II) dependent gene expression requires accessory factors termed transcriptional coregulators. One coregulator that universally contributes to PolII-dependent transcription is the Mediator, a multisubunit complex that is targeted by many transcriptional regulatory factors. For example, the Caenorhabditis elegans Mediator subunit MDT-15 confers the regulatory actions of the sterol response element binding protein SBP-1 and the nuclear hormone receptor NHR-49 on fatty acid metabolism. Here, we demonstrate that MDT-15 displays a broader spectrum of activities, and that it integrates metabolic responses to materials ingested by C. elegans. Depletion of MDT-15 protein or mutation of the mdt-15 gene abrogated induction of specific detoxification genes in response to certain xenobiotics or heavy metals, rendering these animals hypersensitive to toxin exposure. Intriguingly, MDT-15 appeared to selectively affect stress responses related to ingestion, as MDT-15 functional defects did not abrogate other stress responses, e.g., thermotolerance. Together with our previous finding that MDT-15:NHR-49 regulatory complexes coordinate a sector of the fasting response, we propose a model whereby MDT-15 integrates several transcriptional regulatory pathways to monitor both the availability and quality of ingested materials, including nutrients and xenobiotic compounds.In eukaryotes, RNA polymerase II (Pol All organisms adapt their physiology to external input, such as altered food availability or toxic challenges. Many of these responses are driven by changes in gene transcription. In general, sequence specific DNA-binding regulatory factors are considered the specificity determinants of the transcriptional output. Here, we show that, in the roundworm Caenorhabditis elegans, one subunit of a >20 subunit, evolutionarily conserved, non-DNA binding co-factor termed Mediator, specifies a portion of the metabolic responses to a mixture of ingested material. This protein, MDT-15, is required for appropriate expression of genes that protect worms from the effects of toxic compounds and heavy metals. Our previous findings showed that the same protein also cooperates with other regulators to coordinate lipid metabolism. We suggest that MDT-15 may “route” transcriptional responses appropriate to the ingested material. This physiological scope appears broader and more sophisticated than that of any individual regulatory factor, thus coordinating systemic metabolic adaptation with ingestion. Given the evolutionary conservation of MDT-15 and the Mediator, a similar regulatory pathway may ensure health and longevity in mammals. II). These complexes also contain transcriptional cofactors that serve various functions, such as chromatin remodeling and chromatin modification. Within this complex machinery, the sequence specific regulatory factors are generally thought to be the primary determinants that specify transcriptional output in response to a certain signal.Eukaryotic gene transcription requires the concerted interplay of many factors. DNA-binding factors nucleate specific regulatory complexes on individual genes, culminating in assembly of functional RNA polymerase II  that regulate FA and cholesterol metabolism In a previous study we found that the To connect the function of MDT-15 in transcription to its precise physiologic role, we sought to more broadly define MDT-15's sphere of influence. As an initial step, we set out to globally discover new MDT-15-dependent genes in an unbiased fashion, and thus to identify previously unrecognized biological processes that lie downstream of MDT-15.C. elegans Mediator subunit MDT-15 impacts expression of select genes involved in fatty acid (FA) metabolism i.e. allowed to synchronously develop to larval stage L4; see mdt-15 RNA-interference  animals.We previously showed that the mdt-15(RNAi) animals included 12 previously identified MDT-15 target genes involved in lipid metabolism mdt-15 itself, thus validating our experimental approach   worms harvested at the L4 stage. We found that 50 of 97 genes (52%) exhibited more than two-fold deregulation in mdt-15(tm2182) mutants (mdt-15(RNAi) worms. In contrast, 28 genes deregulated more than two-fold in mdt-15(RNAi) worms were not affected to this extent in mdt-15(tm2182) mutants, although most genes were still downregulated to some extent (and only two were upregulated in mdt-15(tm2182) worms). Thus, mdt-15(tm2182) mutants recapitulate many but not all of the gene expression defects exhibited by mdt-15(RNAi) worms. Overall, the gene expression changes in mdt-15(tm2182) worms appear less severe than those presented by mdt-15(RNAi) worms, suggesting that mdt-15(tm2182) may represent a hypomorphic allele. Alternatively, as the mutation is predicted to produce a truncated MDT-15, and as mdt-15 mRNA levels appear normal in mdt-15(tm2182) worms (mdt-15(tm2182) mutant as a valuable tool to study MDT-15 function.To corroborate the data obtained with RNAi depletion of endogenous MDT-15 we obtained a strain carrying a mutation in the ife span , reducedife span , clear aife span  in wild- mutants ; 47 of t2) worms , it is pe.g. FA-Δ9-desaturases and FA-oxidases). These results confirm the importance of MDT-15 in regulation of lipid metabolic genes.To identify pathways and molecular functions common to the 187 genes observed by microarray analysis to be downregulated after MDT-15 depletion, we employed the DAVID gene ontology (GO) annotation tool Drosophila melanogaster proteins harboring DUF227 domains are implicated in insecticide resistance D. melanogaster DUF227 genes are induced by Phenobarbital Unexpectedly, we also discovered other enriched domains subject to MDT-15 dependence, specifically UDP-glucuronosyl/UDP-glucosyltransferases , glutathione S-transferases , short-chain dehydrogenase/reductase SDRs , and FAD-domains  likely involved in heavy metal detoxification. In summary, 47 of 187 genes (25%) that our microarray analysis identified as significantly downregulated in mdt-15(RNAi) worms may be involved in metabolism or elimination of toxic substances (i.e. 20%), suggesting that both processes compose important sectors within MDT-15's sphere of influence , three alcohol and aldehyde dehydrogenases, two ABC-transporters, and two acyl-CoA-synthetases (ACSs). These are all members of protein families associated with detoxification. Interestingly, the ACS proteins represent a class of lipid metabolizing enzymes, some members of which can metabolize xenobiotics bstances . This nunfluence .mdt-15(RNAi) worms revealed fewer enriched functions and domains  worms to published sets of genes exhibiting tissue specific expression in C. elegansmdt-15(RNAi) worms are intestine-selective (22%); in contrast, no MDT-15 target genes were enriched in muscle or germ line, and only 2% were pharynx-specific  worms also exhibited enrichment of intestine-selective genes (data not shown). This selective overrepresentation among MDT-15 targets of intestinally expressed genes fits well with the roles of MDT-15 in lipid metabolism MDT-15 is highly expressed in the intestine specific . CompariC. elegans ugt, gst, dhs, and cyp genes (encoding CYP450s) are important for detoxification, several of these genes are transcriptionally induced when wild type worms are exposed to toxic chemicals  with control or mdt-15 RNAi bacteria seeded on plates that contained toxic compounds (fluoranthene or β-naphthoflavone). We chose fluoranthene because it is a naturally occurring carcinogen, and β-naphthoflavone because it is a known inducer of numerous CYP450s control(RNAi) worms (mdt-15(RNAi) worms; for example, fluoranthene induced mRNA levels of gst-5 ten-fold in control(RNAi) worms, but only two-fold in mdt-15(RNAi) worms (e.g. ugt-25) exhibited MDT-15 dependent activation, suggesting that other regulatory pathways also contribute to gene induction by toxins. Moreover, when mdt-15(tm2182) mutants were exposed to fluoranthene for 40 hr (i.e. L1–L4), we observed defects in fluoranthene-induced mRNA accumulation for 13 of the 21 genes, corroborating the results obtained with RNAi (control(RNAi) and mdt-15(RNAi) worms (panel A) and wild type and mdt-15(tm2182) worms (panel C). Taken together, these data demonstrate that MDT-15 is required not only for basal, but also for toxin-activated transcription of select detoxification genes.To test whether MDT-15 is required to induce these detoxification genes, we fed N2 wild-type worms for 40 hr (i) worms . Importai) worms . Howeverith RNAi . The selcyp genes to be MDT-15 dependent . This may be attributable, at least in part, to the low expression levels of many cyp genes. We therefore used qPCR to examine directly the expression of 43 C. elegans cyp genes, and to explore a potential role for MDT-15 in their regulation  worms  worms . These rmdt-15(RNAi) worms to express and appropriately induce detoxification genes is an indirect effect associated with arrested development and premature death. To address this issue, we determined relative mRNA levels of toxin-responsive genes in mdt-6(RNAi) worms. We focused on MDT-6 because, (i) like MDT-15, it is a Mediator subunit, (ii) it is required for stage- and gene-specific transcription in C. elegans, and (iii) mdt-6 knockdown evokes larval arrest and premature death, phenotypes reminiscent of but distinct from those of MDT-15 depletion mdt-6 RNAi affected neither basal, nor fluoranthene- or β-naphthoflavone-induced expression of detoxification genes  worms, we employed a conditionally sterile strain, CF512 (fer-15(b26)II; fem-1(hc17)III) fer-1 and fem-15 mutations do not compromise the detoxification per se, we first compared the toxin response in CF512 worms to that in N2 worms. We found that, at the L4 stage, CF512 worms exhibited a very similar response to fluoranthene and β-naphthoflavone as did N2 worms . Under these conditions, 12 of the 20 tested genes exhibited MDT-15 dependence in the absence of fluoranthene. Furthermore, 13 genes were fluoranthene-responsive in these worms, and for four of these genes the induction was at least partially MDT-15 dependent, resembling our results obtained in N2 L4 worms (mdt-15(RNAi) and mdt-15(tm2182) worms reflects a direct action of MDT-15, rather than a secondary consequence of reduced viability or arrested development.To assess whether the defects in gene induction by toxins reflected abnormal growth of N2 worms . Next, wL4 worms . Thus, wnhr-49(RNAi) and sbp-1(RNAi) worms exhibited reduced mRNA levels of fat-5, -6, and -7 genes (mdt-15(RNAi) worms, the overall induction of detoxification genes by toxins strongly correlated with the induction in control(RNAi) worms ]; Given that MDT-15 binds to, and collaborates with NHR-49 and SBP-1 to express certain metabolic genes, it seemed conceivable that these two regulatory factors might also be responsible for expression of MDT-15 dependent detoxification genes. To test this hypothesis, we knocked down endogenous NHR-49 or SBP-1 in wild-type worms. As previously demonstrated, -7 genes . In cont-7 genes . Indeed,i) worms . This ismdt-15 depletion or mutation would render worms hypersensitive to toxins. To test this, we grew conditionally sterile CF512 worms on control and mdt-15 RNAi bacteria (RNAi from the L1 stage on) in the presence of various concentrations of toxins, and monitored their development and morphology until day four of adulthood. After this prolonged exposure to mdt-15 RNAi, worms were slightly thinner than control(RNAi) worms, consistent with previous results mdt-15 RNAi, but not control RNAi, to evoke an arrest as small, scrawny adults (mdt-15(tm2182) mutants exhibited a similar adult arrest phenotype when grown on high concentrations of fluoranthene  and zinc (Zn2+), but not by copper (Cu2+) 2+and Zn2+, but not by Cu2+, thus establishing it as a novel metal-responsive gene  with control or mdt-15 RNAi bacteria seeded on plates containing Cd2+, Zn2+ or Cu2+. We then probed for induction of the aforementioned genes, and other known heavy metal responsive genes , by qPCR. Consistent with published results, we observed mRNA accumulation of cdr-1, mtl-1, and mtl-2 in the presence of Cd2+, and of mtl-1 and cdr-1 in the presence of Zn2+  worms (mdt-15(tm2182) mutants were also severely defective for both basal and Cd2+-induced mtl-1, mtl-2, cdr-1, and T18D3.3 expression, and for basal Y37A1B.5 expression  overlaps in statistically significant manner with a list of Cdve genes . To furt of Zn2+ . Importai) worms . Moreovepression . Finally3.3 mRNA . Togethemdt-15 RNAi, and then challenged them with high concentrations of Cd2+ for five hr. We used a worm strain harboring a transcriptional Pmtl-2::GFP reporter, CL2122 mtl-2 promoter activity in vivo. Reminiscent of the results obtained with chronic metal exposure, Cd2+ caused induction of GFP-fluorescence. Importantly, the GFP signal was weaker in worms grown on mdt-15 RNAi, and Cd2+-dependent induction was not observed in these worms  worms upregulate known and putative heat-shock proteins . Thus, in contrast to the aforementioned ingestion-related stresses, MDT-15 might play a negative regulatory role in heat-induced transcription. To test this possibility we grew wild-type N2 worms for 40 hr (i.e. L1–L4) on control and mdt-15 RNAi bacteria at ambient temperature (20°C), subsequently applied heat-shock , and then quantified the mRNA levels of several hsp genes. At 20°C mdt-15(RNAi) worms exhibited increased mRNA levels of T05E11.3, hsp-16.2, and hsp-17 compared to control(RNAi) worms; we detected similar increases for the mRNAs of the HSPs F44E5.4 (HSP70), hsp-16.1, and hsp-16.49  worms (mdt-15(RNAi) worms. Instead, mdt-15(RNAi) worms induced these genes somewhat above the levels exhibited by control(RNAi) worms. Moreover, mdt-15(tm2182) mutants exhibited a qualitatively similar, yet milder gene expression defect  rather than a direct effect of deregulation of hsp gene modulators. Taken together, these results demonstrate that MDT-15 is not generally required for stress-activated gene expression, but rather may constitute an element in a regulatory system that specifically monitors availability and integrity of ingested material  and mdt-15(RNAi) worm populations at 35°C. We found that both populations exhibited similar thermotolerance phenotypes. This was the case in N2 worms exposed to mdt-15 RNAi bacteria from L1–L4 stage (mdt-15 RNAi from the L1 stage on (data not shown). Therefore, the altered heat-shock protein gene expression following mdt-15 depletion is not sufficient to increase resistance to elevated temperatures. This may be due to other gene expression defects in mdt-15(RNAi) worms. Alternatively, abnormally high heat-shock protein expression in mdt-15(RNAi) worms may not confer a marginal benefit over heat-shock protein in control(RNAi) animals.As L4 stage , and alsC. elegans Mediator subunit MDT-15 specifies a portion of the alternative metabolic responses to a complex mixture of ingested material.A prevailing view concerning transcriptional regulation is that DNA-binding regulatory factors are the primary determinants of specificity and coordination within transcriptional regulatory networks. Our results support an additional mechanism in which a specific component of the Mediator complex serves such functions. Specifically, we demonstrate that the C. elegans experiences a diversity of conditions, such as insufficient nutrient supply, potentially detrimental substances (endo- and xenobiotics), and harmful microorganisms. Toxic hydrophobic compounds and heavy metals represent a complex challenge as they can produce their adverse effects both acutely, and during chronic accumulation over prolonged exposure periods. Therefore, efficient detoxification mechanisms have evolved to ensure metabolism and elimination of harmful molecules. Here, we provide several lines of evidence that the conserved Mediator subunit MDT-15 is a key contributor towards regulation of systemic detoxification. First, even in the absence of toxic challenge, mdt-15 depletion or mutation results in downregulation of many detoxification-related genes. In addition, MDT-15 is required to induce select detoxification genes in response to hydrophobic toxins and heavy metals. Finally, depletion or mutation of MDT-15 renders worms hypersensitive to at least one toxic compound. Of note, MDT-15 affects expression of all three classes of detoxification genes, i.e. activation enzymes such as CYP450s (class I), transferase enzymes such as the UGTs and GSTs (class II), and transporters such as pgp-7 and pmp-5 (class III mdt-15(RNAi) worms, because (i) larvally arrested mdt-6(RNAi) and sbp-1(RNAi) worms did not deregulate detoxification genes, (ii) the heat-shock response was unaffected, suggesting that stress-driven transcription can function, and (iii) numerous fluoranthene and cadmium-induced genes were also deregulated in worms exposed to mdt-15 RNAi only after the completion of larval development. Taken together, our data strongly suggest that MDT-15 critically contributes to at least two detoxification pathways in C. elegans.Like most soil-dwelling animals C. elegans. Analogous to metabolic regulation, MDT-15 may collaborate with NHR family regulatory factors to induce specific detoxification programs. Strikingly, the C. elegans genome is lavishly equipped with candidate NHRs MDT-15 integrates expression of metabolic genes by interacting with at least two metabolic regulatory factors, NHR-49 and SBP-1 II transcription, a simple view is that it might function through a single functional domain, or alternatively, that it may somehow operate with little specificity. Instead, the emerging view is that individual Mediator subunits appear to exhibit restricted specificity: For example, in D. melanogaster S2 cells, MED23 is specifically required for heat-induction of hsp26, whereas MED16 is critical to upregulate genes in response to LPS As the Mediator complex contributes to most PolThe apparent routing between detoxification and energy metabolism via MDT-15 is notable as others have found that contaminants such as cadmium affect the expression of energy metabolism genes C. elegans life span, a phenotype partially attributable to reduced FA desaturation C. elegansIt is intriguing that many of the biological processes susceptible to MDT-15 regulation are linked to aging. We previously found that MDT-15 depletion dramatically shortens C. elegans, both activities are directly linked to eating. Although we cannot rule out that some toxins may be absorbed through the worm cuticle, this barrier is generally impervious C. elegans, as its feeding during the growth periods may be accompanied by unavoidable co-ingestion of unfavorable substances. As worms may be unable to evade potential harm, or physically separate detrimental compounds, the need for an efficient defense system arises. We speculate that MDT-15 might be an active component of such a regulatory network. Accordingly, in fed worms MDT-15 may cooperate with SBP-1 to drive fat storage and adipogenesis MDT-15 affects expression of energy metabolism genes that respond to food supply C. elegans strains N2-Bristol (WT), CF512  dvIs15  nhr-8(ok186)mdt-15(tm2182) worms  four times with wild-type N2 worms; after isolating homozygotes we verified the presence of the original genomic deletion by PCR with the following primers: 5′-aatgttgctgctcaacgtgc-3′ (forward primer), and 5′-cgatctcttccaattggtcc-3′ (reverse primer). For total mRNA isolation, worm embryos were allowed to hatch on unseeded nematode growth media (NGM)-lite plates overnight at 20°C, and then grown from L1–L4 stages (for 40 hr) on NGM-lite plates containing 25 µg/mL carbenicillin, 4 mM IPTG (Alexis 582-600), and 12.5 µg/mL tetracycline seeded with the appropriate RNAi bacteria. For validation of candidate MDT-15 targets, we grew worms as follows: control RNAi for 36 hr, control RNAi 24 hr followed by mdt-15 RNAi for 12 hr, control RNAi 12 hr followed by mdt-15 RNAi for 24 hr, or mdt-15 RNAi for 36 hr. Toxins were dissolved in DMSO and added at the following final concentrations (unless indicated otherwise): fluoranthene (Sigma F4418) at 5 µg/ml, and β-naphthoflavone (Sigma N3633) at 6 µg/ml. Heavy metals were dissolved in water and added at the following final concentrations: 3CdSO4*8H2O (Sigma C3266) at 25 µM, ZnCl2 at 100 µM, and CuSO4 at 10 µM. Life span and thermotolerance assays were performed as described mdt-15, mdt-6, nhr-49 and sbp-1 RNAi clones have been described The E. coli strain HT115 carrying the appropriate RNAi vector. Worms were transferred onto 2% (w/v) agarose pads for microscopic examination. We captured images on a Retiga EXi Fast1394 CCD digital camera  attached to a Zeiss Axioplan 2 compound microscope , and used Openlab 4.0.2 software  for image acquisition.Worms were grown on NGM-lite RNAi plates seeded with mdt-15 RNAi for 36 hr to yield synchronized L4 stage populations. Notably, this RNAi protocol did not result in visible death or sickness at the very stage worms were harvested for RNAi isolation. L4 larvae were harvested, washed 5 times in M9, and frozen in liquid nitrogen. RNA was isolated by the Trizol method C. elegans microarrays. We performed five independent biological repeats; one of the arrays was a dye swap. We extracted spot intensities with SpotReader (Niles Scientific), using the default parameters to flag bad spots. To normalize the data, we used RMA background correction and the “NormalizeWithinArrays” function of limma with the print tip loess correction P-value<0.05 to be differentially expressed http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE9720.Starting with developmentally synchronized L1 stage larvae, N2 worms were grown on control or 2+-responsive genes P-value than using the entire set of genes on the array. The expected fraction of genes that overlap is the fraction of MDT-15 regulated genes multiplied with the fraction of Cd2+-reponsive genes. We used the binomial distribution to calculate statistical significance To determine overlaps between our set of MDT-15 dependent genes and the set of CdC. elegans RNA have been described (http://www.ucsf.edu/krylab/Stefan-WormRNA_isolationqPCR.pdf). QPCR was performed in an ABI7300 PCR machine, and the data analyzed using the Ct method (Applied Biosystems Prism 7700 Users Bulletin No. 2 http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf). Relative mRNA levels were normalized to act-1 mRNA levels. Primers for qPCR were designed using Primer3 Isolation, purification, and reverse transcription of Figure S1Flowchart outlining identification and confirmation of new MDT-15 targets, and overlap between genes deregulated following MDT-15 depletion vs. mutation.mdt-15(RNAi) worms and mdt-15(tm2182) mutants. (B) Overlap and separation of genes deregulated in mdt-15(RNAi) worms and mdt-15(tm2182) mutants.(A) The flowchart depicts the workflow regarding identification (by microarrays) and confirmation (by qPCR) of MDT-15 targets. Validation by qPCR was performed in both (3.99 MB TIF)Click here for additional data file.Figure S2Worms carrying a mutation in the MDT-15 gene exhibit short lifespan, reduced brood size, and toxin hypersensitivity.tm2182 indicates the location of the genomic tm2182 mutation. For protein products, light grey indicates the ORF, with the dark grey box highlighting the KIX domain. The tm2182 mutation is predicted to result in the production of a truncated protein, as indicated. (B) Life span analysis of wild-type N2 and mutant mdt-15(tm2182) worms reveals short life span of the mutant worms. Mean life spans (measured in days after reaching adulthood) were 19.7 for N2 worms , and 13.7 days for mdt-15(tm2182) mutants ; P-value <0.0001 (log rank test). The data shown here were obtained with worms cultured at 20°C; similar results were obtained when worms were grown at 25°C (data not shown). (C) Total brood count analysis of wild-type N2 and mutant mdt-15(tm2182) worms reveals reduced brood size of the mutant worms. The mdt-15(tm2182) mutants gave rise to fewer eggs, and most eggs failed to hatch. Total brood size for mutant worms is the sum of unhatched eggs and larvae. Bar graphs represent the total number of progeny±SEM . (D) N2 and mdt-15(tm2182) worms were grown on plates harboring fluoranthene at 10 µg/ml. After four days, animals were scored for developmental defects. Micrographs show representative animals grown on toxin or DMSO (solvent), as indicated. The size bar represents 64.5 µm. Exposure of mdt-15(tm2182) worms to fluoranthene results in small, scrawny adults; on DMSO, mdt-15(tm2182) animals are only slightly thinner than N2 worms.(A) Structure of the MDT-15 gene, and its three mRNA and two protein products. For mRNAs, non-coding exons are black, coding exons are white and introns are indicated as lines. The box labeled (5.81 MB TIF)Click here for additional data file.Figure S3.MDT-15 is required to induce select detoxification genes.control(RNAi)  or wild-type N2 worms (C) on the X-axis vs. the log of the fold-induction of the same gene in worms exposed to the indicated RNAi bacteria , or in mutant worms (C) on the Y-axis. R-values indicate the correlation coefficients between the two samples. Panel A represents the detoxification genes characterized in control(RNAi) worms and worms with mutated or depleted MDT-15 (R<0.6 for fluoranthene exposure) than between wild-type/control(RNAi) worms and worms with either mutated or depleted MDT-6, NHR-49 or NHR-8 (R>0.8 for fluoranthene exposure).The scatter plots depict pairwise comparisons of detoxification gene inductions by fluoranthene or β-naphtoflavone in different worm strains. Each data point represents the log of the fold-induction of an individual gene in (4.30 MB TIF)Click here for additional data file.Figure S4MDT-15 is dispensable for the heat-shock response.mdt-15(tm2182) worms (red) exposed to 35oC for various times, as indicated. Each bar represents the average relative mRNA level of the indicated gene . Relative mRNA levels are normalized to act-1 mRNA levels; the error bars represent SEM.Quantification of the mRNA levels of select heat-shock protein genes in L4-stage N2 worms (blue) and (2.89 MB TIF)Click here for additional data file.Table S1mdt-15(RNAi) worms (compared to control(RNAi) worms).List of genes deregulated in mdt-15(RNAi) animals, whereas mdt-15(RNAi) animals. Genes are ordered by highest statistical significance of over/underexpression in mdt-15(RNAi) animals vs. control(RNAi) animals.The data represent the output of “limma” analysis from five independent mRNA isolations and microarray hybridizations. “ID” refers to the identity of individual spots on the arrays, “M” represents the log of the fold change, “A” represents the averaged spot intensity, and “Function” refers to the predicted or actual function according to WormBase (WS157). In the “Name” column, detoxification related genes are bold; previously recognized MDT-15-dependent genes are italic; HSP-like proteins are underlined. (0.59 MB DOC)Click here for additional data file.Table S2Validation of candidate MDT-15 targets.mdt-15(RNAi) worms vs. control(RNAi) worms, calculated from the average relative mRNA levels from three independent biological replicates . Genes whose MDT-15 dependence (as judged from the microarray experiments) was confirmed by this qPCR analysis are in bold (a gene was classified as MDT-15-dependent if its mRNA level was reduced at least two-fold at any of the three time points). The control genes ama-1 and nhr-23 are not MDT-15 dependent. Note that genes are classified into groups with similar biological functions (left column).QPCR quantification of mRNA levels of 85 candidate MDT-15-dependent genes. Values represent fold changes±SEM in (0.20 MB DOC)Click here for additional data file.Table S3mdt-15(tm2182) worms.Validation of candidate MDT-15 targets in mutant mdt-15(tm2182) worms vs. N2 worms, calculated from the average relative mRNA levels from three independent biological replicates . 50 genes downregulated more than two-fold in mdt-15(tm2182) worm are in bold. The hacd-1 and cpt-3 genes are upregulated in mutant mdt-15(tm2182) worms, whereas MDT-15 itself and control genes ama-1 and nhr-23 are not significantly regulated, consistent with data from our previous study QPCR quantification of mRNA levels of 97 known and candidate MDT-15-dependent genes. Values represent fold changes±SEM in mutant (0.18 MB DOC)Click here for additional data file.Table S4Occurrence of gene families and protein domains in microarray results based on GO terms.mdt-15(RNAi) worms (vs. control(RNAi) worms), ranked by lowest P-value. 100 genes  were not classified by gene functional category analysis; 79 genes were not classified by the protein domain analysis. “Count” indicates the number of genes amongst MDT-15 targets that fit the respective term. “%” indicates the percentage of these amongst all MDT-15 targets. “P-value” indicates the statistical significance of the overrepresentation of an individual category; we used a P-value of 0.05 as cutoff.Occurrence of gene ontology terms and protein domains (based on InterPro annotation) in genes that, by microarray analysis, were found to be upregulated in (0.05 MB DOC)Click here for additional data file.Table S5Many genes downregulated after MDT-15 depletion are intestine-enriched.Comparison of genes downregulated after MDT-15 depletion  and groups of genes expressed in tissue-restricted manner. Columns contain WormBase accession numbers of MDT-15 target genes with respective tissue specific expression. Intestine-, muscle-, and germline-enriched genes are from Pauli et al. (0.09 MB DOC)Click here for additional data file.Table S6cyp genes (encoding CYP450s) is reduced in mdt-15(RNAi) worms.Basal and toxin-induced expression of some mdt-15(RNAi) worms vs. control(RNAi) worms, calculated from the average relative mRNA levels from three independent biological replicates . FLA = fluoranthene; NF = β-naphthoflavone; D = DMSO. Columns on the right indicate the fold-induction by toxins vs. DMSO in the same genetic condition; several genes exhibit reduced fold-induction upon MDT-15 depletion. cyp genes whose expression is toxin-induced are listed in the top section, unresponsive cyp genes in the middle section; in both sections, bold font indicates MDT-15 dependence. Note that some genes are MDT-15-dependent for basal and induced transcription.QPCR quantification of mRNA levels of cyp genes. Values represent fold changes±SEM in (0.16 MB DOC)Click here for additional data file.Table S7Expression of toxin-induced MDT-15 targets is largely unaffected in mdt-6(RNAi) worms.control(RNAi) worms, calculated from the average relative mRNA levels from three independent biological replicates . FLA = fluoranthene; NF = β-naphtoflavone.QPCR quantification of mRNA levels of MDT-15-dependent detoxification genes. Values represent fold changes±SEM in mdt-6(RNAi) worms vs. (0.08 MB DOC)Click here for additional data file.Table S8The toxin response is largely unaffected in L4 stage CF512 and nhr-8(ok186) worms.QPCR quantification of mRNA levels of MDT-15 dependent detoxification genes. Values represent fold changes±SEM in CF512 or nhr-8(ok186) worms vs. N2 worms, calculated from the average relative mRNA levels from three independent biological replicates . FLA = fluoranthene; NF = β-naphtoflavone.(0.10 MB DOC)Click here for additional data file.Table S9Expression of some toxin-induced MDT-15 targets is affected by MDT-15 depletion in adult worms.mdt-15(RNAi) worms vs. control(RNAi) worms, calculated from the average relative mRNA levels from three independent biological replicates . FLA = fluoranthene. Genes whose induction is compromised more than two-fold in mdt-15(RNAi) worms are highlighted bold.QPCR quantification of mRNA levels of MDT-15-dependent detoxification genes in conditionally sterile CF512 worms. Values represent fold changes±SEM in (0.08 MB DOC)Click here for additional data file.Table S10Expression of toxin-induced MDT-15 targets is largely unaffected in nhr-49(RNAi) and sbp-1(RNAi) worms.control(RNAi) worms, calculated from the average relative mRNA levels from three independent biological replicates . FLA = fluoranthene, NF = β-napthtoflavone.QPCR quantification of mRNA levels of MDT-15-dependent detoxification genes in N2 L4 stage worms fed with nhr-49 or sbp-1 RNAi. Values represent fold changes±SEM in nhr-49(RNAi) or sbp-1(RNAi) worms vs. (0.11 MB DOC)Click here for additional data file.Table S11A statistically significant overlap exists between MDT-15 dependent genes and Cd2+-dependent genes.To determine in unbiased fashion whether MDT-15 is involved in regulation of heavy metal detoxification, we determined the overlap between the genes that are deregulated following MDT-15 depletion and genes responsive to Cd2+ Click here for additional data file.


The 18 residue tail abutting the SH3 fold that comprises the heart of the C-terminal domain is the only part of HIV-1 integrase yet to be visualized by structural biology. To ascertain the role of the tail region in integrase function and HIV-1 replication, a set of deletion mutants that successively lacked three amino acids was constructed and analyzed in a variety of biochemical and virus infection assays. HIV-1/2 chimers, which harbored the analogous 23-mer HIV-2 tail in place of the HIV-1 sequence, were also studied. Because integrase mutations can affect steps in the replication cycle other than integration, defective mutant viruses were tested for integrase protein content and reverse transcription in addition to integration. The F185K core domain mutation, which increases integrase protein solubility, was furthermore analyzed in a subset of mutants.in vitro and during infection. Further deletion yielded two viruses, HIV-11-276 and HIV-11-273, that displayed approximately two and 5-fold infectivity defects, respectively, due to reduced integrase function. Deletion mutant HIV-11-270 and the HIV-1/2 chimera were non-infectious and displayed approximately 3 to 4-fold reverse transcription in addition to severe integration defects. Removal of four additional residues, which encompassed the C-terminal β strand of the SH3 fold, further compromised integrase incorporation into virions and reverse transcription.Purified proteins were assessed for in vitro levels of 3' processing and DNA strand transfer activities whereas HIV-1 infectivity was measured using luciferase reporter viruses. Deletions lacking up to 9 amino acids  displayed near wild-type activities 1-270, HIV-11-266, and the HIV-1/2 chimera were typed as class II mutant viruses due to their pleiotropic replication defects. We speculate that residues 271-273 might play a role in mediating the known integrase-reverse transcriptase interaction, as their removal unveiled a reverse transcription defect. The F185K mutation reduced the in vitro activities of 1-279 and 1-276 integrases by about 25%. Mutant proteins 1-279/F185K and 1-276/F185K are therefore highlighted as potential structural biology candidates, whereas further deleted tail variants (1-273/F185K or 1-270/F185K) are less desirable due to marginal or undetectable levels of integrase function.HIV-1 Retrovirus replication proceeds through a series of steps that initiate upon virus entry into a cell, followed by particle uncoating and reverse transcription. To support productive replication, the resulting double stranded cDNA must be integrated into a cell chromosome. The integrated DNA provides an efficient transcriptional template for viral gene expression and ensures for segregation of viral genetic material to daughter cells during division. Due to its essential nature, the integrase (IN) encoded by HIV-1 is an intensely studied antiviral drug target -2-hydroxy-1-propanesulfonate (CHAPS)] containing 25 mM imidazole-0.5 mM phenylmethanesulphonylfluoride was sonicated. After centrifugation for 30 min at 39,000 g, the supernatant was incubated with 0.6 ml of buffer A-25 mM imidazole-equilibrated Ni2+-nitrilotriacetic acid (Ni-NTA) agarose beads  at 4°C for 3 h. The beads were washed twice with 20 volumes of buffer A-25 mM imidazole followed by washing with 30 volumes of buffer A-35 mM imidazole. IN-His6 was eluted with buffer A-200 mM imidazole. IN containing fractions identified by Na dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were pooled and dialyzed overnight against buffer D . The His-Tag was removed using 40 U of thrombin  per mg of protein for 3 h at room temperature, which left the heterologous LVPR sequence at each C-terminus. After removal of thrombin by incubation with Benzamidine beads , IN was concentrated using Centricon-10 Concentrators  and dialyzed against buffer D for 4 h. Protein concentration was determined by spectrophotometer, and aliquots flash frozen in liquid N2 were stored at -80°C. Quantitative image analysis  of Coomassie-stained gels revealed that each IN preparation was minimally 90% pure.rain PC2  transforRecombinant LEDGFp75 expressed in bacteria was purified as previously described . LEDGFp7Anti-IN monoclonal antibody 8G4  was puri32P]TTP  using Sequenase version 2.0 T7 DNA polymerase  to label the phosphodiester within the pGTOH dinucleotide that is cleaved during 3' processing [32P]ATP  using T4 polynucleotide kinase  -2-hydroxy-1-propanesulfonate; CTD: C-terminal domain; DMEM: Dulbecco's modified Eagle's medium; DTT: dithiothreitol; FBS: fetal bovine serum; IN: integrase; LRT: late reverse transcription; LTR: long-terminal repeat; Ni-NTA: NiThe authors declare that they have no competing interests.MJD constructed molecular clones, purified recombinant IN proteins, and conducted in vitro integration assays. BM performed the brunt of virological measurements including infectivity, LRT, and 2-LTR circle Q-PCRs. LK purified 8G4 antibody, performed western blotting, and performed some IN purifications and enzyme assays. MCS performed Alu-PCR and quantified virus release from transfected HeLa cells. FDN devised the western blotting procedure, and trained and supervised BM. DEH supplied essential reagents. AE conceived of the study, supervised and interpreted experimental results, and wrote the manuscript. All authors read and approved the final manuscript.


Lim-HD proteins control crucial aspects of neuronal differentiation, including subtype identity and axonal guidance. The Lim-HD proteins Lhx2/9 and Lhx1/5 are expressed in the dorsal spinal interneuron populations dI1 and dI2, respectively. While they are not required for cell fate acquisition, their role in patterning the axonal trajectory of dI1 and dI2 neurons remains incompletely understood.Using newly identified dI1- and dI2-specific enhancers to trace axonal trajectories originating from these interneurons, we found that each population is subdivided into several distinct groups according to their axonal pathways. dI1 neurons project axons rostrally, either ipsi- or contra-laterally, while dI2 are mostly commissural neurons that project their axons rostrally and caudally. The longitudinal axonal tracks of each neuronal population self-fasciculate to form dI1- and dI2-specific bundles. The dI1 bundles are spatially located ventral relative to dI2 bundles. To examine the functional contribution of Lim-HD proteins to establishment of dI axonal projections, the Lim-HD code of dI neurons was altered by cell-specific ectopic expression. Expression of Lhx1 in dI1 neurons caused a repression of Lhx2/9 and imposed caudal projection to the caudal commissural dI1 neurons. Complementarily, when expressed in dI2 neurons, Lhx9 repressed Lhx1/5 and triggered a bias toward rostral projection in otherwise caudally projecting dI2 neurons, and ventral shift of the longitudinal axonal fascicule.The Lim-HD proteins Lhx9 and Lhx1 serve as a binary switch in controlling the rostral versus caudal longitudinal turning of the caudal commissural axons. Lhx1 determines caudal turning and Lhx9 triggers rostral turning. GFP expression is restricted to dorsal neurons 11.5 -26. Two To further characterize the cell type specificity of the enhancers, the embryos were co-electroporated along with a Cre-dependent nuclear GFP (nGFP). To determine the identity of reporter-expressing cells, embryos were analyzed at stage 23 to 24 by co-staining with dI-specific antibodies to Lhx2/9 (dI1), Lhx1/5 , Isl1 (dI3), Pax2  and Engrailed1 (V1) Figure . nGFP ex+ .Of the neurons that express nGFP under the control of #169, 56% (n = 166) are dI2, as indicated by the co-localization to the dorsal Lhx1/5+ Figure  and Lhx1s Figure  and the s Figure ; 35.5% as Figure . Of the Ngn1 gene was studied in the chick neural tube  and [ATGCTAGCGGTGTTGTGGTTGACAGCAG]. EdI2/V1 was amplified utilizing the primers [ATGAGCTCGCTCTCTCTGCCTACCTCAGC] and [ATGCTAGCAACCTAGTGCCCTTGCACAC]. The enhancers were cloned into 5'plasmids . The 13G project .in vitro transcription ). In situ hybridization was performed on electroporated sections and was combined with immunohistochemistry. Prior to the in situ hybridization, sections were incubated with primary GFP antibody, and then a standard in situ protocol was applied, followed by a secondary fluorescent antibody treatment. Alternatively, adjacent sections were collected on different slides. One set of slides was used for in situ hybridization, and the other for immuno-detection of GFP.Antisense digoxigenin-labeled probes were prepared by bHLH: basic helix loop helix; BLC: bifurcating longitudinal commissural; E: embryonic day; FTC: forked transverse commissural; GFP: green fluorescent protein; ILC: intermediate longitudinal commissural; LF: lateral funiculus; MLC: medial longitudinal commissural; nGFP: nuclear GFP; VF: ventral funiculus.The authors declare that they have no competing interests.OA performed the experiments presented in Figures EdI1/2 enhancer drives expression in dI1 and dI2 neurons. The EdI1/2 enhancer element was cloned upstream of Cre recombinase and electroporated with a conditional nuclear GFP (CAGG-loxP-STOP-loxP-nGFP) plasmid. Chick embryos were electroporated at stage 16 and fixed at stage 23. Cross-sections of electroporated neural tube were stained with dI-specific antibodies. Neurons expressing nGFP are Lhx1/5+/Pax2- (A) and Lhx2/9+/Isl1-. The boxed areas are represented as in their different channels at the right side of each panel. The arrows point to the dI2 neurons that express nGFP, and the arrowheads to dI1 neurons expressing nGFP. Scale bars: 50 μm.Click here for fileRostral versus caudal turning of dI2 axons. The contra-lateral side of (A-D) cervical and (E-H) sacral levels of four different embryos expressing GFP in dI2 neurons  or dI2/V1 neurons . The vast majority of the axons at the cervical level are dI2rost, and at the sacral level dI2caud. FP, floor plate. Scale bar: 150 μm ; 200 μm ; 250 μm (D).Click here for fileMost dI2 neurons are commissural. (A-C) Cervical and (D) brachial levels of four different neural tubes obtained from chick embryos were electroporated at stage 16 with three plasmids: dI1/2::Cre, dI2/V1::Gal4 and UAS::LoxP-STOP-LoxP-GFP. Only dI2 neurons expressed GFP. Only in one embryo (A) are ipsi-lateral longitudinal axons that project rostrally visible. For quantification, EGFP brightness intensity at the ipsi- and contra-lateral sides was measured utilizing NIH image software. Scale bar: 150 μm ; 200 μm .Click here for fileThe level of ectopic Lhx9 protein is similar to the levels of the endogenous protein. Lhx9 was expressed (A) uniformly (pCAGG-Lhx9-IRES-nGFP), or (B) in dI2 neurons (EdI2/V1::Cre + pCAGG-LoxP-STOP-LoxP-Lhx9-IRES-GFP). Sections were stained with Lhx2/9 antibody. The levels of the exogenous Lhx9 are similar to the endogenous levels. Arrows point to cells with high level expression, and arrowheads to cells with low level expression. White indicates endogenousand yellow exogenous Lhx9 proteins. Scale bar: 100 μm.Click here for fileLhx2 mediates a caudal-to-rostral change in the turning of dI2 axons. Lhx2 + taumyc were expressed ectopically in dI2 neurons, utilizing the Cre/lox system and the EdI2/V1 enhancer (EdI2/V1::Cre + pCAGG-LoxP-STOP-LoxP-Lhx2-IRES-taumyc). (A) At the caudal sacral level dI2Lhx2 axons turn caudally. At the rostral sacral level axons turn rostrally and caudally, forming a crisscross pattern  . Rostral to the lumbar level dI2Lhx2 axons turn rostrally . (B) A schematic illustration of the phenotype of dI2Lhx2/9 axonal cues. c, cervical level; b, brachial level; FP, floor plate; l, lumbar level, s, sacral level; t, thoracic level. Scale bars: 150 μm (A); 100 μm .Click here for fileLhx2 + Lhx9 mediate a caudal-to-rostral change in the turning of dI2 axons. Lhx9 + GFP and Lhx2 + taumyc were expressed ectopically in dI2 neurons, utilizing the Cre/lox system and the EdI2/V1 enhancer (EdI2/V1::Cre + pCAGG-LoxP-STOP-LoxP-Lhx9-IRES-taumyc + pCAGG-LoxP-STOP-LoxP-Lhx2-IRES-taumyc). (A) At the caudal sacral level dI2Lhx2/9 axons turn caudally . At the rostral sacral level axons turn rostrally and caudally, forming a crisscross pattern . Rostral to the lumbar level dI2Lhx2/9 axons turn rostrally . (B) The electroporated axons co-express GFP and taumyc. (C) A schematic illustration of the phenotype of dI2Lhx2/9axonal cues. c, cervical level; b, brachial level; FP, floor plate; l, lumbar level, s, sacral level; t, thoracic level. Scale bars: 150 μm ; 75 μm (A1–A3).Click here for file


Expression of the bcl-2 proto-oncogene was studied immunohistochemically in 251 invasive ductal breast carcinomas  and the results were correlated with clinicopathological data and prognostic variables. Sixty-three (25%) tumours were scored bcl-2 negative and 188 (75%) tumours were bcl-2 positive. No relationship could be observed between bcl-2 status and tumour grade, pTNM staging or menopausal status. A strong positive relationship was demonstrated between bcl-2 immunoreactivity and oestrogen receptor status (P < 0.001) and progesterone receptor status (P < 0.001). No prognostic value was demonstrated for bcl-2 expression on disease-free survival and overall survival in axillary node-negative breast cancer patients. However, in axillary node-positive breast cancer patients multivariate analysis demonstrated absence of bcl-2 expression to be independently related to shortened disease-free survival (P = 0.003) and shortened overall survival (P < 0.001). Our results suggest a potential important role for bcl-2 expression as a modulator of response to adjuvant therapy in breast cancer.


P< 0.04); simultaneous analysis of TS and p53 indicated 7% OR for p53-positive/TS-positive tumours vs 46% for p53-positive/TS-negative tumours (P< 0.03). Logistic regression analysis confirmed a significant association between TS tumour status and clinical response to chemotherapy (hazard ratio (HR): 2.91; 95% confidence interval (CI) 8.34–1.01; two-sided P< 0.05). A multivariate analysis of overall survival showed that only a small number of metastatic sites was statistically relevant . Our study suggests that immunohistochemical expression of p53 and TS could assist the clinician in predicting response of colorectal cancer patients to modulated MTX-5-FU therapy. © 2000 Cancer Research CampaignThe purpose of this work was to analyse the ability of p53 and thymidilate synthase (TS) primary tumour expression to retrospectively predict clinical response to chemotherapy and long-term prognosis in patients with advanced colorectal cancers homogeneously treated by methotrexate (MTX)-modulated–5-fluorouracil (5-FU-FA). A total of 108 advanced colorectal cancer patients entered the present retrospective study. Immunohistochemical p53 (pAb 1801 mAb) and TS (TS106 mAb) expression on formalin-fixed paraffin-embedded primary tumour specimens was related to probability of clinical response to chemotherapy, time to progression and overall survival. p53 was expressed in 53/108 (49%) tumours, while 54/108 (50%) showed TS immunostaining. No relationship was demonstrated between p53 positivity and clinical response to chemotherapy (objective response (OR): 20% vs 23%, in p53+ and p53– cases respectively) or overall survival. Percent of OR was significantly higher in TS-negative with respect to TS-positive tumours (30% vs 15% respectively;


Myr-ZAP-70 transduced cells partially reversed the in vivo defects of RhoH-associated thymic development and TCR signaling. Together, our results suggest that RhoH regulates TCR signaling via recruitment of ZAP-70 and Lck to CD3ζ in the immunological synapse. Thus, we define a new function for a RhoH GTPase as an adaptor molecule in TCR signaling pathway.RhoH is an hematopoietic-specific, GTPase-deficient Rho GTPase that plays a role in T development. We investigated the mechanisms of RhoH function in TCR signaling. We found that the association between Lck and CD3ζ was impaired in RhoH-deficient T cells, due to defective translocation of both Lck and ZAP-70 to the immunological synapse. RhoH with Lck and ZAP-70 localizes in the detergent-soluble membrane fraction where the complex is associated with CD3ζ phosphorylation. To determine if impaired translocation of ZAP-70 was a major determinant of defective T cell development,  RhoH, an hematopoietic-specific Rho GTPase,was first identified as a fusion transcript with Bcl-6 in B-cell diffuse large cell lymphomaκB and p38 kinase by other Rho GTPases in Jurkat T cellsRhoh-/- HPCsRhoH has also been proposed to function as a negative regulator of other Rho GTPases, particularly Rac. For instance, RhoH is a potent inhibitor of the activation of NFRhoh-/- mice have impaired TCR signaling, resulting in defective thymocyte selection and blocked maturation of T cellsWe previously demonstrated that Rhoh-/- T cellsRhoh-/- T cells, Lck showed impaired association with CD3ζ and decreased localization to the immunologic synapse. This was not associated with defects in its kinase activity as measured by in vitro kinase assay. Further biochemical data suggest that RhoH regulates CD3ζ phosphorylation by recruiting ZAP-70 and Lck to the cell membrance fraction to form the TCR complex for TCR signaling activation. In these studies, we found that membrane-targeted ZAP-70 could partially rescue the defective TCR signaling in RhoH-deficient T cells. Taken together, these data suggest that RhoH regulates TCR signaling via recruiting ZAP-70 and Lck to CD3ζ in the immunological synapse for activation of downstream signaling molecules.TCR signaling is controlled by complex cross-regulation of several key downstream signaling components. ZAP-70 has been reported to promote the phosphorylation of CD3ζ and the association of CD3ζ with Lck, as ZAP-70 deficiency impairs the TCR-induced CD3ζ phosphorylation-/-Rhoh mice were generated by standard DNA homologous recombination and backcrossed in a C57BL/6J background as described previouslytg/+; Rhoh-/- mice expressing p14 TCR (Vα2/Vβ8) transgene in a compound 129Sv and C57BL/6J background were generated as described previouslyRag2-/- mice were purchased from Taconic Animal Models . All experiments involving animals were approved by the Institutional Animal Care and Use Committee of the Cincinnati Children's Research Foundation. Single cell suspensions were generated from dissected thymus and lymph node. T cells were purified by negative selection using the Pan T Cell Isolation Kit . The purity of isolated T cells in all experiments was greater than 90%, as assessed by flow cytometry with anti-mouse CD3ε Ab. For TCR activation, thymocytes or T cells were incubated with anti-mouse CD3ε mAb  and anti-mouse CD28 mAb  (both at 5 µg/107 cells). Cross-linking of mAbs was accomplished using anti-Hamster IgG Abs . After incubation for 2 min at 37°C, cold phosphate-buffered saline  was added to stop the activation.129Sv ATGGGCAGCAGCAAGAGCAAGCCCAAGCCCGATCCCGCGGCGCAC-3′5′-GACGAATTC (MGSSKSKPK coding sequence was underlined), (reverse) 5′-ACCGTCGACTCAGCCACATGCAGCCTCGGCCACCTGT-3′. Amplified PCR products were subcloned in pSF91 bicistronic retroviral vector expressing enhanced green fluorescence protein (EGFP) at EcoRI and SalI sites4 coprecipitation procedure . Retroviral supernatants were collected every 12 hours. Titer of recombinant retrovirus was determined by infecting NIH3T3 cells using serial dilutionMyr-ZAP-70 was constructed by the addition of the avian src myristoylation sequence (MGSSKSKPK) at the N terminus of ZAP-70 cDNA+ cells were sorted by a fluorescence-activated cell sorting (FACS) Vantage Sorter  and were injected intravenously into the sub-lethally irradiated (300 Rads using a 137Cs irradiator) Rag2-/- recipient mice. At 8 weeks post transplantation, single cell suspensions were prepared from thymus and subjected to flow cytometric analysis.Low density bone marrow (LDBM) cells were harvested 4 days after 5-fluorouracil  injection. Retrovirus-mediated transduction of mouse LDBM cells was performed as described previouslyThe following antibodies were used in immunoblot analyses at 1∶1000 dilution: anti- ZAP-70 (1E7.2) and anti-phosphotyrosine (4G10) ; anti-HA (3F10) ; anti-β-actin (AC15) ; anti-CD3ζ (3F67) and anti-Lck (3A5) ; anti-phospho-Src Tyr416 (2101), anti-phospho-ZAP-70 Tyr319 (2701), anti-phospho-ZAP-70 Tyr493 (2704), anti-Lck (2752), anti-ZAP-70 (99F2), anti-phospho-LAT Tyr191 (3584)anti-phospho-p44/p42 Thr202/Tyr204 (9101) and anti-p42/p44 (9102) . Primary antibodies were detected with horseradish peroxidase-conjugated goat anti-mouse , or goat anti-rabbit , donkey anti-rat (Santa Cruz Biotechmology) or donkey anti-goat secondary (Santa Cruz Biotechmology) antibodies (1∶3000 dilution) with enhanced chemiluminescence detection  The following fluor-conjugated monoclonal anti-mouse antibodies (mAb) were used for flow cytometry: CD4 (RM4-5), CD8a (53-6.7), and CD3ε mAb (2c11) .-/-Rhoh p14 transgenic mice and positively selected for CD8+ T cells by a magnetic bead cell selection technique . For positive selection of CD8+ T cells, lymph node T cells were labeled with biotinylated anti-CD8a mAb. These cells were then further incubated with anti-biotin magnetic beads (Miltenyi Biotec) and purified in accordance with the manufacturer's recommendations. Antigen-presenting (APC) cells (CHB.2B B cell lymphoma cell line)-/-Rhoh p14 transgenic mice and then layered onto poly-L-lysine-coated coverslips. After 5 min, cells were fixed with BD Cytofix/Cytoperm ) and stained with Rhodamine (TRITC)-labeled phalloidin , anti-ZAP-70  or anti-Lck  followed by anti–rabbit Alexa488 or anti-mouse Alexa555 (Molecular Probes). For the localization of Lck and ZAP-70 in -/-Rhoh T cells expressing Myr-ZAP-70, pan-T cells were isolated from LN by using Pan-T Cell Isolation Kit (Miltenyi Biotec). T cells were transduced with retroviruses co-expressing EGFP and Myr-ZAP-70 or EGFP alone as described previously+ T cells were sorted with a FACSVantage and then were cultured without anti-CD3ε Ab stimulation  and 10 ng/ml IL7 (Peprotech)) for 2 days. EGFP+ T cells were incubated with biotin-labeled anti-mouse CD3ε mAb  and anti-mouse CD28 mAb , and then conjugated with Dynabeads Biotin Binder . T-microbeads conjugates were layered onto poly-L-lysine coated coverslips and incubated at 37°C for 5 min. Cells were fixed with BD Cytofix/Cytoperm (BD Biosciences) and stained with anti-ZAP-70 or anti-Lck Ab  followed by anti–rabbit Alexa555 (Molecular Probes). Z series fluorescence images were captured with a Leica DMIRB fluorescence microscope equipped with a 40x/0.55 NA air objective lens , a Hamamatsu C4742 digital camera  and a deconvolution system driven by Openlab software . Deconvoluted images were processed using Adobe Photoshop 6.0 . At least 100 cells were analyzed per condition.Lymph node cells were isolated from WT and 2, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, and complete protease inhibitor cocktail (Roche)). After centrifugation for 5 min at 900 g, took 200 µl post-nuclei (PN) fraction. PN fraction was separated into particulate membrane-containing (P100) and cytosol (C) fractions by centrifugation for 30 min at 100,000× g. Take 200 µl supernatant as C fraction. Pellet was solubilized with 200 µl Mg2+ lysis/wash buffer (Millipore) and separated by additional 30 min at 100,000× g. The supernatant was kept as detergent-soluble membrane (SM) fraction. The pellet was further solubilized with 200 µl 0.05% sodium dodecyl sulfate (SDS)-containing Mg2+ lysis/wash buffer, and then centrifuged for 30 min at 13,000× g. The supernatant was kept as detergent-insoluble membrane (IS) fraction. Equal volumes of each fraction were used for immunoprecipitation and immunoblotting. For immunoprecipitation, each fraction was 5-fold diluted with SDS-containing Mg2+ lysis/wash buffer .Cells were lysed by brief sonication in 200 µl ice-cold buffer A . Jurkat T cells were transfected by electroporation . The expression vector for kinase-dead ZAP-70 (pCDNA3-KA-ZAP-70) and constitutively active ZAP-70 (pCDNA3-AA-ZAP-70) was kindly provided by Dr. A. Weiss2+ lysis/wash buffer (Upstate Biotechnology) containing mixture of protease and phosphatase inhibitors . Cellular lysates (500 µg) were immunoprecipitated, using the anti-Lck , anti-HA  or anti-ZAP-70  antibodies. The immunoprecipitated mixture or 20 µg total lysates were separated on a SDS polyacrylamide gel, and transferred to a polyvinylidene difluoride membrane (PVDF) (BioRad). The membranes were probed with antibodies indicated in each experiment. For Lck kinase assay, anti-Lck immunoprecipitate products were washed three times with lysis buffer and once with kinase buffer  and resuspended with 30 µL kinase buffer. Reaction was started by addition of 10 µCi [γ-32P] ATP and left for 10 min. Reaction mixtures were subjected to SDS-PAGE and analyzed by autoradiography.Wild-type and Each experiment was performed two-four times. Representative data or images from one of the repeated experiments were shown in the figures. Statistical significance was determined by a Student t-Test and data with p-value less than 0.05 are considered as significant difference.Src family phosphotyrosine kinase (PTK), LckRhoh-/- T cells demonstrate defective CD3ζ phosphorylation and ZAP-70 recruitment to the TCR complex which results in the impaired TCR signaling and T cell developmentin vitro kinase activity was similar in wild type (WT) and Rhoh-/- thymocytes  results in the formation of the immunological synapse (IS). ZAP-70 has been reported to promote CD3ζ phosphorylation by recruiting Lck to TCR complex in a kinase activity-independent mannerh ZAP-70 . In contjugation . This de T cells , while oEngagement of the TCR induces the translocation of several molecules such as Lck, ZAP-70 and CD3ζ to the detergent-insoluble membrane fraction of T cells that contains lipid raftsSince ZAP-70 is defective in translocation to the TCR complex and the detergent-insoluble membrane fraction in RhoH-deficient T cellsBinding of ZAP-70 to the ITAM motifs of CD3ζ has been suggested to induce a conformational change of ZAP-70 that facilitates the phosphorylation of Tyr315 and Tyr319 in interdomain B of ZAP-70Since CA-Lck alone did not phosphorylate RhoH efficiently, we next investigated whether ZAP-70 could phosphorylate RhoH with or without expression of Lck. As noted above, co-expression of CA-Lck, ZAP-70 and RhoH led to increased phosphorylation of RhoH which is immunoprecipitated with ZAP-70 , lane 3.We next investigated whether Lck and ZAP-70 activity is required for the association of ZAP-70 with RhoH and phosphorylation of RhoH in T cells . ExpressRhoh-/- mice was due to defective ZAP-70 membrane localization, we generated a retroviral vector expressing Myr-ZAP-70 or control vector and utilized these vectors to transduce Rhoh-/- low density bone marrow cells (LDBM). First to determine the functional consequences of Myr-ZAP-70, we transduced Ba/F3 cells. Expression of the Myr-ZAP-70 in Ba/F3 cells led to an increase in the proportion of ZAP-70 protein in the detergent-soluble (27% to 40%) and detergent-insoluble (6% to 22%) membrane fractions of these cells (Transient or stable membrane association of ZAP-70 mediated by the membrane-permeable synthetic ligand FK1012A or by a myristoylation sequence -tagged ZAP70 (Myr-ZAP-70) can activate downstream signaling of TCRse cells . We subsse cells .-/-Rhoh bone marrow cells transplanted into -/-Rag2 mice. Rhoh-/- bone marrow (BM) cells were transduced with a bi-cistronic retroviral vector co-expressing enhanced green fluorescent protein (EGFP) and HA-RhoH, Myr-ZAP-70 or EGFP alone (as control). Transduced and EGFP+-sorted BM cells were transplanted via intravenous injection into the sub-lethally irradiated Rag2-deficient recipient mice. Development of CD8 single positive (SP) thymocytes at 8 weeks post-transplantation in -/-Rag2 mice infused with Myr-ZAP-70-transduced RhoH-/- BM cells was significantly increased compared with mice transplanted with Rhoh-/- BM cells transduced with the empty-vector and reached levels equivalent to mice transplanted with cells transduced and expressing the RhoH cDNA . Total thymic cellularity was also increased in mice transplanted with Myr-ZAP-70-transduced Rhoh-/- BM cells .Next, we analyzed the engraftment and T cell development of Myr-ZAP-70-expressing hoH cDNA . In addi-/-Rhoh thymocytes expressing Myr-ZAP-70. As previously noted-/-Rhoh thymocytes showed impaired activation of LAT, CD3ζ and p44/42  T cells with retroviruses co-expressing EGFP and Myr-ZAP-70 or EGFP alone. EGFP+ sorted LN T cells were incubated with anti-CD3ε and CD28 Abs, and then conjugated with Dynabeads. Lck was polarized towards the microbead interface of -/-Rhoh T cells expressing Myr-ZAP-70, while Lck was widely dispersed over the membrane of -/-Rhoh T cells expressing only EGFP -/-Rhoh mice have T lymphopenia and demonstrate reduced TCR-induced T cell proliferation in vitro. Proteomic studies demonstrated that RhoH interacts with ZAP-70 in TCR signaling, providing an initial insight into the molecular mechanism of defective T cell signaling in -/-Rhoh cells.Previous studies have implicated RhoH in T cell development and TCR signaling, inside-out integrin signaling and adhesion in T cells and antagonism of the activity of the Rac GTPase pathways in cells of hematopoietic lineages-/-Rhoh T cells show defective ZAP-70 and Lck localization to the IS and defective downstream signaling of the TCR pathway. Our studies demonstrate protein interactions between Lck, ZAP-70 and RhoH and that the translocation of both ZAP-70 and Lck to the IS is decreased in -/-Rhoh T cells. Thus, RhoH appears to be involved in the coordinated movement of proteins in the IS. We are currently studying which domains in addition to the ITAM-like motifs of RhoH are critical for these interactions.Here we show that RhoH plays a critical role in facilitating the localization of multiple TCR signaling components-likely via the ZAP-70 interaction-to the membrane, particularly the detergent insoluble fraction of the membrane that contains lipid rafts. Lck and ZAP-70 have been shown to translocate to microclusters of receptors and signaling molecules in the peripheral regions of the T-APC interface within lipid rafts during initiation of the TCR activation signal. The clusters form a central supramolecular activation cluster (c-SMAC) of the IS -/-ZAP-70 mice, suggesting a role for ZAP-70 as an upstream facilitator of Lck localization and functionPrevious studies have implicated the translocation of Lck, ZAP-70 and the TCR into the IS in effective TCR signaling Taken together, these data suggest that RhoH may act as an adaptor molecule for ZAP-70 and Lck recruitment which, in turn, phosphorylates RhoH to enhance the heterotrimeric association of these molecules. In support of this hypothesis, CA-Lck is required for the activation of ZAP-70 and co-expression of CA-Lck and ZAP-70 enhanced the phosphorylation and binding affinity of RhoH to ZAP-70. Since CA-Lck alone did not phosphorylate RhoH efficiently, we also investigated the potential role of ZAP-70 in RhoH phosphorylation. Co-expression of kinase-dead Lck and ZAP-70 did not result in measurable phosphorylation of RhoH. However, a constitutive active mutant of Lck induced phosphorylation of RhoH and enhanced interaction among ZAP-70/Lck/RhoH in Jurkat T cells, whereas kinase-dead Lck inhibited the phosphorylation of RhoH and CD3ζ. Constitutive active ZAP-70 also enhanced ZAP-70/RhoH complex formation through Lck activation because AA-ZAP-70 showed enhanced CD3ζ phosphorylation and association with Lck in Jurkat T cells. Co-expression of kinase-dead ZAP-70 with CA-Lck resulted in reduced phosphorylation of RhoH, and the KA-ZAP-70 did not co-immunoprecipitate with RhoH and showed lower binding affinity to Lck. These data suggest that functional ZAP-70 is required for the Lck-mediated RhoH phosphorylation and optimal association of RhoH with ZAP-70.-/-Rhoh thymocytes expressing Myr-ZAP-70. As expected, a higher proportion of Myr-ZAP-70 compared with wild-type ZAP-70 showed association with the cell membrane in T cells. Myr-ZAP-70 partially rescued in thymocyte development and TCR signaling in the absence of RhoH. Membrane-bound ZAP-70 can activate TCR minimally without stimuli, but still requires functional Lck activity -/-Rhoh cells, we suggest that RhoH function is required for the membrane localization of ZAP-70, particularly to the IS. In the experiments reported here, there could be two reasons why Myr-ZAP-70 did not completely restore the TCR signaling in -/-Rhoh cells completely. First, the sustained TCR activation by membrane-bound ZAP-70 might induce a refractory state that reduces TCR signaling efficiency as previously reported-/-Rhoh T cells expressing Myr-ZAP-70, Lck was polarized towards the interface of cells and microbeads coated with anti-CD3ε and anti-CD28 antibodies, while Lck remained dispersed over the entire membrane in -/-Rhoh T cells expressing only EGFP. These data strongly suggest that RhoH functions with ZAP-70 to also facilitate the translocation of Lck into the TCR complex. Taken together with the data showing partial rescue of T cell development after Rhoh-/- cells expressing Myr-ZAP-70 are transferred into Rag2-/- mice, these data suggest that this function of RhoH is physiologically important.Supporting a critical role for RhoH in functionally important localization of ZAP-70 to the membrane, anti-CD3/28 Abs-induced phosphorylation of CD3ζ and LAT was partially restored in Other investigators have confirmed that RhoH promotes the ZAP70-dependent phosphorylation of the LAT signalosome-/-, Rac1-/-;Rac2-/-, ZAP-70-/-Rhoh and -/-Vav1  Lysates of Ba/F3 cells transduced with retroviral vectors expressing ZAP-70 or Myristoylated ZAP-70 (Myr-ZAP-70) were separated by centrifugation into cytosol (C), detergent-soluble (SM) and detergent-insoluble (IM) membrane fractions. Equal volumes of each fractions were immunoblotted for ZAP-70 and β-actin as a loading control. (B) LDBM cells from wild type mice were infected with retroviral vectors expressing ZAP-70, Myr-ZAP-70 or EGFP alone. The transduced cells were fixed and stained with anti-ZAP-70 (red). Bars, 3 µm.(4.38 MB TIF)Click here for additional data file.


Compared with controls, mean IGFBP-2 SDS were significantly elevated in patients with Dukes B (P< 0.001), Dukes C (n= 13 P< 0.001) and advanced disease (n= 42 P< 0.0001), with a significant trend from early to advanced disease (one-way ANOVA P< 0.001). Furthermore, IGFBP-2 SDS were positively related to tumour size (P= 0.01) and fell significantly in patients following curative resection (P= 0.04), suggesting that circulating levels reflect tumour load. We tested the potential tumour marker characteristics of IGFBP-2 SDS against three endpoints: metastasis alone; local pelvic recurrence alone; and metastasis and recurrence combined. The sensitivities for IGFBP-2 alone (≥ + 2SD) were modest at 55%, 46%, and 52%, but in combination with CEA, increased substantially to 90%, 77% and 86%, respectively. We conclude that the serum IGF-II and IGFBP-2 profiles may provide insights into underlying biological mechanisms, and that serum IGFBP-2 may have an adjunct role in cancer surveillance in patients with colorectal cancer. © 2000 Cancer Research CampaignThis study explored the relationships of serum insulin-like growth factors, IGF-I and IGF-II, and their binding proteins (IGFBP)-2 and IGFBP-3, with key clinicopathological parameters in 92 patients with colorectal cancer (cases). Comparisons were made with 57 individuals who had a normal colonoscopy (controls). Serial changes were examined in 27 cases. As IGF-related peptides are age- and sex-dependent, absolute concentrations were converted to standard deviation scores (SDS). Mean IGF-II SDS were elevated in Dukes A (


Drug scheduling alterations can improve the therapeutic index of both 5-fluorouracil and anthracyclines. We investigated a regimen of weekly doxorubicin and continuous infusional 5-fluorouracil (AcF) in loco-regionally recurrent and metastatic breast cancer. The aims of this phase II study were to use low-dose weekly anthracyclines in a patient group where liver metastases are a frequent problem, to optimise scheduling of 5-fluorouracil using continuous infusion and to conserve alkylating agent use for late intensification in responding patients. Fifty-six patients received 5-fluorouracil 200 mg m-2 day-1 and doxorubicin 20-30 mg m-2 week-1 for at least 6 weeks. Sixty-two percent were chemonaive. Patients were evaluated for dose intensity, response, toxicity and survival. Of the assessable patients, 76% achieved UICC response criteria . WHO grade 3+ toxicities were: alopecia, 98%; mucositis, 62%; neutropenia, 22%; and grade 3 palmar-plantar syndrome, 24%. Median survival was 13 months, with visceral metastasis conferring a significantly worse outcome (P = 0.03). Grade 3+ mucositis was more frequent with planned doxorubicin dose intensity > or = 25 mg m-2 week-1 (P = 0.04). AcF is highly active in breast cancer with acceptable toxicities and can be used before alkylating agent-based high-dose therapy.


Huntingtin (htt) is a multi-domain protein of 350 kDa that is mutated in Huntington's disease (HD) but whose function is yet to be fully understood. This absence of information is due in part to the difficulty of manipulating large DNA fragments by using conventional molecular cloning techniques. Consequently, few studies have addressed the cellular function(s) of full-length htt and its dysfunction(s) associated with the disease.Adaptable, RNAi Insensitive &Synthetic). It includes synthetic cDNA coding for full-length human htt modified so that: 1) it is improved for codon usage, 2) it is insensitive to four different siRNAs allowing gene replacement studies, 3) it contains unique restriction sites (URSs) dispersed throughout the entire sequence without modifying the translated amino acid sequence, 4) it contains multiple cloning sites at the N and C-ter ends and 5) it is Gateway compatible. These modifications facilitate mutagenesis, tagging and cloning into diverse expression plasmids. Htt regulates dynein/dynactin-dependent trafficking of vesicles, such as brain-derived neurotrophic factor (BDNF)-containing vesicles, and of organelles, including reforming and maintenance of the Golgi near the cell centre. We used tests of these trafficking functions to validate various pARIS-htt constructs. We demonstrated, after silencing of endogenous htt, that full-length htt expressed from pARIS-htt rescues Golgi apparatus reformation following reversible microtubule disruption. A mutant form of htt that contains a 100Q expansion and a htt form devoid of either HAP1 or dynein interaction domains are both unable to rescue loss of endogenous htt. These mutants have also an impaired capacity to promote BDNF vesicular trafficking in neuronal cells.We describe a flexible synthetic vector encoding full-length htt called pARIS-htt (We report the validation of a synthetic gene encoding full-length htt protein that will facilitate analyses of its structure/function. This may help provide relevant information about the cellular dysfunctions operating during the disease. As proof of principle, we show that either polyQ expansion or deletion of key interacting domains within full-length htt protein impairs its function in transport indicating that HD mutation induces defects on intrinsic properties of the protein and further demonstrating the importance of studying htt in its full-length context. Serum and Glucocorticoid-induced kinase (SGK) and is dephosphorylated by calcineurin Q23-C[HA-TC] (pARIS-mCherry-httQ23) and pARIS-httpcDNA3.2-N[His-mCherry]Q100-C[HA-TC] (pARIS-mCherry-httQ100) were used to transfect HEK cells and protein expression was analyzed by immunoblotting with various htt-specific antibodies recognizing different epitopes within the protein, including htt-4C8 whose epitope maps to positions 443-457 of the amino acid sequence and the htt-2C1 antibody raised against the carboxy-terminal region (2788-2990) Q23-C[HA-TC] (pARIS-mCherry-httQ23) and pARIS-httpcDNA3.2-N[His-mCherry]Q100-C[HA-TC] (pARIS-mCherry-httQ100).We use the following nomenclature to describe the first step constructs in the Entry vector: pARIS-htt-N[His-mCherry]Q23-C[HA-TC] and pARIS-htt-N[His-mCherry]Q100-C[HA-TC]. These constructs were transposed to pcDNA3.2 to generate pARIS-httpcDNA3.2-N[His-mCherry]Q23-Δ633-672-C[HA-TC] hereafter denoted pARIS-mCherry-httQ23-Δdyn. To generate the htt construct deleted for the HAP1 binding domain, the deletion was first generated within the F2 fragment (pUC19-F2Δ170-268), transposed to Entry-based pARIS-htt by insertion of SacI/SacII (fragment 2) generating pARIS-htt-N[His-mCherry]Q23-Δ170-268-C[HA-TC] and next transposed to pcDNA3.2 to generate pARIS-httpcDNA3.2-N[His-mCherry]Q23-Δ170-268-C[HA-TC] hereafter denoted pARIS-mCherry-httQ23-ΔHAP1. Requests for constructs may be sent to the following e-mail address: paris-htt.constructs@curie.fr.To generate the htt construct deleted for the dynein-interacting domain, the deletion was first generated within the F3 fragment (pUC19-F3Δ633-672), transposed to Entry-based pARIS-htt by insertion of SacII/KpnI (fragment 3) generating pARIS-htt-N[His-mCherry]Q23-Δ633-672-C[HA-TC] and next transposed to pcDNA3.2 to generate pARIS-htt2 and cultured in DMEM supplemented with 10% bovine calf serum, 1% L-glutamine and 400 μg/ml geneticin . Mouse neuronal cells, STHdh+/+ cells derived from immortalized striatal progenitor cells were grown as previously described [HEK and Cos7 cells were grown at 37°C in 5% CO2 in Dulbeco's modified Eagle's medium (DMEM) supplemented with 10% bovine calf serum, 1% L-glutamine and antibiotics (50 units/ml penicillin and 50 μg/ml streptomycin). HeLa cells stably expressing GFP-mannosidase II , were grown at 37°C in 5% COescribed .For pARIS-htt expression analysis, HEK cells were transfected with pARIS-mCherry-httQ23, pARIS-mCherry-httQ100 or equivalent amount of empty vector, using the calcium phosphate method . WesternFor immunofluorescence experiments, Cos7 cells seeded in 12-well plates with 18 mm coverslips were transfected with pARIS-mCherry-httQ23, pARIS-mCherry-httQ100 or equivalent amount of empty vector using FuGENE reagent  according to the manufacturer's instructions. Immunostaining was done after 48 h.For gene replacement experiments, HeLa cells stably expressing GFP-mannosidase II were seeded in 12-well plates with 18 mm coverslips. Sequential transfection was performed as following: attached cells were first transfected using Lipofectamine 2000 (Invitrogen) with siRNA-htt or scRNA. After 24 h, cells were transfected again with pARIS-mCherry-httQ23, pARIS-mCherry-httQ100, pARIS-mCherry-httQ23-Δdyn or pARIS-mCherry-httQ23-ΔHAP1. Cells were processed for western blotting or immunostaining 24 h after. DNA, siRNA and Lipofectamine 2000 quantities were used according to the manufacturer's instructions.To perform co-immunoprecipitation experiments, HEK cells were transfected using Lipofectamine 2000 with siRNA-htt as described above. After 24 h the cells were transfected with pARIS-mCherry-Htt constructs and/or HAP1-GFP using the calcium phosphate method. Immunoprecipitation assays were performed after 24 h.For videomicroscopy experiments mouse neuronal cells were electroporated with Kit L Nucleofector according to the supplier's manual . BDNF-eGFP and pARIS-htt DNA or equivalent amount of empty vector were added to the electroporation mix. After electroporation, cells were seeded in 12 well plates with 18 mm coverslips.Transfected HeLa GFP-mannosidase II cells were treated with 4 μM nocodazole for 30 min at 4°C and 90 min at 37°C to allow a complete depolymerization of microtubules. Cells were washed twice with DMEM prior to methanol fixation (2 min at -20°C).Anti-huntingtin antibodies used in this study htt-4C8, htt-2C1 and 1C2 were previously described ,31, α-tuTransfected cells were harvested and lyzed in 50 mM Tris-HCl, pH 7.5, containing 0.1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 50 mM NaF, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 0.1% (v/v) β-mercaptoethanol, 250 μM PMSF, 10 mg/ml aprotinin and leupeptin. Cell lysates were centrifuged at 20,000 g for 10 min at 4°C. Equal amounts of protein were subjected to SDS-PAGE on 6% polyacrylamide gels and transferred to nitrocellulose membranes . Blocked membranes (5% milk in TBS-0.1% Tween-20) were incubated with mouse anti-huntingtin antibodies , mouse anti-polyQ expansion (1C2), rat anti-HA, mouse anti-GFP, mouse anti-DIC or mouse anti-α-tubulin antibodies and washed three times with TBS-0.1% Tween-20 for 10 min. Membranes were then labelled with secondary IgG-HRP antibodies raised against each corresponding primary antibody. After three washes, the membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate  according to the instructions of the supplier. Membranes were exposed to Amersham Hyperfilm™ MP  films and developed.Immunoprecipitations were performed as described  with minAfter methanol fixation cells were blocked for 1 h at RT with PBS-BSA 3% and incubated with primary antibodies for 1 h prior staining with Alexa Fluor secondary antibodies. Nuclei were stained with DAPI (Roche). The mounting medium was 0.1 g/ml Mowiol 4-88  in 20% glycerol.Images on fixed samples were acquired at RT with a Leica SP5 laser scanning confocal microscope equipped with a 63 × oil-immersion objective or with a Leica DM RXA microscope with a PL APO oil 63 × NA of 1.4 objective coupled to a piezzo and a Micromax RTE/CCD-1300-Y/HS camera controlled by Metamorph software . Z-stack step was of 0.2 μm. All stacks were treated by automatic batch deconvolution using the PSF of the optical system, Meinel algorithm with parameters set at 7 iterations, 0.7 sigma and 4 frequencies.http://imagejdocu.tudor.lu/doku.php?id=plugin:analysis:3d_object_counter:start). The quantification was achieved tagging each identified object within the z-stacks (around 30 z-stacks per image), treating each Golgi particle as an individual object. Statistics about each object were calculated, volume as: number of voxels of the object × x calibration × y calibration × z calibration. The overall measurements were obtained from 3 independent experiments and analyzed to determine the mean volume per particle for each condition.Images of fixed cells were acquired as described . Only HeLa cells stably expressing GFP-mannosidase II and transfected with our pARIS-htt constructs were analyzed. Once deconvolved, images were analyzed with ImageJ software using 3D object counter plugin  for Image J.Mouse neuronal transfected cells were grown on glass coverslips and mounted in a Ludin's chamber. The microscope and the chamber were kept at 33°C. Live videomicroscopy was carried out using a Leica DM IRBE microscope and a PL APO oil 100 × objective with a numerical aperture of 1.40-0.70, coupled to a piezo device (PI) and recorded with Photometrics CoolSNAP HQ2 camera  controlled by Metamorph software. Images were collected in stream set at 2 × 2 binning with an exposure time of 50-150 ms (frequency of 2 s) with a Z-step of 0.3 μm. Deconvolution was performed as described for fixed samples. All dynamic parameters of intracellular transport were obtained from three independent experiments with a total of about 1500-5000 measures from 18-39 independent cells. Dynamics were characterized by tracking positions of eGFP vesicles as a function of time with an especially developed plugin  and Serial Cloner 2.0  software.Click here for fileSequence text files of pARIS-htt constructs used in the study. It includes sequences of pARIS-mCherry-httQ23/Q100 plasmids in Entry and pcDNA vectors.Click here for file


H1) region genes and the immunoglobulin (IgM)heavy-chain constant region of an isogenic Xenopus hybrid, X. laevis/X, gilli, LG7, havebeen sequenced. The LG7 clone represents the first Xenopus hybrid that is homozygousfor the IgH locus. The VH1 family was specifically investigated because VH1 genes areused by the antibodies produced during the Xenopus antidinitrophenol (DNP) response,These VH1 germ-line sequences establish a so-called ”dictionary— that is available forstudying somatic hypermutational mechanisms in immunized frogs.Twenty-eight heavy-chain variable (V


SET-NUP214 fusion resulting from a recurrent cryptic deletion, del(9)(q34.11q34.13) has recently been described in T-cell acute lymphoblastic leukemia (T-ALL) and in one case of acute myeloid leukemia (AML). The fusion protein appears to promote elevated expression of HOXA cluster genes in T-ALL and may contribute to the pathogenesis of the disease. We screened a panel of ALL and AML cell lines for SET-NUP214 expression to find model systems that might help to elucidate the cellular function of this fusion gene.SET(TAF-Iβ)-NUP214 fusion gene transcript. RT-PCR analysis specifically recognizing the alternative first exons of the two TAF-I isoforms revealed that the cell lines also expressed TAF-Iα-NUP214 mRNA. Results of fluorescence in situ hybridization (FISH) and array-based copy number analysis were both consistent with del(9)(q34.11q34.13) as described. Quantitative genomic PCR also confirmed loss of genomic material between SET and NUP214 in both cell lines. Genomic sequencing localized the breakpoints of the SET gene to regions downstream of the stop codon and to NUP214 intron 17/18 in both LOUCY and MEGAL cells. Both cell lines expressed the 140 kDa SET-NUP214 fusion protein.Of 141 human leukemia/lymphoma cell lines tested, only the T-ALL cell line LOUCY and the AML cell line MEGAL expressed the SET-NUP214 fusion gene. Of special note is that the formation of the SET exon 7/NUP214 exon 18 gene transcript requires alternative splicing as the SET breakpoint is located downstream of the stop codon in exon 8. The cell lines are promising model systems for SET-NUP214 studies and should facilitate investigating cellular functions of the the SET-NUP214 protein.Cell lines LOUCY and MEGAL express the recently described  SET-NUP214 (TAF-1/CAN) gene fusion has previously been described as result of a chromosomal translocation t in a case of acute undifferentiated leukemia .HOXA cluster genes are described as targets of the SET-NUP214 fusion protein [SET-NUP214 expression decreases HOX gene expression and inhibits proliferation in the SET-NUP214 positive T-ALL cell line LOUCY [SET-NUP214 also expressed above average levels of HOXA9. Confirming a positive correlation between SET-NUP214 and HOX gene expression, quantitative real-time PCR revealed more than 1000× higher HOXA9 levels in the SET-NUP214 positive cell line LOUCY than in six other T-ALL cell lines tested (data not shown). HOXA9 expression levels were also high in cell line MEGAL, but not above many SET-NUP214 negative AML cell lines (data not shown) which may be due to the fact that HOXA cluster genes are often highly expressed in myeloid leukemias [eukemias ,8.SET-NUP214 gene in the T-ALL cell line LOUCY and in the AML cell line MEGAL by genomic sequencing. In both cell lines, the centromeric fusion is located downstream to the stop codon of SET. Therefore, alternative splicing might turn out to be obligatory for expression of SET-NUP214 mRNA.We demonstrated the presence of the The 141 continuous cell lines investigated in this study were either taken from the stock of the cell bank (DSMZ – German Collection of Microorganisms and Cell Cultures) or were generously provided by the original investigators. Detailed references and cultivation protocols have been described previously .SET/NUP214 mRNA expression was performed applying RT-PCR. RNA was prepared using the Trizol reagent . For mRNA quantification, reverse transcription was performed using the SuperScript II reverse transcriptase kit . Previous studies identified SET exon 7/NUP214 exon 17 and SET exon 7/NUP214 exon 18 fusions in T-ALL and AML patients [SET exon 6 and NUP214 exon 20 for SET-NUP214 expression screening. Analyses were repeated with previously described primers from SET exon 7 and NUP214 exon 18 [SET exon 6 forward: 5'-GAA GAG GCA GCA TGA GGA AC-3'; NUP214 exon 20 reverse: 5'-TAC TTT GGG CAA GGA TTT GG-3'; SET exon 7 forward: 5'-TGA CGA AGA AGG GGA TGA GGA T-3'; NUP214 exon 18 reverse: 5'-ATC ATT CAC ATC TTG GAC AGC A-3'. The same NUP214 exon 18 reverse primer was used in combination with alternative exon 1 forward primers to detect TAF-Iα-NUP214 and TAF-Iβ (SET)-NUP214 mRNA isoforms: TAF-Iα exon 1 forward: 5'-TAA ACG CCA GTC TCC ACT CC-3', TAF-Iβ (SET) exon 1 forward: 5'-AGC TCA ACT CCA ACC ACG AC-3'. For the determination of genomic SET and NUP214 breakpoints in cell lines LOUCY and MEGAL, genomic PCR was performed with the following sets of primers: (i) SET exon 7 forward: 5'-TGA CGA AGA AGG GGA TGA GGA T-3'; NUP214 exon 18 reverse: 5'-ATC ATT CAC ATC TTG GAC AGC A-3'. (ii) SET intron 8/exon 8 forward: 5'-TCA GGA GGA TGA AGG AGA AGA-3'; NUP214 intron 17/18 reverse: 5'-GAG GTG GCA GAG AGG TGG TA-3'; (iii) SET exon 8 forward: 5'-CTG CCA CTC AAT GGG AGA AT-3'; NUP214 intron 17/18 reverse: 5'-ACA AGA ATT ACC CGG GTG TG-3'; PCR was performed in a total volume of 50 μl with a DNA thermal cycler  for 35 cycles under standard conditions. Products were electrophoresed in 1.2% agarose gels and observed under UV light. PCR products were ligated into the pGEM-T Easy Vector System  and sequenced .Screening of cell lines for patients ,10. We aFISH was performed as described previously . TilepatHOXA9 (Hs00365956_m1) expression levels with TBP as endogenous control. For copy number analysis of genomic DNA, we performed relative quantitative PCR with the following oligonucleotides: ABL1 forward: 5'-CAC CGT TAA TTG GGA CTG TGT G-3'; ABL1 reverse: 5'-AAT GGT AGA GTG GTG CTC CTT G-3'; CRAT forward: 5'-CCT GTC CAG TTG GTC ACA CTC-3'; CRAT reverse: 5'-GCC TTT CTA GCT TGA TGC CTC-3'; NUP214 forward: 5'-GGC CAG GTT GGA TTT CAT AC-3'; NUP214 reverse: 5'-CTC ATG ATC CAG GGT GAC AG-3'; SET forward: 5'-TAG ACA GCG CCT AGC ACA TC-3'; SET reverse: 5'-TCC CTT CCA GTC CTG TTA ATG. PCR reactions were performed using SYBR-green chemistry under standard conditions. Values were calculated by the 2-ΔΔCt method. As endogenous control, the repetitive element LINE1 was used.Quantitative PCR was carried out using a 7500 Applied Biosystems real-time PCR system following the manufacturer's protocol . TaqMan probes (Applied Biosystems) were used to quantify human 6 cells were pelleted and washed with ice-cold phosphate-buffered saline (PBS), resuspended and boiled for 10 min in 25 μl SDS sample buffer containing 15% glycerol, 125 mM Tris-HCl pH 6.8, 5 mM EDTA, 2% SDS, 0.1% bromophenol blue and 1% β-mercaptoehanol. The samples were separated on 7% or 12% gels depending on the size of the wild-type proteins to be detected. Blotting and staining conditions were as described previously [Analysis of SET-NUP214 protein expression was performed as follows: 1 × 10The authors declare that they have no competing interests.HQ designed the study and wrote the paper. BS developed and performed the genomic quantitative PCR. SR co-wrote the manuscript. JR performed Western blot analyses, MZ carried out PCR analyses. RML performed the cytogenetic part of the study. HGD provided and cultivated cell lines and critically read the manuscript. All authors read and approved the manuscript.