transcriptional unit, allowing the selection of transfected clones by the Hygromycin resistance and the mitochondrial DsRED signal.(B) Example of a TTDA-GFP expressing cell transfected with a mixture of two different XPC small-interference RNA-expressing constructs.(C) Strip-FRAP_abc of UV-irradiated TTDA-GFP expressing cells knocked-down for XPC (green curve), compared with untreated TTDA-GFP expressing cells (blue curve) and UV-treated TTDA-GFP expressing cells (red curve). Relative fluorescence (fluorescence post-bleach divided by fluorescence pre-bleach) plotted against time in seconds.(557 KB PPT)Click here for additional data file.Figure S3FRAP curve of TTDA-GFP expressing cells untreated (green line) and treated see with Act(48 KB PDF)Click here for additional data file.


Cathelicidins are mammalian proteins containing a C-terminal cationic antimicrobial domain. Porcine PR-39 cathelicidin affects leukocyte biology. Mechanisms of action may involve alteration of heparan sulfate proteoglycan-dependent functions in inflammatory cells. It was tested  whether PR-39 affects human neutrophil migration and if such effects involve heparan sulphate proteoglycans. Neutrophils were from forearm venous blood of healthy donors. Migration was tested in modified Boyden chamber assays. Involvement of heparan sulfate proteoglycans was tested by their chemical modification and by the use of specific antibodies. PR-39 induced migration in neutrophils in a concentration dependent manner. Modification of heparan sulfate proteoglycans with sodium chlorate inhibited migration whereas chemotaxis toward the chemoattractant formyl-Met-Leu-Phe was not affected. Removal of heparan sulfates or chondroitin sulfates from the surface of  neutrophils by heparinase or chondroitinase inhibited migration toward  PR-39. In conclusion, antimicrobial PR-39 stimulates human neutrophil chemotaxis in a heparan sulfate proteoglycan-dependent manner. Involvment of  syndecans is likely as both heparinase and chondroitinase were abrogating. Data suggest active participation of heparan sulfate proteoglycans of neutrophils in cathelicidin peptide-mediated regulation of the antimicrobial  host defense. Two major antimicrobial peptide families in mammals are the defensins . PR-39 induced human neutrophil chemotaxis in a concentration dependent manner with a maximum effect at 10 μmol/L (data not shown). To investigate the role of intact HSPG on the surface of neutrophils in PR-39-induced cell migration, neutrophils were pretrated for 50 min with heparinase I or chondroitinase  at 37°C, followed by two washing steps. As glypicans carry heparan sulfate side chains but not chondroitin sulfate side chains, whereas syndecans carry both [To determine whether PR-39 induces human neutrophil chemotaxis, we tested rry both , experimin vivo expression of syndecans has been reported as being affected by PR-39 [Because PR-39-induced chemotaxis was inhibited by chondroitinase and heparinase I, we suggested the involvement of syndecans but not glypicans by PR-39. Moreover, by PR-39 . TherefoIn the present study, PR-39 stimulated human neutrophil chemotaxis in a bell-shaped dose-response curve. PR-39 is known to induce chemotaxis in porcine neutrophils . As PR-3For LL-37, the only human cathelicidin identified so far, it was proposed that chemotactic responsiveness of leukocytes involves formyl peptide receptor-like 1, and activation of this formyl peptide receptor-like 1 cross-deactivates LL-37 responsiveness . In mastAntithrombin-III, a prototypical glycosaminoglycan ligand, has only recently been identified to exert direct effects on cells of the innate immune system via HSPG ,32. AlreThe syndecan family of cell surface proteoglycans has been implicated in a number of biological processes, including blood coagulation, cell adhesion, signal transduction and wound repair . OriginaPR-39 has been shown to interact with a domain within the integrin mediated signaling protein, namly p130(Cas) . IdentifThe data provided do not permit the conclusion that only syndecan-4 mediates the chemotaxis of human neutrophils to PR-39. Other syndecans or glypicans may also participate in coordinating the chemotactic response, as additional cell surface proteoglycans harboring HS and/or CS might also be involved.To summarize, besides proposed involvement of formyl peptide receptor-like 1, no signalling pathway for cathelicidins in neutrophils had been identified so far. Our results provide strong evidence for interactions of PR-39 with proteoglycans on the surface of leukocytes. Biochemical and functional tests identify syndecan-4 as a putative acceptor site for PR-39 which contains consensus sequences for glycosaminoglycan recognition. Our findings may be of particular relevance if PR-39 proofs have a therapeutic potential in neutrophil-mediated inflammatory diseases.BSA – Bovine serum albuminfMLP – formyl-Met-Leu-PheHSPG – Heparan sulfate proteoglycanPR-39 – Prolin-arginine-rich 39 peptideThe author(s) declare that they have no competing interests.AD carried out the chemotaxis experiments and drafted the manuscript. BM and NCK carried out the enzyme digestion and antibody inhibition studies, respectively. CRR, GR and JRP participated in the design of the study. CJW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


The binding of viral-specific antibodies to cell-surface antigens usually results in down modulation of the antigen through redistribution of antigens into patches that subsequently may be internalized by endocytosis or may form caps that can be expelled to the extracellular space. Here, by use of confocal-laser-scanning microscopy we investigated the kinetics of the modulation of respiratory syncytial virus (RSV) antigen by RSV-specific IgG. RSV-infected human epithelial cells (HEp-2) were incubated with anti-RSV polyclonal IgG and, at various incubation times, the RSV-cell-surface-antigen-antibody complexes (RSV Ag-Abs) and intracellular viral proteins were detected by indirect immunoflourescence.Interaction of anti-RSV polyclonal IgG with RSV HEp-2 infected cells induced relocalization and aggregation of viral glycoproteins in the plasma membrane formed patches that subsequently produced caps or were internalized through clathrin-mediated endocytosis participation. Moreover, the concentration of cell surface RSV Ag-Abs and intracellular viral proteins showed a time dependent cyclic variation and that anti-RSV IgG protected HEp-2 cells from viral-induced death.The results from this study indicate that interaction between RSV cell surface proteins and specific viral antibodies alter the expression of viral antigens expressed on the cells surface and intracellular viral proteins; furthermore, interfere with viral induced destruction of the cell. Antibody-induced modulation of antigen is a complex biological phenomenon closely resembling other receptor-ligand interactions. Following exposure to specific antibodies, surface antigens are usually redistributed on the cell surface and are internalized or expelled into the extracellular medium ,2. TheseParamyxoviridae family. Its genome encodes two non-structural and nine structural proteins, three of which are transmembrane surface glycoproteins: The G protein is involved in the virus attachment; the F protein mediates fusion of virus with cell membranes [Respiratory syncytial virus (RSV) is an enveloped pneumovirus classified within the embranes , and SH embranes . Cells iembranes .Worldwide, RSV is the most important viral pathogen of serious lower-respiratory tract illness in infants and young children. RSV infects nearly 70% of infants in their first year of life; by the age of 24 months old virtually all children will have been infected at least once and about half will have experienced at least two infections ,13,14. RIndividuals previously infected with RSV can be subsequently re-infected (within months) with either an identical or antigenically closely related virus despite the presence of serum antibodies ,13,29. RThe current study was designed to examine whether RSV-cell-surface-antigen-antibody complexes (RSV Ag-Abs) in epithelial cells undergo aggregation into patches that subsequently either form caps or are internalized through endocytosis. Furthermore, kinetic assays were used to determine the concentration level and fate of viral proteins in RSV-infected cells that had been incubated with anti-RSV antibodies.By determining the RSV Ag-Abs in plasma membrane and viral proteins in the cytoplasm, we investigated the effect of the presence of RSV-specific IgG on infected epithelial cells over a period of time (1 hour) and on the viability of these cells. Here, we present evidence that anti-RSV IgG induced redistribution of cell surface viral glycoproteins and that internalization of RSV Ag-Abs was partially inhibited by incubation in hypertonic medium, thus suggesting the participation of a clathrin-mediated mechanism. We also observed a time-dependent, cyclic fluctuation in the concentration of RSV Ag-Abs in cell surface and in intra-cellular viral proteins. Moreover, anti-RSV IgG protected HEp-2 cells from viral-induced cell death.Human epidermoid carcinoma larynx cell line HEp-2 from our laboratory  was grown in Dulbecco's modified medium  which was determined to be mycoplasma free by using a mycoplasma detection kit (Boheringer Mannheim). Long strain RSV has been used as the prototype virus in our laboratory for over ten years. The procedures for propagating cells and viruses and for assaying viral infectivity have been described elsewhere .6 TCID50/ml; 400 μg protein/ml) that had been purified by linear sucrose gradient. Pre-immune sera was obtained from the rabbits before immunization. Anti-RSV serum characteristics were evaluated by its neutralization activity in viral infectivity [Polyclonal anti-RSV sera were obtained in our laboratory from male New Zealand rabbits after three intramuscular immunizations with RSV  that had been grown on glass cover slips, previously treated with poly-L lysine (1 μg/ml at room temperature (RT)) and washed with phoshate-buffered saline (PBS), for 30 min., were infected with RSV at a multiplicity of infection (m.o.i.) of 50  and thereafter washed in PBS. Infected cells were permeabilized and fixed with cold methanol (5 min) and cold acetone (30 sec) and the viral antigen was visualized by indirect immunofluorescence by using goat anti-RSV  as first antibody and rabbit anti-goat fluorescence conjugate  as second antibody, as previously described [HEp-2 cells (1 × 10escribed . FluoresTo RSV-infected cells (three cover slips), anti-RSV IgG, diluted 1:5 in D-MEM containing 1% glutamine, was added to cover the cell monolayer and the mixture was incubated at 37°C. At 0, 10, 20, 30, 40, 50, or 60 minutes of incubation, cell samples were taken, then washed in PBS to remove excess of anti-RSV IgG, and fixed with 4% paraformaldehyde in PBS. The RSV Ag-Abs was visualized by indirect immunofluorescence as previously described. Controls were, RSV-infected cells (three cover slips) incubated with pre-immune polyclonal IgG, mock-infected cells (three cover slips) incubated with anti-RSV IgG and (three cover slips) with pre-immune polyclonal IgG.Cells grown in cover slips were infected and incubated at 37°C with anti-RSV IgG, at 0, 10, 20, 30, 40, 50, or 60 minutes washed permeabilized and fixed. Then intracellular viral proteins were detected by adding anti-RSV IgG and incubated 1 h at 37°C. Afterwards, cells were washed and viral proteins visualized by indirect immunofluorescence as described. Controls were as described for visualization of RSV Ab-Ags on the cell surface.The sucrose inhibition assay was used . BrieflyFluorescent samples were examined in a Bio-Rad MRC 600 confocal laser scanning system that was linked to an Axioskop Zeiss microscope with Plan-Neuflour 40×/0.75P H2 objective. Krypton-argon laser light was used to excite fluorescein isothiocyanate  with emission BHS filter. Data were processed with Bio-Rad CoMOS with Z-step of 1.08 nm.To infected HEp-2 cells, anti-RSV IgG was added, and cell viability was determined at 2, 24, 48, and 72 h of incubation. Sterile 0.2% ethylenediamine tetra acetate (EDTA) in 0.9% saline was added to each cell monolayer; after incubation (15 min), the cells were suspended in D-MEM containing 2% FBS and trypsin (5 μg/ml). Trypan blue solution was added to the cell suspension and the viable cells were counted by light microscopy. As controls, both infected cells incubated with pre-immune IgG and mock-infected cells incubated with anti-RSV IgG were used.RSV proteins, present in viral infected HEp-2 cells, were visualized in permeabilized cells by indirect immunofluorescence, at various times after infection. We observed (results not shown) that viral proteins could be visualized at 6 to 8 h post infection ; however, for 90 to 95% of the cells, a fluorescence intensity of 2–3 was observed at 12 h p.i.  assay, antibody-bound viral glycoproteins were observed as a rim, with fluorescence intensity of 1 Fig. . After 1In the 30-minute-incubation assay, RSV Ag-Abs content and fluorescence intensity decreased, with the fluorescence (intensity of 2) being once again observed as rim Fig. . In the After establishing that anti-RSV IgG had induced an incubation-time-dependent fluctuation in the concentration of cell-surface RSV Ag-Abs, we decided to determine whether a similar effect would occur in intracellular viral proteins, therefore, anti-RSV IgG was added to permeabilized cells and observed by indirect immunoflourescence. Results were obtained by examining the distribution of fluorescence on at least 100 cells. All assays were run independently at least three times.At time 0, the cytoplasm of permeabilized infected cells Fig.  was fluoAt 40 minutes, a clear reduction in the concentration of viral proteins was evident, as the fluorescence was negligible Fig. , whereasOur results showed that anti-RSV IgG incubation induced the removal and re-appearance of intracellular viral proteins in a cyclic manner Fig. , an effeThe strategy consisted of inhibition of receptor-mediated endocytosis by exposing the cells to hypertonic medium containing sucrose. Clathrin-coated pits derived from clathrin-coated vesicles are not formed in hypertonic medium . To thisAs shown in Figure At 10-minute incubation, intracellular fluorescently labelled proteins were localized in one area of the cytoplasm of the rounded cells; patches with fluorescence (intensity of 1) were distributed randomly Fig. . At 20-mde novo took place. During the assays, the concentration of viral proteins varied, although the fluctuation was less pronounced than that in the former determinations . In comparison to the value obtained at 2 h p.i., the cell survival at 48 and 72 h p.i. was reduced to 6.5% and 1.45%, respectively. No cell viability change was evident in mock-infected HEp-2 cells incubated with either anti-RSV IgG or pre-immune IgG. The data suggest that anti-RSV polyclonal IgG protected infected cells from viral-induced death.In this report, we determined the effect of incubation RSV infected epithelial cells with polyclonal RSV IgG on expression and localization of viral proteins and cell viability. Kinetics of cell-surface viral glycoproteins and intracellular viral proteins was monitored through confocal microscopy and cell viability determined by trypan blue exclusion.Our data show that the binding of specific antibodies to RSV glycoproteins anchored to the plasma membrane of human epithelial cells led to their redistribution and aggregation Fig . SubsequThe lower concentration of RSV Ag-Abs on cell surface can be explained through cap formation, with subsequent release to the extracellular medium or by endocytosis of RSV Ag-Abs. Reduction of RSV Ag-Abs concentration on the cell-membrane was evidenced at both 30 and 50 minutes of incubation with anti-RSV IgG Fig.  and 2E iThe interpretation that caps were released into the medium was supported by the simultaneous presence of cells with caps and areas without detectable viral glycoproteins Fig. . This obde novo and/or RSV Ag-Abs internalized through a mechanism(s) different from clathrin-mediated endocytosis.Our data, obtained by incubation in hypertonic media, showed that the concentration of intracellular viral proteins decreased over the course of the determinations, thereby suggesting that endocytosis through clathrin-mediated mechanism was inhibited Fig. . The remde novo is particularly considered because, throughout the course of these experiments, we observed that infected cells incubated with anti-RSV IgG were protected from viral-induced death. This observation was confirmed through comparative cell-survival determinations between infected HEp-2 cells incubated either with anti-RSV IgG or with pre-immune IgG. The loss of cell- surface viral glycoproteins during culturing RSV infected cells for longer than 72 h in the presence of anti-RSV IgG might explain the lack of syncytia formation and hence avoid death of the cell [Protein synthesis the cell .de novo of viral proteinsThe increase in RSV Ag-Ab concentration may be explained as resulting from the internalization of the RSV Ag-Abs, with subsequent dissociation of the complexes and recycling of the liberated viral antigen back to the cell surface  and/or oOur data suggest that in the human epithelial cell line we used both caps formation and endocytosis took place Fig.  and 3. IAlthough viral proteins on the cell plasma membrane were detected at m.o.i. of 10 to 20, the glycoprotein-antibody complexes were clearly observable as patches at higher multiplicity, implying that a defined content of RSV proteins was required for clustering into patches. This observation is similar to that of reports on monocytes infected with pseudorabies virus, in which glycoprotein capping occurred only after the patch size exceeded a minimal threshold size .During consecutive assays, cell-surface- and intracellular-protein concentrations fluctuated in a cyclic manner, thus suggesting a continued removal and replacement of cell-surface and intracellular proteins. In the current work, determination was made at 10-minute intervals; however, by using either shorter or longer time intervals, the fluctuation cycles in the concentration of viral-protein-antibody complexes may be optimized.alphaherpesvirinae family has been reported that specific tyrosine family of motifs , in the cytoplasmic tails of the viral transmembrane glycoproteins activate clathrin-mediated endocytosis [Exactly how RSV Ag-Abs initiate the redistribution process, in capping or in internalization, is not fully understood; however, in the ocytosis . Therefoocytosis .The present findings indicate that specific antibody bounded to the surface of RSV infected cell modify cell-surface viral determinants, the intracellular viral polypeptide concentration and interfere with viral induced destruction of the cell.Paramyxoviridae family [Alterations of RSV intracellular viral proteins expression by anti-RSV IgG interaction with cell-surface viral glycoproteins is reminiscent with reports in the measles virus system, where expression of intracellular viral proteins is modify by the interaction of specific antibodies with cell-surface viral glycoproteins . Measlese family .How these processes are involved in the viral life cycle and viral pathogenesis is unknown, however, increase in viral replication with concomitant enhance of the disease -19 mightTo our knowledge, this is the first report on antibody-induced modulation of respiratory syncytial virus antigens. Moreover, the system we described allows study long time interaction between RSV infected cells and antiviral antibodies.RSV Respiratory Syncytial VirusCOPD Chronic Obstructive Pulmonary DiseaseRSV Ag-Abs RSV-cell-surface-antigen-antibody complexesThe author(s) declare that they have no competing interests.RES and RT contributed in the experimental work, analysis of results and discussion of results. LV performed the majority of the experimental work and BG conceived and wrote the manuscript. All authors read and approved the final manuscript


The Syrian hamster embryo cell lines, SHOK and MC-1, were used as recipient cells for DNA transfection assay to detect transforming genes in experimental mouse tumours. A mouse repeat sequence was utilised to check whether each transformed focus included mouse genomic DNA in the Hamster background. We investigated five mouse tumours that are related to X-ray radiation, and detected activated c-K-ras, c-mos, and c-cot oncogenes which induced foci of hamster cells. These results show that SHOK and MC-1 cells have unique properties for detecting transforming genes in experimental mouse tumours.


A genome-wide survey of the transcriptional activity of TE-related genes that were associated with fifteen developmental stages and stress conditions revealed clear, albeit low, general transcription of TE-related genes. Oryza sativa) has created an opportunity to conduct a comprehensive evaluation of the transcriptional activities of these potentially mobile elements and their related genes.Transposable element (TE)-related genes comprise a significant portion of the gene catalog of grasses, although their functions are insufficiently characterized. The recent availability of TE-related gene annotation from the complete genome sequence of rice (cis elements in the promoters, appear to contribute to the transcriptional activation of representative TE-related genes.We conducted a genome-wide survey of the transcriptional activity of TE-related genes associated with 15 developmental stages and stress conditions. This dataset was obtained using a microarray encompassing 2,191 unique TE-related rice genes, which were represented by oligonucleotide probes that were free from cross-hybridization. We found that TE-related genes exhibit much lower transcriptional activities than do non-TE-related genes, although representative transcripts were detected from all superfamilies of both type I and II TE-related genes. The strongest transcriptional activities were detected in TE-related genes from among the MULE and CACTA superfamilies. Phylogenetic analyses suggest that domesticated TE-related genes tend to form clades with active transcription. In addition, chromatin-level regulations through histone and DNA modifications, as well as enrichment of certain Our findings reveal clear, albeit low, general transcription of TE-related genes. In combination with phylogenetic analysis, transcriptional analysis has the potential to lead to the identification of domesticated TEs with adapted host functions. Oryza sativa) genome sequence allowed further functional classification of the coding sequences of this important crop and model of grass species [Oryza Repeat Database [The completion of the rice  TE-related genes in at least one of our 15 samples. This is in contrast to findings in non-TE-related genes, 85.8% of which are expressed in at least one sample and 22.6% in all samples, using the same selection criteria. Expressed TE-related genes tend to exhibit transcription in a relatively small number of samples. The percentages of expressed TE-related genes in a wide range of samples are markedly lower than those of non-TE-related genes Figure . For thoTo probe quantitatively for the transcriptional activity of TE-related genes, the expression intensities of those 1,084 transcribed TE-related genes and an similar number of randomly selected transcribed non-TE-related genes are visually juxtaposed after clustering Figure . Even thTo gauge the reliability of our microarray data for TE-related genes, we first compared rice cDNA and EST collections with our data. We found 496 TE-related genes in the cDNA/EST collection in TIGR database . These cIn addition to taking an inventory of transcribed TE-related genes in various tissues and under multiple growth conditions, the availability of high-quality complete genome sequence provided an opportunity to elucidate how transcriptional activities evolve following sequence divergence. To this end, phylogenic trees were generated for all major TE-related gene superfamilies and were integrated with their members' expression profiles.Karma with active transposition has been reported [Karma was also identified in the rice genome [The type I TE-related genes can be classified into two groups according to the presence or absence of long terminal repeats (LTRs). TE-related genes without LTRs belong to the long interspersed elements (LINEs) type, which may encode retrotransposase and mobilize noncoding short interspersed elements (SINEs). Only 34 LINE-type TE-related genes were identified in rice Table . We founreported ; its trae genome , which lcopia and Ty3/gypsy, which are both ubiquitous throughout plants and believed to have contributed significantly to the evolution of genome structure and function [gypsy elements outnumbering Ty1/copia elements [Tos17 is the best characterized and is known to exhibit active transposition in tissue culture [Tos17 not only in cultured cells but also in a wide range of organs , suggesting that tissue culture may provide a way to propagate somatic transposition events to progeny. Sireviruses are a plant-specific lineage of the Ty1/copia retrotransposons that interact specifically with proteins related to dynein light chain 8 [LTR-type TE-related genes belong to two superfamilies, namely Ty1/function . Both faelements . Our exp culture . We foun chain 8 . We founcopia and Ty3/gypsy superfamilies.A large number of type I TE-related genes have not yet been further classified Table . We detehAT-like, PIF/Pong-like, Mariner-like, and Helitron-like superfamilies have transcriptional activities similar to or lower than those of type I TE-related genes.Type II TE-related genes are in general more actively transcribed than type I TE-related genes. Different from type I, type II TE-related genes are highly variable among major superfamilies with respect to transcriptional activity. Whereas CACTA and MULE superfamilies are actively transcribed, Mutator-like superfamily (MULE) is one of the first groups of identified transposases with a few reported transcriptionally active members in rice [MuDR-like, Jittery-like, and TRAP-like [TRAP-like branch may have recently been amplified, and high similarity among family members has resulted in lack of unique oligo probes with which to examine their expression profiles. Interestingly, we have found at least three clades with clear active transcription in MuDR-like and Jittery-like branches , which are ubiquitous in the rice genome [PIF/Pong-like elements were recently reported: maize PIF and rice Pong [PIF, namely OsPIF1 [Pong elements in the rice genome, which are represented by a single probe in the microarray. This probe detected transcription activity in tillering shoot and drought-exposed panicles only .Both e genome . Low proice Pong ,23,60. Iber size ; this suHelitron-like, is relatively under-characterized in the rice genome [Helitron-like transposons have the potential to move and shuffle genes or exons in maize [Helitron-like transposon among the seven examined ones with transcriptional activity in any samples .A recently identified unique type II TE superfamily, e genome . Strikinin maize ,14. In rWe were unable to further classify another 787 type II TE-related genes into any superfamilies Table . InteresGenes or exons can be transduplicated by MULEs ,63, whicTEs, including TE-related ORF encoding genes, are under multiple levels of epigenetic control, including DNA methylation and histone modifications . In ArabTos17 and Tos3 of the Ty1/copia superfamily; Ty3/gypsy elements Os09g15460, Os03g32070 and OSR30; MULE superfamily DNA transposons MUG1, FAR1-like and Os11g05820; CACTA DNA transposons Os10g31320, Os09g29980 and Os04g08710; and DAYSLEEPER-like from the hAT-like superfamily. Seedling shoot samples were used for all analyses discussed here. To verify transcription independently, we used PCR to amplify reverse-transcribed cDNA (RT-PCR). Transcript accumulation assayed by RT-PCR is in general consistent with microarray results . The cellulose synthase gene has strong transcription in almost all samples we profiled. In contrast, the retrotransposon target does not exhibit transcription in any sample.It has been shown that small RNAs derived from repetitive genome sequences repress transcription by means of RNA interference in bidopsis . Small Rbidopsis ,67. Sixtbidopsis , whereasArabidopsis, as well as in several other eukaryotes, that some adjacent genes tend to have co-expression patterns [P < 10-16, by Welch two-sample t-test). In the case of opposite orientation, an increase of 9% in transcription level was found (P < 10-16). By comparing the effects of transcribed upstream gene orientation in these two scenarios, we found that the same orientation corresponded to 6% more expression than the other scenario (P < 10-7). There is no clear distinction between the two scenarios for TE-related genes with untranscribed upstream genes . The orientation of downstream non-TE-related genes did not significantly affect the transcription of upstream TE-related genes.It was recently reported in patterns -71. Readpatterns ,72,73. Wcis elements in transcription. To this end, we searched for enrichment of cis elements in the promoter regions of transcribed TE-related genes. We grouped TE-related genes based on the number of samples with transcription and searched for frequency of occurrence of all reported cis elements within each group. Among 439 reported elements in plants [copia, Ty3/gypsy, or the CACTA superfamily. TATA box was identified, which is usually found in the 5'-upstream region of eukaryotic genes and is critical for accurate initiation of transcription [Arabidopsis [To explore further the possible underlying mechanisms that control the transcription of TE-related genes, we attempted to identify possible involvement of n plants , nine ofcription . The T-bbidopsis . We alsoTEs account for an overwhelming proportion of plant genomes. To ensure the viability of their host and hence their own survival, the transposition of TEs should be tightly controlled . TranscrArabidopsis and Drosophila that the activity of TE elements may reduce as the copy number increases [Using 70-mer oligonucleotide microarrays covering more than 2,000 rice TE-related genes, we surveyed the transcription profiles under a wide range of organ samples under various conditions. Considering that TE-derived cellular genes are relatively rare, autonomous TEs probably contribute to most of these TE-related genes. Genome profiling revealed that 25% to 30% of the TE-related genes were transcribed in one sample, which was much lower than the corresponding percentage of non-TE-related genes Figures  and 2. Mncreases ,78. TherPIF/Pong-like, Mariner-like and Helitron-like type II TE-related genes, were more tightly controlled at the transcription level. Type I TE-related genes are amplified through a copy-and-paste mechanism [PIF/Pong-like and Mariner-like superfamilies regulate the activity of MITEs, which dominate the rice genome [Helitron-like superfamily go through a unique rolling cycle replication to rapidly amplify themselves [Among TE-related genes, a smaller proportion of type I than type II genes were transcribed Figure , a discrechanism . PIF/Pone genome . Membersemselves .Many TE-related genes exhibit organ-specific, growth stage-specific, and stress-specific expression profiles in our collection of samples. These genes exist in all superfamilies, as shown in Figures It is important to note that transcriptional activity does not necessarily correspond to transpositional activity. Transcription is just the first of several steps required for the transposition of type I and type II TEs ,80. ActiArabidopsis and rice. Interestingly, domesticated TE genes tend to be within actively transcribed TE gene clades. The rice homologs of the two reported cases of domesticated transposons in Arabidopsis, namely FAR1/FHY3 and DAYSLEEPER, were located in two actively transcribed clades. MUG1, a putative domesticated gene revealed by cross-species sequence comparison analysis, was shown to be transcribed from our data and located within an actively transcribed clade. These examples may suggest that actively transcribed clades of TE-related genes are a rich source for domesticated TE genes. In fact, several other actively transcribed clades have been observed, especially for the MULE and CACTA superfamilies, from our analysis  and printed onto poly-L-lysin coated microscope slides using a contact microarrayer. The same recommended set of 12 unique negative control 70-mer oligonucleotides based on heterologous genes  were incMicroarray experiments and detailed rice sample preparation were described previously -47. SampMicroarray spot intensity signals were acquired using Axon GenePix Pro 3.0 software package . To identify and remove systematic sources of variation, including dye and spatial effects, spot intensities from the GenePix Pro output files of all repeats of a given sample pair were normalized using limma, a software package for the analysis of gene expression microarray . This noTE family classification was according to TIGR annotation . Hand anCluster analysis was applied to all TE-related genes and 1,353 randomly selected non-TE-related genes showing expression in at least one sample. Average normalized log-transformed expression intensities were subjected to cluster analysis. For hierarchical clustering, Pearson correlation was used to compute similarities, and a complete linkage clustering algorithm was used. Cluster analysis was performed using the software Cluster  and visuTotal RNA was extracted from independently prepared rice seedling shoots using Qiagen RNeasy kit . After DNase I treatment, total RNA was used for cDNA synthesis using Superscript II  in accordance with the manufacturer's protocol. PCR primers were designed according to sequence using Primer3 . The ampChIP was carried out as described elsewhere  using seMethylation of DNA was assessed by McrBC digestion following a previously published protocol . GenomicThe genome sequences 2 kilobases upstream of the annotated translation start site were retrieved from the TIGR database. Both DNA strands were searched for known plant motifs using the PLACE database . Enrichmcopia superfamily. Additional data file gypsy superfamily. Additional data file Mariner superfamily. Additional data file Helitron superfamily.The following additional data are available with the online version of this paper. Additional data file L1. Bootstrap values were calculated from 300 replicates. The sample numbers are identical to those in Table The phylogenetic tree was generated from a multiple alignment of conceptually translated sequences by using the neighbor-joining methods and rooted with human Click here for fileL1. Bootstrap values were calculated from 300 replicates. Sample numbers are identical to those in Table The phylogenetic tree was generated from a multiple alignment of conceptually translated sequences by using neighbor-joining methods and rooted with human Click here for fileSoymar1. Bootstrap values were calculated from 300 replicates. Sample numbers are identical to those in Table The phylogenetic tree was generated from a multiple alignment of conceptually translated sequences by using neighbor-joining methods and rooted with soybean Click here for fileSoymar1. Bootstrap values were calculated from 300 replicates. Sample numbers are identical to those in Table The phylogenetic tree was generated from a multiple alignment of conceptually translated sequences by using neighbor-joining methods and rooted with soybean Click here for fileC. elegance CeHEL1. Bootstrap values were calculated from 300 replicates. Sample numbers are identical to those in Table The phylogenetic tree was generated from a multiple alignment of conceptually translated sequences by using neighbor-joining methods and rooted with Click here for file


Non-invasive predictive assays which can confirm the presence or absence of hypoxic cells in human tumours show promise for understanding the natural history of tumour oxygenation, and improving the selection of patient subsets for novel radiotherapeutic strategies. Sensitiser adducts have been proposed as markers for hypoxic cells. Misonidazole analogues radiolabelled with iodine-123 have been developed for the detection of tumour hypoxia using conventional nuclear medicine techniques. In this pilot study, we have investigated one such potential marker, 123I-iodoazomycin arabinoside (123I-IAZA). Patients with advanced malignancies have undergone planar and single-photon emission computed tomographic (SPECT) imaging after intravenous administration of 123I-IAZA. We have observed radiotracer avidity in three out of ten tumours studied to date. Normal tissue activity of variable extent was also seen in the thyroid and salivary glands, upper aerodigestive tract, liver, intestine, and urinary bladder. Quantitative analysis of those images showing radiotracer avidity revealed tumour/normal tissue (T/N) ratios of 2.3 , 1.9  and 3.2  at 18-24 h post injection. These preliminary data suggest that the use of gamma-emitter labelled 2-nitroimidazoles as diagnostic radiopharmaceuticals is feasible and safe, and that metabolic binding of 123I-IAZA is observed in some, but not all tumours. The inference that tumour 123I-IAZA avidity could be a non-invasive measure of tumour hypoxia deserves independent confirmation with needle oximetry.


Fixed and mobile-bearing in total knee arthroplasty are still discussed controversially. In this article, biomechanical and clinical aspects in both fixed and mobile-bearing designs were reviewed. In biomechanical aspect, the mobile-bearing design has proved to provide less tibiofemoral contact stresses under tibiofemoral malalignment conditions. It also provides less wear rate in in-vitro simulator test. Patients with posterior stabilized mobile-bearing knees had more axial tibiofemoral rotation than patients with posterior stabilized fixed-bearing knees during gait as well as in a deep knee-bend activity. However, in clinical aspect, the mid-term or long-term survivorship of mobile-bearing knees has no superiority over that of fixed-bearing knees. The theoretical advantages for mobile-bearing design to provide a long-term durability have not been demonstrated by any outcome studies. Finally, the fixed-bearing design with all-polyethylene tibial component is suggested for relatively inactive, elder people. The mobile-bearing design is suggested for younger or higher-demand patients due to the potential for reduced polyethylene wear and more normal kinematics response after joint replacement. For younger surgeon, the fixed-bearing design is suggested due to less demand for surgical technique. For experienced surgeon, one familiar surgical protocol and instrumentation is suggested rather than implant design, either fixed-bearing or mobile-bearing. Total knee arthroplasty (TKA) has become a standard operative procedure to relieve pain and restore function in patients with osteoarthritis or rheumatoid arthritis. Current total knee prosthesis (TKP) devices can be subdivided into two groups based on different fundamental design principals: fixed-bearing knees, where the polyethylene tibial insert locked with tibial tray, and mobile-bearing designs which facilitate movement of the insert relative to the tray . In eachat the bone-implant interface predisposing to component loosening. Conversely, prosthesis with a low conformity bearing surface produces less constraint force that decreasing component loosening, but generates high contact stress leading to early failure of the polyethylene [Implant loosening and polyethylene wear in fixed-bearing knee prostheses were recognized as major causes of late failure. Fixed-bearing prosthesis with a high conformity bearing surface provides low contact stress, but produces high torque ethylene ,3. Furthethylene .Mobile-bearing knee prosthesis was introduced with the aim to reduce polyethylene wear and component loosening . The mobAlthough mobile-bearing design has hypothetical advantages over fixed-bearing knee, both designs show excellent survival rates of up to 95% in 10-year follow-up -11 and cMany biomechanical studies have demonstrated that contact stress on the tibial polyethylene component is closely related to polyethylene wear ,15. Mats3/million cycles when subjected to standard kinematics with force-controlled anterior-posterior translation. This showed a two-fold reduction in volumetric wear rate in comparison to the fixed-bearing PFC Sigma knees subjected to intermediate kinematics (9.8 ± 3.7 mm3/million cycles). The authors [One major goal of mobile-bearing knee is to reduce the overall wear damage by increasing the contact area, while minimizing the constraint and encouraging nature knee motion, by allowing the polyethylene bearings to move freely on polished plates on the upper tibia . McEwen  authors  attribut authors ,21. In c authors . Mobile- authors . In thatIn vitro cadaveric kinematics study under controlled laboratory condition was conducted by Most et al . In theiIn vivo kinematics studies by using fluoroscopy have been conducted on subjects with normal knee joints and on patients who had implantation of a fixed-bearing or mobile-bearing TKP -30. The Patients who had a posterior stabilized fixed-bearing TKA routinely demonstrated posterior femoral rollback in the lateral component during knee flexion, although it was less in magnitude than that in the normal knees . HoweverAxial tibiofemoral rotation after TKA is another important parameter for knee's kinematics. A multicenter analysis to determine in vivo axial tibiofemoral rotational magnitude and patterns in 1,027 knees was reported . In thatOsteolysis has been well documented as a major complication after TKA -37. The Despite many studies demonstrated advantages of using a mobile-bearing knee ,9,38, esSome conventional fixed-bearing TKAs have been proved to be clinical successful. Survivorship of the Genesis  TKA was 96% at 10 years follow-up . Ritter However, in the current situation, surgeons face more and more young, active patients who need TKA. Patient's expectations for a more functional and longer-lasting result following TKAs continue to drive advances in both implant design as well as the surgical technique. The mobile-bearing knee prosthesis was designed to reduce contact stress and constraint force, which potentially, provided long-term durability. Buechel et al  reportedAlthough long-term survivorship for fixed-bearing and mobile-bearing knees have been reported, there are few studies to compare the performance of fixed-bearing and mobile-bearing in patients with bilateral TKAs. Bhan et al  reportedThe exact indications for using the mobile-bearing knee are still unclear. Even though the mobile-bearing design has theoretically favourable features compared with fixed-bearing system, these have not been proven biomechanically and to extend the implant longevity in clinical aspect. Based on our experience in the past thirty-year, the fixed-bearing design with all-polyethylene tibial component is suggested for relatively inactive, elder people. For younger or higher-demand patients, the mobile-bearing design is suggested due to the potential for reduced polyethylene wear and more normal kinematics response after joint replacement. For the younger surgeon, using the fixed-bearing design is suggested due to less demand in surgical technique. For the experienced surgeon, one familiar surgical protocol and instrumentation is suggested rather than implant design, either in fixed-bearing or mobile-bearing.The author(s) declare that they have no competing interests.CHH designed the main framework and also performed final check for this manuscript. JJL carried out the paper survey and drafted the manuscript. CKC performed the final check in this manuscript, especially in biomechanical aspect. All authors read and approved the final manuscript.


Arabidopsis thaliana and have established a searchable Arabidopsis coiled-coil protein database.Long alpha-helical coiled-coil proteins are involved in diverse organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems and tissues, motors, levers, rotating arms, and possibly springs. Mutations in long coiled-coil proteins have been implemented in a growing number of human diseases. Using the coiled-coil prediction program MultiCoil, we have previously identified all long coiled-coil proteins from the model plant Here, we have identified all proteins with long coiled-coil domains from 21 additional fully sequenced genomes. Because regions predicted to form coiled-coils interfere with sequence homology determination, we have developed a sequence comparison and clustering strategy based on masking predicted coiled-coil domains. Comparing and grouping all long coiled-coil proteins from 22 genomes, the kingdom-specificity of coiled-coil protein families was determined. At the same time, a number of proteins with unknown function could be grouped with already characterized proteins from other organisms.MultiCoil predicts proteins with extended coiled-coil domains (more than 250 amino acids) to be largely absent from bacterial genomes, but present in archaea and eukaryotes. The structural maintenance of chromosomes proteins and their relatives are the only long coiled-coil protein family clearly conserved throughout all kingdoms, indicating their ancient nature. Motor proteins, membrane tethering and vesicle transport proteins are the dominant eukaryote-specific long coiled-coil proteins, suggesting that coiled-coil proteins have gained functions in the increasingly complex processes of subcellular infrastructure maintenance and trafficking control of the eukaryotic cell. The coiled-coil was one of the earliest protein structures described and first discovered in the two-stranded coiled-coil protein alpha-keratin . Coiled-The foremost feature of coiled-coil domains appears to be their ability to act as "cellular velcro" to hold together molecules, subcellular structures, and even tissues. They can act as protein-protein interaction motifs, for examples as dimerization domains in transcription factors and receptor kinases -18. TheyBesides associating with and interconnecting other molecules and macromolecular structures, long coiled-coil domains exhibit a number of structural and mechanical functions . TypicalOn a primary structure level, amino acid sequences with the capacity to form left-handed alpha-helical coiled-coils are characterized by a heptad repeat pattern in which residues in the first and fourth position are hydrophobic, and residues in the fifth and seventh position are predominantly charged or polar . This paIn the post-genomics era, such structure-prediction algorithms can now be applied to whole proteomes. Based on the prediction algorithm COILS, roughly 10% of all proteins encoded by eukaryotic genomes contain coiled-coil domains whereas prokaryotic genomes contain only 4–5% . Using tIn a comparative genomics approach, we determined the coiled-coil content of 22 predicted whole proteomes using the prediction pipeline and processing software developed to create the ARABI-COIL database . The 22 Prediction and selection of coiled-coil proteins was performed using the MultiCoil algorithm  and the Bacillus subtilis, MultiCoil predicted no coiled-coil proteins with domains longer than 250 amino acids in the bacterial genomes analyzed. However, archaeal and eukaryotic genomes contain proteins predicted to form coiled-coils of this length. Strikingly, prediction of coiled-coil domains over 400 amino acids in length was completely absent in bacterial genomes, but present in eukaryotes as well as two archaea, Sulfolobus solfataricus and Archeoglobus fulgidus. These numbers however do not take discontinuous coiled-coil prediction into account, as evident in the case of prokaryotic SMC proteins . 2–3% of the genes in these archaea were found to code for coiled-coil proteins. In contrast to eubacteria, all of the coiled-coil size-classes analyzed are represented in this group, with proteins predicted to form coiled-coils longer than 400 residues present in Methanococcus jannaschii and Archeoglobus fulgidus proteomes , and seven gram-negative bacterial genomes  were analyzed.Bacterial genomes for this study were chosen from different families to represent a wide range of prokaryotic species. Three gram-positive bacterial genomes  or sequences largely covered by coiled-coil (at least 60% of the sequence) were included in the combined pilot sequence set comprising 527 unique sequences. A maximum P-score of 1.0e-20 was used as the critical threshold when selecting only the most prominent sequence similarities in this test group. In all, 12,013 pair-wise P-score values were selected, defining as many unique relationships from the 277,729 possible pair-wise relationships. Sequences were then grouped using Kruskal's minimum cost spanning tree algorithm using the P-score value as the edge weight for the selected P-score values. 166 independent non-overlapping sequence subsets (subtrees) were defined in this manner. The largest grouping consisted of 270 sequences, representing over half of the sequences in the pilot sequence set and including functionally distinct families such as for example myosins, golgins, and SMC proteins. Distinct clusters of long coiled-coil proteins besides this large, heterogeneous group were formed by the animal and yeast tropomyosins (two separate clusters), the laminins, the CASP/CDP-family and the nuclear lamins.To prevent clustering based on the inherent coiled-coil repeat similarities, amino acids predicted to form coiled-coil domains were computationally masked out before being subjected to sequence similarity comparison Figure . The cluAfter determining the consistency of clusters formed after masking coiled-coil domains with well-known coiled-coil protein families such as the SMC proteins, myosins and kinesins, we proceeded to cluster all 3576 predicted long coiled-coil sequences from the 22 genomes. The clustering algorithm was further improved to first preclude transitively similar sequences by requiring all sequences in each cluster to satisfy the P-score threshold for all pair-wise relationships within the cluster and secondly to identify "bridge" sequences meeting these criteria for multiple clusters . A P-score threshold of 10e-06 was selected as the appropriate balance of sequence coverage and cluster discrimination. Table Synechocystis  and fission yeast (Schizosaccharomyces pombe) as eukaryotic, unicellular organisms. Protein clusters found to be specific for yeast were typically small , flies (Drosophila melanogaster), and mammals (Mus musculus and Homo sapiens) were analyzed. Clusters that appeared to be specific to animals  and a monocot (Oryza sativa) plant genome were analyzed. Clusters of long coiled-coil proteins specific to Arabidopsis and rice contained mostly sequences of so far unknown function , membrane tethering and vesicle transport proteins appear to be specific for eukaryotes in our clustering analysis, indicating another major class of specialized coiled-coil proteins that evolved after the formation of eukaryotic cells. It has been previously suggested that the higher content of long coiled-coil domains in metazoa compared to plants and protists indicates the presence of extensive coiled-coil matrices in animal cells and tissues . One of Earlier surveys of coiled-coil sequences in GenBank had suggested that invertebrate genomes contain more coiled-coils than vertebrates, and that animal genomes contain four times more "extended" coiled-coils (>75 amino acids) than plant genomes . While wHeliobacter pylori with a higher percentage than C. elegans, and an exceptionally low coiled-coil content in Mycobacterium tuberculosis. Our analysis was consistent with these previous observations and resulted in 5.6% coiled-coil for Heliobacter pylori versus 5.4% in C. elegans and only 1.8% in Mycobacterium tuberculosis, the lowest percentage for all 22 genomes analyzed here.Comparable with the Arabidopsis coiled-coil protein database ARABI-COIL, this study takes a more restrictive approach to identifying coiled-coil proteins than previous genome-wide approaches to predict coiled-coil proteins ,43. In cMultiCoil provides a more stringent coiled-coil prediction than other programs such as COILS, resulting in less false positive predictions. In tests on the PDB database of solved protein structures, two-thirds of the sequences predicted by COILS did not contain coiled-coils . By compIn this study, we focused on proteins potentially involved in structural functions. As the emphasis was placed on proteins with long or multiple coiled-coil domains, it is possible that our selection criteria resulted in the exclusion of homologs of proteins with short stretches of coiled-coil that barely qualified for the analysis. The selection criteria applied in this study have been shown to exclude 97% of the known bZIP proteins from Arabidopsis . Other eWhen clustering sequences with long coiled-coil domain in the pilot analysis, the majority of proteins with long coiled-coil domains was grouped together in one large cluster. Many of the proteins with unknown functions in this group were annotated as "myosin-like", however only about 20% of the proteins in the cluster actually contained a myosin motor domain. In the other cases, the only similarity to myosin was the presence of a long coiled-coil domain similar to the myosin coiled-coil tail. This illustrates the ease with which long coiled-coil domains can lead to misannotations in databases with annotations based on sequence similarity searches.Masking the coiled-coil domains before sequence comparison and clustering significantly increased the specificity of the clustering analysis, however protein sequences with high coiled-coil coverage were lost in the subsequent clustering as the masking left little to no sequence for comparison. Examples are the animal and yeast tropomyosins, many of which were predicted to contain 100% coiled-coil coverage, paramyosin, and the plant cytoskeletal protein CIP1 with more than 80% coiled-coil coverage .Our genome-wide identification of coiled-coil proteins and subsequent clustering provides data suggesting evolutionary conservation or uniqueness of coiled-coil proteins among 22 fully sequenced genomes. We found SMC, MukB, SbcC and Rad50 proteins to be the proteins with the longest coiled-coil domains occurring in prokaryotes, whereas eukaryotic proteomes also contained proteins with stretches of coiled-coil longer than the SMC rod domains. The high conservation of the SMC proteins and their structural relatives involved in chromosome maintenance and repair demonstrates the universal importance and conservation of DNA housekeeping mechanisms.Long coiled-coil proteins specific to eukaryotes are predominantly involved in subcellular infrastructure maintenance and trafficking control. Table The repeat nature of the coiled-coil motif makes it difficult to clearly determine sequence homology relationships between long coiled-coil proteins. Functional studies will have to reveal whether so far uncharacterized prokaryotic and plant coiled-coil proteins fulfill similar functions to metazoan counterparts.Proteome sequence sets of fully sequenced genomes were downloaded from the European Bioinformatics Institute (EBI)  for orgaPrediction and selection of coiled-coil proteins was performed using the underlying schema and software systems developed to create the ARABI-COIL database . In summTo eliminate interference of the coiled-coil repeat motif with sequence homology analysis, coiled-coil domains were "masked" before subjecting the sequences to Smith-Waterman sequence similarity searches. Mask information was created based on the processed MultiCoil prediction data generated to populate the MySQL databases for each genome. A Java-based program was applied to the FASTA sequences selected for Smith-Waterman comparison to replace all amino acids predicted to be contained in coiled-coil domains with the letter X, effectively masking coiled-coil domains.Smith-Waterman comparison was conducted using the TimeLogic Smith-Waterman implementation at OSC and the Blosum62 scoring matrix on all unique sequences in the combined sequences set. Sequences with masked coiled-coil domains were used as query on unmasked sequence sets as target. A P-score cut-off of 1.0e-03 was used as a threshold for selecting sequence similarity relationships. For sequences to be characterized as pair-wise similar and recovered for use in the clustering analysis, the P-score value must be less than this threshold based on the query-target Smith-Waterman comparison.After completing the pair-wise similarity calculation using the Smith-Waterman algorithm and extracting sequence pairs and associated P-scores, sequences were grouped using a modified version of Kruskal's minimum cost spanning tree algorithm . The algThe clustering was tested in a pilot analysis on a combined sequence set including 527 prokaryotic long coiled-coil proteins and eukaryotic proteins containing extended coiled-coil domains of at least 250 amino acids in length or at least 60% of the protein sequence in a coiled-coil. Edges with P-scores greater than 1.0e-03 to 1.0e-15 were ignored when combining sub-trees in the algorithm. The success of the clustering was estimated by observing the clustering behavior of well-known coiled-coil protein families, such as SMC proteins and myosins. After testing the effects of masking the coiled-coil domains and optimizing cut-offs for P-scores during clustering, the complete coiled-coil sequence set containing 3576 long coiled-coil proteins from the 22 genomes was processed similarly. Different P-score thresholds were explored in efforts to increase specificity in the multi-genome sequence set while preserving comprehensive coverage. Employing Kruskal's algorithm, the 3576 sequence set resulted in 156 clusters covering 3567 sequences using a threshold of 1.0e-03, 467 clusters covering 3551 sequences using a threshold of 1.0e-6 and 850 clusters covering 3520 sequences using a threshold of 1.0e-15. .Even with the improved selectivity of the clustering demonstrated in the pilot investigation using masked coiled-coil sequences, the overall effectiveness of the resulting clustering still required refinement to achieve sufficient specificity. The use of Kruskal's algorithm for subset selection enabled transitively similar sequences to be included in specific clusters. (Transitively similar sequences are sequences in which sequence A is similar to sequence B and sequence B is similar to sequence C thereby clustering sequence A and C which would otherwise not belong to the same cluster.) One drawback of this simplified clustering is that a given sequence need only be similar to at least one other sequence in the cluster. This limitation resulted in clusters containing sequences which, while closely related to at least one other sequence in a cluster, were not closely related to every sequence within the cluster.The algorithm was consequently improved to specifically preclude transitively similar sequences by requiring all sequences in a given cluster to satisfy the P-score threshold for all pair-wise relationships in the cluster. The new algorithm dramatically improved specificity, with the same 3576 masked sequence set generating 1213 non-overlapping clusters covering 3567 sequences, 1263 non-overlapping clusters covering 3551 sequences, and 1384 non-overlapping clusters covering 3520 sequences with the improved algorithm for the same corresponding P-score threshold values. The P-score threshold of 1.0e-06 was selected as the appropriate balance of sequence coverage and cluster discrimination required.The interest in identifying sequences which qualified for more than one cluster and bridged multiple clusters of protein families drove a second modification of the clustering algorithm. By design, the modified Kruskal's algorithm created mutually orthogonal, non-overlapping clusters while precluding transitively similar sequences from populating the same cluster. The 'greedy' algorithm was modified to specifically identify transitively similar sequences between clusters, enabling a unique ability to identify "bridge" sequences which satisfy participation criteria in multiple clusters or protein families. The modification amounted to simply validating each sequence's individual ability to satisfy participation criteria for a cluster based on the non-overlapping cluster partitioning.The software used to conduct the actual cluster analysis in the study is available for download at the Ohio Bioscience Library .Multiple sequence alignments and phylogenetic trees were generated for clusters of interest using sequences with masked coiled-coil domains and ClustalW version 1.82 incorporating the Blossum scoring matrix . PhylogeCASP, CDP/cut alternatively spliced productCC, coiled-coilCDD, conserved domain databaseCDP, CCAAT displacement proteinCENP, centromer proteinCIP1, COP1-interactive protein 1CHUP1, chloroplast unusual positioning 1CLIP, cytoplasmic linker proteinDAM, disheveled associated activator of morphogenesisDIA1, Diaphanous-related formin 1DOC1, downregulated in ovarian cancer 1EBI, European Bioinformatics InstituteERM, ezrin/radixin/moesinFPPs, filament-like plant proteinsHsp, heat shock proteinIF, intermediate filamentKCBP, kinesin-like calmodulin-binding proteinKIP1, kinase interacting protein 1KLP, kinesin-like proteinMCP, methyl-accepting chemotaxis proteinMKRP, mitochondrial kinesin-related proteinMLP, myosin-like proteinNuMA, nuclear mitotic apparatusORF, open reading frameOSC, Ohio Supercomputer CenterPAKRP, phragmoplast-associated kinesin-related proteinPP1, protein phosphatase 1RBP, Retinoblastoma-binding proteinROCK, Rho-associated coiled-coil containing kinaseSLAP, sarcolemmal-associated proteinSMC, structural maintenance of chromosomesS/W, Smith-Waterman sequence comparisonTACC, transforming acidic coiled-coilTpr, translocated promoter regionVIPP1, vesicle-inducing plastid protein 1XML, extensible markup languageAR coordinated this study, analyzed the data, and prepared the manuscript. SJS participated in MultiCoil and Smith-Waterman output processing and ClustalW analysis. EAS generated MultiCoil and Smith-Waterman outputs, developed software for pre- and post-processing and coiled-coil masking, and wrote the code for the clustering algorithm. IM proposed and supervised the study and edited the manuscript. All authors read and approved the final manuscript.Prokaryotic coiled-coil proteins Tables S1-S14: Protein details of all long coiled-coil proteins predicted in the prokaryotic genomes analyzed in this study. Open file with Acrobat Reader.Click here for fileEukaryotic clusters of interest Figures S1-S6: Phylogenetic trees based on ClustalW alignments of the sequences, displayed using TreeView v.1.6.6. Open file with Acrobat Reader.Click here for fileSequence details for Figures S1-S6, supplement to Synechocystis. All other sequence IDs correspond to the EBI identifiers in the downloaded FASTA files. Max. Coil Length, longest coiled-coil domain in the protein sequence; Coil Coverage, percent of sequence predicted to be in a coiled-coil. Open file with Microsoft Excel.Click here for fileYeast clusters Table S18: Protein information and prediction data for sequences in yeast clusters with two species (Saccharomyces cerevisiae and Schizosaccharomyces pombe) represented. Sequence IDs correspond to the EBI identifiers in the downloaded FASTA files. Max. Coil Length, longest coiled-coil domain in the protein sequence; Coil Coverage, percent of sequence predicted to be in a coiled-coil. Open file with Microsoft Excel.Click here for fileSmall mammalian clusters; supplement to Table Click here for fileSmall plant clusters; supplement to Table Click here for file


Two retrovirus vectors were compared for their ability to express granulocyte-macrophage colony stimulating factor (GM-CSF) in a haematopoietic cell line, FDCP1, which is dependent on GM-CSF for survival. Both a MoMLV-based vector pVneoGM, and a MPSV-based vector, M3neoGM, were found to be capable of transmitting and expressing both GM-CSF and neomycin sequences in the myeloid FDCP1 cell line. Our results also demonstrate that pVneoGM is more efficient at generating GM-CSF independent colonies than M3neoGM. Analysis of cell lines derived after infection confirmed pVneoGM expressed higher levels of GM-CSF. Cell lines generated by infection with pVneoGM responded to levels of exogenous recombinant GM-CSF which did not stimulate growth of the parental cell line, suggesting autocrine stimulation may convey a proliferative advantage under sub-optimal growth conditions. Finally the parental vectors pVneo and M3neo were shown to be capable of expressing the neomycin gene in both murine haematopoietic progenitor and stem cells.


Activation of the type I insulin-like growth factor receptor (IGFIR) promotes proliferation and inhibits apoptosis in a variety of cell types. Transgenic mice expressing a constitutively active IGFIR or IGF-I develop mammary tumors and increased levels of IGFIR have been detected in primary breast cancers. However, the contribution of IGFIR activation in promoting breast cancer progression remains unknown. Mammary epithelial cell lines grown in three-dimensional cultures form acinar structures that mimic the round, polarized, hollow and growth-arrested features of mammary alveoli. We used this system to determine how proliferation and survival signaling by IGFIR activation affects breast epithelial cell biology and contributes to breast cancer progression.Pooled, stable MCF-10A breast epithelial cells expressing wild-type IGFIR or kinase-dead IGFIR (K1003A) were generated using retroviral-mediated gene transfer. The effects of over-expression of wild-type or kinase-dead IGFIR on breast epithelial cell biology were analyzed by confocal microscopy of three-dimensional cultures. The contribution of signaling pathways downstream of IGFIR activation to proliferation and apoptosis were determined by pharmacological inhibition of phosphatidylinositol 3' kinase (PI3K) with LY294002, MAP kinase kinase (MEK) with UO126 and mammalian target of rapamycin (mTOR) with rapamycin.473 of Akt and Ser2448 of mTOR throughout IGFIR structures. Inhibition of PI3K with LY294002 or MEK with UO126 prevented the development of acinar structures from IGFIR-expressing but not control cells. The mTOR inhibitor rapamycin failed to prevent IGFIR-induced hyperproliferation and survival signaling.We found that MCF-10A cells over-expressing the IGFIR formed large, misshapen acinar structures with filled lumina and disrupted apico-basal polarization. This phenotype was ligand-dependent, occurring with IGF-I or supraphysiological doses of insulin, and did not occur in cells over-expressing the kinase-dead receptor. We observed increased proliferation, decreased apoptosis and increased phosphorylation of SerIncreased proliferation and survival signaling as well as loss of apico-basal polarity by IGFIR activation in mammary epithelial cells may promote early lesions of breast cancer. Three-dimensional cultures of MCF-10A cells over-expressing the IGFIR are a useful model with which to study the role of IGFIR signaling in breast cancer progression and for characterizing the effects of chemotherapeutics targeted to IGFIR signaling. Cycles of proliferation and invasion followed by massive apoptosis are central to the physiology of the mammary gland. The branching ductal architecture develops postnatally in response to hormone stimulation during puberty, but does not reach a fully differentiated state until pregnancy and lactation when hormones stimulate the proliferation and differentiation of terminal ductal lobular units that contain many alveoli. Following lactation, the mammary gland undergoes massive apoptosis and returns to its pre-pregnancy state, a process called involution. Insulin-like growth factor (IGF) signaling is essential for these processes.Activation of the type I insulin-like growth factor receptor (IGFIR), by binding of IGF-I or supraphysiological doses of insulin, results in receptor autophosphorylation, association and phosphorylation of adapter proteins such as insulin receptor substrate (IRS)-I, Shc, Crk, Grb-2, and Grb-10, and subsequent activation of downstream signaling pathways, including phosphatidylinositol 3' kinase (PI3K)/Akt and Ras/Raf/mitogen-activated protein kinase (MAPK). Activation of the IGFIR leads to proliferation and anti-apoptotic signaling in a variety of cell lines ,5.in vivo .Three-dimensional cultures of MCF-10A cells were grown in 8-well chamber slides  as described  using grMCF-10A acinar structures were immunolabeled as described  with theDuring the initial phases of MCF-10A acinar development (days 0 to 4) a single cell plated on basement membrane (Matrigel) progresses to a cluster of cells at which time polarization and survival signaling in cells in contact with basement membrane drive the formation of a polarized outer layer of cells. Apoptosis and autophagy clear the inner cells, those that lack contact with the basement membrane, to generate a hollow, polar, growth arrested structure by approximately day 10 -31. To sTo test whether these phenotypes were dependent on the protein kinase activity of IGFIR, we generated stable pools of MCF-10A cells expressing a kinase-dead IGFIR construct, with lysine 1003 in the ATP-binding site mutated to alanine (K1003A) . Figure 473 and phosphorylation of ERK (Thr183 and Tyr185). Treatment of K1003A-10A cells with insulin or IGF-I did not augment phosphorylation of downstream signaling pathways; however, there was still some detectable phosphorylation of Akt and ERK after insulin and IGF-I stimulation, most likely resulting from the mixed population of infected cells.We performed western blots on whole-cell lysates from monolayer cultures of control, K1003A and IGFIR-10A cells treated with insulin (10 μg/ml) or IGF-I (50 ng/ml). Figure p < 0.01, Student's t test; Figure Figure In addition to increased cell proliferation, IGFIR structures displayed decreased cell death. Panels c, d of Figure MCF-10A acinar structures exhibit polarized organization with the apical surface located toward the lumen and basal surfaces in contact with basement membrane -30. We oActivation of the IGFIR results in phosphorylation of the beta subunit, subsequent phosphorylation and recruitment of adapter proteins such as IRS-I and Shc, and activation of two main signal transduction pathways: PI3K/Akt and Ras/Raf/MAPK. Figure L/Bcl-2-associated death promoter homolog) and the forkhead transcription factors . We iewed in . In contiewed in -43. We oWe tested the involvement of the PI3K/Akt and the Ras/Raf/MAPK pathways in promoting proliferation and survival signaling in IGFIR structures by pharmacological inhibition of PI3K with 50 μM LY294002 ,45, MAP Inhibition of MEK with 10 μM UO126 prevented any significant development of IGFIR structures. Any structures present in these cultures appeared as clusters of cells Figure , panel cInhibition of PI3K with 50 μM LY294002 had a similar effect on IGFIR structure formation: any structures present appeared as clusters of cells Figure , panel eThree-dimensional cultures of MCF-10A cells expressing myristoylated Akt formed large, partially filled structures with increased proliferation at early stages of growth . Rapamycin situ, have increased cell numbers and filled and enlarged mammary alveoli that remain contained by basement membrane proteins; cancerous cells overcome the constraints of the basement membrane and invade the surrounding connective tissue in invasive stages; advanced, metastatic stages are characterized by cancer cells leaving the breast, usually via the lymphatic system, and invading other organs [in vitro models [Breast cancer is a progressive disease: pre-invasive lesions, such as atypical hyperplasias and carcinomas r organs . Three bo models . Recapito models .Our data support and extend those reported in a recent paper by Irie and colleagues  demonstrAcinar structures formed from MCF-10A cells expressing a variety of oncogenes have several characteristic features: increased proliferation, decreased apoptosis, and filled lumina. Activation of ErbB2 in three-dimensional cultures of MCF-10A cells resulted in highly proliferative, multi-acinar structures with filled lumina . These sAcinar structures formed from IGFIR-10A cells display notable similarities and differences with each of these phenotypes. First, IGFIR structures displayed increased proliferation as assessed by Ki-67 expression, similar to ErbB2, CSF1R and growth hormone structures. However, the increase in proliferation in IGFIR structures occurs during both early (data not shown) and late stages of acinar development, as opposed to structures formed from over-expression of Akt1 in which proliferation is seen only in early stages. Second, IGFIR structures had decreased apoptosis, assessed by cleaved caspase-3 expression, as would be expected from their filled lumina, a feature that is similar to ErbB2, CSF1R, Akt1 and growth hormone structures. The anti-apoptotic effects could occur through direct actions on apoptotic signaling molecules, such as through suppression of Bim  or repreOver-expression of IGFIR in a breast cancer cell line, MCF-7, and growth on Matrigel resulted in enlarged structures as a result of aggregation of cells due to increased cell-cell adhesion via E-cadherin . AdditioDependence on the continued signaling of a mutated or over-expressed oncogene may be exploited for targeted cancer therapies . AntisenRapamycin prevented acinar morphology associated with over-expression of activated Akt1 and mutated PI3K ,58. AlthEpithelial cell proliferation, survival signaling and invasion of the surrounding stromal tissue are critical processes for normal mammary gland development, suggesting a strong link between normal mammary development and pathogenesis of breast cancer. The increased proliferative and survival signaling we observed in IGFIR structures are compatible with the established role of IGF-I signaling in mammary gland development. IGFIR knockout mice die shortly after birth due to respiratory failure, but transplanted embryonic mammary buds from IGFIR knockout mice in syngeneic recipients showed decreased proliferation of cap cells in the terminal end buds and decreased branching and extension of ducts into the fat pad . SimilarSeveral factors promoting or affecting breast cancer pathogenesis, such as Her2/neu, estrogen and progesterone receptors, E-cadherin, vascular endothelial growth factor, and BRCA1 have been identified and are useful as prognostic indicators or therapeutic targets -67. In tCSF = colony-stimulating factor; CSF1R = colony-stimulating factor receptor; DIC = differential interference contrast; DMEM = Dulbecco's modified Eagle's medium; DMSO = dimethyl sulfoxide; ERK = extracellular-signal regulated kinase; GM130 = Golgi matrix protein of 130 kDa; IGF-I = type I insulin-like growth factor; IGFIR = type I insulin-like growth factor receptor; IRS-I = insulin receptor substrate I; MAPK = mitogen-activated protein kinase; MEK = MAP kinase kinase; mTOR = mammalian target of rapamycin; p70S6K = p70S6 kinase; PBS = phosphate-buffered saline; PI3K = phosphatidylinositol 3' kinase; TO-PRO-3 = TO-PRO-3 iodide.The authors declare that they have no competing interests.GMY participated in the experimental design and interpretation of results, carried out the experimental procedures and drafted the manuscript. WE participated in the experimental design, interpretation of results and revision of the manuscript.


Recent characterizations of methyl transferases as regulators of cellular processes have spurred investigations into how methylation events might influence the HIV-1 life cycle. Emerging evidence suggests that protein-methylation can positively and negatively regulate HIV-1 replication. How DNA- and RNA- methylation might impact HIV-1 is also discussed. Biologically, methylation is executed by discrete enzymes (methyltransferases) which recognize DNA, RNA and proteins. Methylation of DNA, RNA or protein can regulate gene expression, RNA metabolism, and protein activity. Recently, protein methylation has stepped into the lime light due to the identification and characterization of protein arginine methyltransferases (PRMTs) which catalyze additions to arginine residues. PRMTs transfer one or two methyl groups from S-adenosylmethinine (SAM) to the guanidine-nitrogen atoms in arginine, forming methylarginine and/or S-adenosylhomocysteine . PRMTs aArginine methylation impacts cellular processes ranging from transcription, RNA processing, and cell signaling. PRMTs can function as transcriptional co-activators through remodeling chromatin and modifying histone tails. Methylation of histone H3 and H4 by CARM1 and PRMT1 is thought to contribute to a histone code ,6. RNA bRetrovirology [et al. used HIV-1 infected CEM T-cells and provirus transfected HEK293T cells to link protein methylation to virion infectivity. They employed adenosine periodate (AdOx), a general inhibitor of many methyltransferases, to address the effect of methylation on HIV-1 in tissue culture. Willemsen et al. found that AdOx treatment increased virus production from both adherent cells transfected with a proviral molecular clone and T-cells infected with HIV-1. Interestingly, the authors saw decreased p24 levels in infected CEM T-cells treated with AdOx, which was accompanied by substantially increased p24 amounts in the culture supernatants. This provocative observation suggests that methylation may regulate the processing and/or assembly of HIV-1 Gag protein. This interpretation makes sense because they also saw differences in the composition and size of virions produced in the presence or absence of AdOx, a finding consistent with perturbed virion assembly when methylation events are inhibited. Finally, AdOx treatment was found to significantly decrease HIV-1 infectivity; however, this decreased infectivity was overcome when HIV-1 virions were pseudotyped with VSV-G protein. The latter result invokes a methylation step in either HIV-1 envelope-receptor binding and/or virus envelope-uncoating in newly infected cells  poorly et al. stems clearly from PRMT6 methylation of Rev, the results accrued by Willemsen et al. paint a less clear mechanistic picture. Willemsen et al. used a broadly non-specific inhibitor of methylation, AdOx. AdOx is a competitive inhibitor of all SAM dependent methylation which includes protein-, RNA-, and DNA- modifications. Hence, the findings from Willemsen et al. while suggestive of an effect involving HIV-1 envelope cannot formally exclude contributions from Adox's action on unidentified RNA- or DNA- methylation.Collectively, the two new articles extend the emerging notion for methylation regulating HIV-1 biology. While the activity described by Invernizzi et al. [So where do these reports lead us? The HIV-1 genome contains 9 open reading frames which can encode 15 proteins, and virus replication involves a complex interplay between viral and cellular proteins. What remains to be deciphered is the relative contribution of methylation on virus-factors versus cellular macromolecules. In this regard, Kwak et al.  have repBesides protein-methylation, there is compelling evidence that DNA-methylation influences HIV-1 replication. CpG methylation of LTR DNA has been reported to promote viral latency -19. SomeCaenorhabditis elegans mRNAs [Finally, what about RNA-methylation? RNA methyl modifications are involved in functional maturation of many species of cellular RNAs including mRNA, rRNA, tRNA, snRNA and snoRNA. Methylation of the RNA cap (7-methylguanosine cap) is a critical processing step for cellular and viral mRNAs. This action increases mRNA stability and promotes efficient export and translation. Other cap methyl modifications  have been identified in Oncorna viruses, Reo viruses, Sindbis virus, and ns mRNAs -25. The The author(s) declare that they have no competing interests.