In contrast to the enormous progress made in mapping out postsynaptic contributions of receptors, scaffolding structures and receptor trafficking to synaptic transmission and plasticity, the small size of nerve terminals has largely precluded direct analyses of presynaptic modulation of excitability and transmitter release in central synapses. Recent studies performed in accessible synapses such as the calyx of Held, a giant axosomatic synapse in the sound localization circuit of the auditory brainstem, have provided tremendous insights into how central synapses regulate the dynamic gain range of synaptic transmission. This review will highlight experimental evidence that resolves several long-standing issues with respect to intricate interplays between the waveform of action potentials, Ca Binding of Ca2+ to the release sensor (i.e. synaptotagmin) triggers membrane fusion between synaptic vesicles (SVs) and presynaptic active zones (AZs) via engagement of SNAREs. Upon opening of the fusion pore, neurotransmitters are unloaded into the synaptic cleft and activate postsynaptic receptors to generate excitatory or inhibitory postsynaptic potentials (EPSPs or IPSPs), ultimately affecting postsynaptic spike initiation and firing patterns that encode information.Information transfer between nerve cells is largely mediated by fast chemical neurotransmission across their junctions or synapses. Electric pulses or action potentials (APs or spikes) are initiated near the soma of presynaptic neurons and propagated along the axon to the nerve terminal where voltage-gated Ca2+ influx and quantal output is primarily based on classical work in invertebrate synapses from squid, crayfish and Aplysia n, where n denotes Ca2+ cooperativity (3~5) m) for immature and mature synapses o, 1~2 mM), and provided compelling evidence that microdomain and nanodomain coupling modalities are likely distinct physical entities, and may operate in the same or different central synapses depending on their functionalities or developmental stages.Collectively, these observations demonstrate that, at the calyx of Held synapse, there is a developmental transformation from "microdomain," the coupling modality involving cooperative action of many loosely associated N- and P/Q-type VGCCs, to "nanodomain", the coupling modality in which opening of fewer tightly coupled P/Q-type VGCCs effectively gates a fusion event. Incidentally, a recent study by Bucurenciu et al. (2008) showed nanodomain coupling modality also operates at the hippocampal basket cell-granule cell inhibitory synapses . Unlike 2+ imaging and flash photolysis of caged Ca2+ indicators (i.e. DM-nitrophen) with paired patch-clamp recordings to directly measure the sensitivity of the Ca2+ sensors in the developing calyx of Held synapse  in a spatially homogeneous manner in the terminal. Such increases bypass the dynamic coupling process of Ca2+ entry through VGCCs to the release machinery during an AP or a voltage-step, and directly trigger transmitter release correlated with a known [Ca2+]i. With appropriate experimental paradigms [2+]i in individual synapses are estimated. When release rates are plotted against [Ca2+]i, their relationship can be fitted with Ca2+ binding-release models similar to the five-site kinetic model proposed by Schneggenburger and Neher (2000). Using this approach, the sensitivity of the release machinery, or the Ca2+ sensor(s), to Ca2+ can be estimated. Surprisingly, experimental data showed that Ca2+ sensitivity of the release sensor in fact decreases with synaptic maturation while molecular cooperativity (n) remains unchanged. KD values increase from ~81 (P9-P11) to ~123 μM (P16-P19)  nanodomains, promoting highly synchronized release with minimal synaptic delay, temporal jitter, and asynchronous release during transmission at mature calyx synapses. Short Ca2+ transients will also reduce the likelihood of presynaptic Ca2+ overload that can occur during prolonged high-frequency transmission, lowering the energy required for Ca2+ clearance via ion pumps and exchangers o, and the number of VGCCs per AZ, prevents establishment of a realistic model for release site topography. Nevertheless, measurements of the Ca2+ channel/domain cooperativity (m) and the EGTA induced attenuation of transmitter release provide sufficient constraints for establishing theoretical models to recapitulate the nature of Ca2+-secretion coupling at the developing calyx of Held synapse.With accumulating evidence from the developing calyx of Held synapse, it is important to further conceptualize the spatial reorganization of VGCCs and SVs in AZs to interpret experimental data. However, lack of precise knowledge of the subsynaptic distribution patterns of VGCCs, their single-channel conductance at physiological concentrations of [Ca2+ diffusion model of Naraghi and Neher (1997) allows for the calculation of the [Ca2+] at any distance from the mouth of a single VGCC  from any desired arrangement of VGCCs simply by summating their individual contributions. This freedom allows us to assume any distribution of VGCCs around a SV while being able to determine the [Ca2+] at that point. This provides a constraint governing the peak [Ca2+] experienced by the release sensor for a particular set of buffer conditions.The linearized buffered Cand Neher 997 allowis known . A parti2+] at the release sensor, we selected a simple ring of VGCCs, at equal r from the SV, as a simplified topography. It is well established that, at least in the immature synapse, VGCCs are most likely at variable distances from their SV, located in clusters or some other heterogeneous spacing arrangement [2+] arising from multiple VGCCs and sufficiently address whether VGCC-SV coupling differences can account for developmental changes in SV release at this synapse.To determine the  transient in each case  [2+ channel/domain cooperativity values for this developmental stage is around 3 [r = 23 nm to the peak [Ca2+] reproduces our experimental findings well. These models represent a simplified interpretation of release site topography which allows for simplification of the calculations required to reproduce main experimental observations including developmental increases in quantal output and the extent of EGTA-induced attenuation of EPSCs in immature and mature synapses.Given these limits, we simulated a number of possible VGCC-SV separation distances with different numbers of VGCCs contributing to the i transients until the experimental release profiles of native synapses are accurately reproduced. Simulations performed from data recorded in the mouse calyx of Held, as summarized in Figure 2+]i "seen" by the Ca2+ sensors increases from 35 to 56 μM and the SV release rate increases from ~600 SVs/ms to 1000 SVs/ms despite smaller and briefer ICa [2+]i, were also derived from the developing rat calyx of Held synapse [The rise and collapse of local Ca(but see ). Howeveeriments . Ca2+ trH) model  as the iefer ICa . Similar synapse .2+]i values (10~300 μM) described in previous work  gradients are assumed to be at steady-state around an open VGCC and buffer saturation is small, the differential equations describing buffered Ca2+ diffusion can be linearized, considerably simplifying the mathematical description of the system  carried by three Ca2+ buffers can be described by the following;At steady state, the  at any distance r from a single VGCC for N described buffer species.In these equations, B2+] as a function of r for various buffer compositions . Note that the hypothesized endogenous mobile buffer properties, recently uncovered at the immature calyx of Held, have not been included here [Using the above equations, we have shown the [Cas Figure . In addided here . This as


In the chr15q25.1 region spanning the nicotinic receptors CHRNA3 and CHRNA5, we identified multiple SNPs associated with CPD (p<10−3), including rs1051730, which has been associated with nicotine dependence, smoking intensity and lung cancer risk. In parallel, we selected 11,199 SNPs drawn from 359 a priori candidate genes and performed individual-gene and gene-group analyses. After adjusting for multiple tests conducted within each gene, we identified between two and five genes associated with each measure of smoking behavior. Besides CHRNA3 and CHRNA5, MAOA was associated with CPDBI (gene-level p<5.4×10−5), our analysis provides independent replication of the association between the chr15q25.1 region and smoking intensity and data for multiple other loci associated with smoking behavior that merit further follow-up.The contribution of common genetic variation to one or more established smoking behaviors was investigated in a joint analysis of two genome wide association studies (GWAS) performed as part of the Cancer Genetic Markers of Susceptibility (CGEMS) project in 2,329 men from the Prostate, Lung, Colon and Ovarian (PLCO) Trial, and 2,282 women from the Nurses' Health Study (NHS). We analyzed seven measures of smoking behavior, four continuous , and three binary . Association testing for each single nucleotide polymorphism (SNP) was conducted by study and adjusted for age, cohabitation/marital status, education, site, and principal components of population substructure. None of the SNPs achieved genome-wide significance (p<10 Cigarette smoking is a risk factor for more than two dozen diseases and the single biggest cause of preventable mortality worldwide Genome-wide linkage studies have identified chromosomal regions that may harbor loci contributing to one of many smoking behavior phenotypes: age at initiation Until very recently, candidate gene association studies have focused on genes in a few candidate pathways. A ‘reward deficiency syndrome’ has been postulated as one unifying theme to account for the role of diverse neurotransmitters in nicotine dependency CHRNA5, and CHRNA3, and CHRNB4 and was also identified in a recent GWAS of dichotomized smoking intensity A recent genome-wide association study of over 13,000 smokers identified a region on chromosome 15q25.1 associated with smoking intensity (number of cigarettes smoked per day) To identify loci associated with smoking initiation, intensity and cessation we performed a genome-wide association study (GWAS) using data from subjects genotyped as part of the Cancer Genetic Markers of Susceptibility (CGEMS) project, including 2,617 ever-smokers Subjects were drawn from two previous genome-wide association studies (GWAS), performed as part of the Cancer Genetic Markers of Susceptibility (CGEMS) project For both PLCO and NHS, analyses of population stratification were conducted using approximately 10,000 unlinked SNP markers We selected four continuous and three binary smoking behaviors for analysis . The conAll of these behaviors were measured by baseline questionnaire (BQ) in the PLCO (administered from 1994–2001) In the NHS, SMKAGE was measured at BQ; all other behaviors were measured using cumulative information from the BQ (administered in 1976) and subsequent follow-up questionnaires (one every two years). The majority of NHS subjects (2149) had smoking data available up through the 2002 questionnaire. For those few women (133) with no smoking data available from the 2002 follow-up cycle, we used data from the latest available follow-up. Age at initiation was defined as the age when a subject started smoking cigarettes “regularly.” Former smokers were defined as ever-smokers who identified themselves as non-smokers on any questionnaire (and did not identify as a smoker on any subsequent questionnaire). Age at cessation was explicitly asked in NHS BQ. For women who quit smoking after the BQ, age at cessation was inferred as the median age between the questionnaire that defined the woman as a former smoker and the last questionnaire that identified her as a smoker. Prior to 1982, current or former smokers were asked to write in the average number of cigarettes they smoked per day; subsequent questionnaires captured information about smoking intensity in categories . Pack years was estimated as the sum of the products of smoking intensity  at questionnaire k times the interval between questionnaire k and questionnaire k+1 . Smoking duration was calculated as the sum of all intervals where a woman was a smoker. Average cigarettes per day (the CPD variable used in this GWAS) was calculated as pack-years divided by smoking duration.Q and I2 statistics http://hydra.usc.edu/GxE/)Continuous phenotypes were log transformed to achieve approximate normality and SNP genotypes were coded as counts of minor alleles. For each study, we defined any phenotype that was more than three standard deviations from the mean to be an outlier. Outliers that were above (below) the mean were then truncated to the 99th (1st) percentile of the raw distribution. We tested for association between each SNP marker and each continuous phenotype using linear regression, adjusted for study center (PLCO) or geographic region (NHS); age at smoking assessment in five-year bins (baseline for PLCO or last available follow-up for NHS); marital status  or living arrangement ; education ; prostate (PLCO) or breast (NHS) cancer case-control status; and selected principal components of genetic variation. For binary traits, we used unconditional logistic regression, adjusted for the same covariates. These tests were conducted separately for PLCO and NHS. For SNPs that passed QC filters and had minor allele frequency above 1% in both studies, we combined evidence for association across PLCO and NHS using weighted Z-scores CTNNA3, FBXL17, FTO, NRX1, PBX2, TRPC7, VPS13A) and CPD  We selected 359 genes for additional analyses, based on their hypothesized relationship to smoking behavior. 349 of these genes were previously selected by the NICSNP Candidate Gene Committee For each candidate gene we tested the null hypothesis that no SNP within 20 kb upstream of the start of transcription and 10 kb downstream of the stop of transcription (based on NCBI build 35.1) was associated with smoking behaviors using a parametric permutation procedure that allows for covariate adjustment. We compared the smallest observed p-value for any SNP in the candidate gene region to the empirical null distribution of the smallest p-value based on 20,000 random permutations. This approach provides a gene-level p-value that is adjusted for both the number of SNPs in the gene region and their linkage disequilibrium structure.Candidate genes (and the SNPs in the corresponding gene regions) were grouped based on known functional similarity. We used a slightly modified version of the groups developed by the NICSNP Candidate Gene Committee. . To testMultiple SNPs in the 15q25.1 region have been shown to be associated with CPD, nicotine dependence, or risk of lung cancer The distributions of the smoking behaviors and demographic covariates included in the analysis of the NHS and PLCO datasets are presented in 10 p-values for SNP association with smoking behaviors (−7) in any combined analysis pooling evidence for association across the two studies . Including this result, there are 8 gene regions and 3 genomic regions with predicted but not verified coding regions associated with SNPs in the group of SNP smoking behavior results with P<10−5 , located 10,051 base pairs 3′ of PSMA4 and 6,290 base pairs 5′ of CHRNA5 in a region of strong linkage diseqilibrium spanning both genes.We performed genome wide association analyses for seven related smoking behaviors in two datasets totaling 4,611 individuals and 2617 ever smokers. We selected smoking behaviors with established hereditary components −7) associations, we did find additional evidence for an association between genetic variants in the chr15q25.1 region and number of cigarettes smoked per day. Candidate gene analyses also provided suggestive evidence for association between variants in the MAOA gene region and the smoking behavior cigarettes per day.Although we did not discover novel genome-wide significant  common variants with small individual effects on smoking behavior, since our power to detect any one is small. Even with our relatively large sample size, our power to detect a variant similar to the 15q25.1 SNP rs1051730 (which was estimated to explain about 0.7% of the trait variance The lack of genome-wide significant results suggests that common variants have at most a modest influence on smoking behavior. We had adequate power to detect a variant that explained even 2.5% of the variation in cigarettes per day. We had 61% power in the NHS sample and 71% power in the PLCO sample to detect such a variant at the 10CHRNA3 (chr15q25.1) and CHRNA1 (chr2q31.1) are associated in the CGEMS sample with three smoking behaviors: CPD, PKYRS and SMKAGE , was highly statistically significantly associated with CPD (p = 5×10−32); the SNP rs8034191 (LOC123688 IVS2+256) was also highly statistically significantly associated with CPD (p = 2×10−29). These SNPs were evaluated using a total of 26,789 (rs1051730) or 24,891 (rs8034191) smokers from this study and two other reports. The CHRNA5 SNP rs16969968  was significantly associated (p<.01) with CPD in this study but not an earlier, smaller study; combined evidence for association in 3,464 smokers remained significant (p = 2×10−3). Comparative judgments of the relative importance of the individual SNPs are not possible due to the different sample sizes, the strong LD among the SNPs and the inability to adjust for the effects of the other SNPs in this meta-analysis.Finally, we combined our chr15q25.1 results with data from three other published reports  [44], [4−5) between genetic variation in MAOA and a dichotomized measure of smoking intensity (10 or less cigarettes smoked per day versus more than 10). This was the only gene-level result that remained significant after Bonferroni correction for the number of genes tested, which we regard as a conservative multiple-testing correction. This association is notable because of the role of the monoamine oxidases in the regulation of catecholamines and the inhibition of monoamine oxidases A and B by tobacco smoke Our candidate gene analyses identified an association . We have previously discussed nicotinic receptor findings above. The association of rs7050529 (IVS3+286 of TRPC5) with CPD  is notabpermuted<0.05) associations with smoking behaviors  and NFKB1 . FBXL17 is one of 68 members of the human F-box protein superfamily, a large group of ubiquitin ligases FBXL17 was also identified in the NICSNP GWAS FBXL17 SNP identified in this study. One SNP genotyped in this study (rs885624) was in the same LD block as rs10793832 but was not significantly associated with quit status in either this study alone or in the combined analysis (p = 0.39).In our study, the observed association between cell cycle control genes and quit status may be driven by association of SNPs at ADH4 gene-level P = 0.048 (rs3828541), and ADH6, gene-level P = 0.034 (rs3857224) suggests that genetic variation at these ADH loci may influence the establishment of smoking behavior. However this analysis did not control for alcohol consumption and so this finding should be considered preliminary.The finding that the alcohol dehydrogenases genes were significantly associated with the smoking behavior EVNV in this analysis , and conduct a genome-wide association scan for differences in genetic effect between men and women. Such analyses are important, because the effect for some loci may differ between men and women or be restricted to one gender, e.g., due to differences in the environment. However, no SNPs achieved genome-wide significance for association with any smoking behavior in either study, and no SNP achieved genome-wide significance for heterogeneity in effect between men and women (between studies).This study has several strengths. We performed a GWAS and candidate gene study investigating a variety of smoking behaviors with public health importance for the first time in a sample unselected for smoking behaviors and/or smoking attributable disease. We confirm important findings from recent GWAS and candidate gene studies of nicotine dependence and CPD. Our sample size is relatively large, yet still not large enough to reliably detect variants with modest effects on smoking behaviors. The absence of selection bias in the cohort bases for the samples enhances generalizability to U.S. non-Hispanic whites although a modest limitation is that the education level in both cohorts is above average. By limiting analyses to subjects of European ancestry and adjusting for principal components of population structure, we minimized risk of false positives due to population stratification, but are not be able to detect SNP alleles associated with smoking behavior that are common in non-Europeans but rare among European-Americans. The smoking behavior characteristics for the two studies are quite similar after taking into account expected differences by gender , and theThe diverse smoking behaviors we investigated represent the spectrum of key events in an individual's smoking history from initiation  thru establishment of dependency , to outcome (current versus former cigarette smoking status), with potential genetic influence at each stage. The finding that selected genes are associated with multiple phenotypes may represent both correlations among the phenotypes but also pleiotropic effects of the genes, and is a strength of the integrative approach Table S1(0.04 MB XLS)Click here for additional data file.Table S2(0.03 MB DOC)Click here for additional data file.Table S3(0.05 MB DOC)Click here for additional data file.Figure S1(0.43 MB TIF)Click here for additional data file.Figure S2(0.31 MB TIF)Click here for additional data file.


Analysis of a 2.6 million longSAGE sequence tag resource generated from nine human embryonic stem cell lines reveals an enrichment of RNA binding proteins and novel ES-specific transcripts. To facilitate discovery of novel human embryonic stem cell (ESC) transcripts, we generated 2.5 million LongSAGE tags from 9 human ESC lines. Analysis of this data revealed that ESCs express proportionately more RNA binding proteins compared with terminally differentiated cells, and identified novel ESC transcripts, at least one of which may represent a marker of the pluripotent state. Embryonic stem cells (ESCs) can be derived from the inner cell mass of blastocysts and are defined by their ability to be propagated indefinitely as undifferentiated cells with the potential, upon appropriate stimulation, to generate cell types representing all three embryonic germ layers . Since tMicroarray-based approaches have been used to define the transcriptomes of numerous human ESC lines, including BG01, BG02, WA01, WA07, WA09, WA13, WA14, TE06, UC01 and UC06 -22. ThesLongSAGE libraries were constructed using total RNA purified from nine different human ESC lines cultured as undifferentiated cells by serial passaging on mouse embryonic fibroblast (MEF) feeder layers  Table 11. To enaTo investigate the similarities and differences between the libraries, we performed hierarchical clustering using Pearson correlation coefficients . For thiP = 1.8 × 10-7 and 1.0 × 10-6, respectively, by one-sided t-tests).To assess the representation of known genes in the nine human ESC transcriptomes, we compared our data to other human sequence tag-based resources -18. HighP < 3.0 × 10-5; Additional data file 4). The most significantly affected transcript  encoded Secreted frizzled-related protein-1 (Sfrp1), a well characterized antagonist of WNT signaling. Our analysis suggested that the two isoforms of Sfrp1 we identified either retained or lost the 3' untranslated region -PCR . InteresNlaIII for SAGE and Dpn1 for MPSS) and the fact that different mRNA preparations were used in each study. To further explore this lack of concordance, we compared the longSAGE and MPSS-derived gene lists to a common gene list derived from Affymetrix expression arrays generated from the same RNAs used to construct our LongSAGE libraries [To generate a list of transcripts common to all libraries (excluding the HSF-6 library because of the differentiation markers found therein), we first identified tags from each library that uniquely mapped to transcripts within RefSeq  and the ibraries . The AffLongSAGE offers opportunities for discovering novel transcripts. These can be identified as tags that map uniquely to the genome but not to any available transcript resources. To look for these, we used the 2.5 million tag meta-library, which contained 379,645 unique tag sequences. Grouping LongSAGE tags that mapped to genomic locations in close proximity to one another  resultedMAPK2 gene, none of the identified ORFs demonstrated Ka/Ks ratios suggestive of purifying selection [To further characterize these putative novel, low-abundance ESC library specific transcripts, we compared the ESC meta-library to publicly available data derived from 247 non-ESC SAGE libraries that together contained 654,491 unique tag sequences. This comparison identified 20,047 tag sequences found only in the human ESC meta-library . For subsequent analyses, we focused on those tags that uniquely mapped at least 2 kb away from any known gene. This analysis reduced the number of tags to 634 , of which 301 were found within genomic regions exhibiting sequence conservation between human and mouse or rat . We used rapid amplification of cDNA ends (RACE)  to cloneelection . Hence, Foxb1 gene within its first intron. Foxb1 encodes a winged helix transcription factor involved in the development of the vertebrate central nervous system and Foxb1-/- mice display phenotypes consistent with a requirement for this gene in both embryonic and postnatal stages of development [Foxb1 transcripts except for a single Foxb1 tag in the HSF-6 library. This general lack of Foxb1 expression in ESCs and the genomic location of the Foxb1 gene within the first intron of HA_003240 are consistent with the notion that Foxb1 expression is repressed by expression of HA_003240, possibly by steric inhibition of the transcription initiation complex [Oct4 encodes a transcription factor that regulates a number of key human ESC markers, including Nanog, through co-operative binding with a Sox family member [Foxb1 locus is intriguing and suggests an interesting mechanism for negatively regulating Foxb1 expression in Oct4-expressing cells.Four RACE clones were found to have genomic coordinates that overlapped with those of known transcripts . One of these .To more fully characterize a transcript identified by a singleton tag , we attempted to recover a full length transcript using 5' and 3' RACE and primers annealing within the terminal exon of the putative transcript. Alignment of the resulting candidate full length sequence to the human genome revealed a transcript that contained two introns  with matches to other ESTs, of which 7 were found only in data derived from pluripotent human ESC lines. One RACE clone that overlapped an EST derived from pluripotent human ESC lines (HA_003152) was also found to be expressed in all nine ESC lines studied here. BLAT  alignmenOct4, Lin28 and Msx1 in the same RNA preparations. Figure Oct4 and Lin28 in the human ESCs stimulated to differentiate into embryoid bodies and an up-regulation of expression of the early differentiation marker Msx1. Significant reduction of expression was observed in four of the six transcripts tested, including HA_003152, whose expression was undetectable at d30  to compare transcript levels in RNA purified from human ESCs maintained under conditions that promote their maintenance in an undifferentiated state to RNA extracts obtained from human ESCs that had been stimulated to differentiate into embryoid bodies. To provide a comparative dataset we selected five additional novel transcripts for qPCR. In all cases, qPCR amplicons were designed to cross exon-exon boundaries. As controls we also monitored expression of 0 Figure . These tAs part of the ongoing effort to elucidate mechanisms regulating ESC self-renewal, we generated 2.5 million LongSAGE tags from nine human ESC lines. Comparison of these data to libraries prepared from differentiated tissues identified a group of ESC-library specific transcripts and an enrichment of transcripts encoding mitochondrial and RNA binding proteins (by comparison to differentiated cells). RNA binding proteins play a role in the regulation of mRNA processing and examination of non-canonical longSAGE tags in the human ESC libraries suggest that these cells express a distinct collection of gene isoforms. One such isoform may bypass translational down regulation through the expression of a transcript lacking predicted miRNA target sequences.An emerging theme in digital gene expression profiling is the identification of a large class of transcripts that map uniquely to the genome, but cannot be localized to any known or computationally predicted transcripts. Tags in this class are predominantly found at relatively low levels. Analysis of the 2.5 million LongSAGE tags generated in the course of this study revealed 14,588 such tag sequences, a subset of which were found exclusively in human ESCs. As a first step towards understanding the relevance of these transcripts to ESC biology we generated 5' RACE clones for 52 novel apparently ESC-specific transcripts. Analyses of these transcripts revealed that the majority do not appear to encode proteins and do not overlap existing pseudogene predictions. One transcript was found to be expressed across all nine ESC lines we profiled and matched ESTs generated by others from ESCs. Its restricted expression pattern suggests that it may represent a novel transcriptional marker for the maintenance of pluripotentiality. In addition to the discovery of this potential marker, we also identified four novel transcripts that may participate in the regulation of expression of known genes, one of which is known to play a direct role in differentiation. Our analyses indicate that there are many previously undiscovered transcripts expressed in human ESCs and support the contention that sampling of SAGE libraries to depths beyond currently accepted practice is required to fully explore the coding potential of the mammalian transcriptome. To assess possible functions associated with such rare transcripts, we are actively pursuing the cloning and characterization of the remaining novel human ESC-specific transcripts identified in this study.Detailed information regarding the human ESC lines used in this study can be found at the NIH Stem Cell Information website . The pasNine LongSAGE  librariein silico to form 14 bp tags. A total of 2,508,608 tags corresponding to 222,337 unique 14 bp tag sequences  were utilized in this analysis. These tags were directly compared to all unique tags from the human SAGE libraries to generate a list of tags found solely in the ESC meta-library.LongSAGE tags of at least 99.9% accuracy  software  as folloNlaIII restriction sites (CATG). Of these, our analysis defined a subset of 19.4 million genomic tag sequences that were unique within the genome.LongSAGE tags were mapped to known and computationally predicted transcripts using versions of the following databases available as of March, 2005: RefSeq , RefSeqXA second table was generated that stored information about exons: genome sequence contig, transcript orientation, exon number, exon boundary type and nucleotide positions of exon boundaries for all approximately 267,000 exons annotated on release 35 of the Reference Sequence genome. The LongSAGE tag sequences were compared to the unique genomic tag table, yielding sets of genomic positions for all tags in the library. These in turn were compared to the table of exon information, producing a mapping for each tag relative to annotated exons.t-test comparing two samples was used. The null hypothesis was that the two samples arose from populations with the same mean and standard deviation. The values within each sample were the number of GO categories represented in each library of the set, nine in the ESC set and four in the normal set. To account for variation due to library size, only the transcripts with the top 1,000 expression values were included. A one-sided p value was reported. Microsoft Excel was used to perform the computation.For the GO category comparisons, a standard p value for the null hypothesis that the two tag frequencies arose from Poisson distributions with the same mean. This was derived using a normal approximation to the Poisson as described by Kal et al. [p < 0.05 were selected. Tag counts were converted to tags per million, and transcripts that differed by less than three-fold were eliminated. All pairs of tags existing within the same transcript were then listed if the differential expression for the two tags was in the opposite direction.To select differentially expressed LongSAGE tags, the ESC and CGN meta-libraries were compared on a tag per tag basis to obtain a l et al. . All trafree™ kit; Ambion, Austin, TX, USA) treated RNA using the BD SMART RACE cDNA Amplification kit following the manufacturer's recommended protocol . Gene specific 5' RACE primers were designed using custom scripts and Primer 3 [®-TOPO® vector using the TOPO TA Cloning® Kit for Sequencing (Invitrogen). Plasmid vectors were electroporated into bacterial cells, and recombinant clones were selected on agar plates containing appropriate antibiotics as described [First strand 5' and 3' RACE ready cDNA was synthesized from 2.0 μg of DNase I  treated total RNA using the SuperScript Choice System following the manufacturer's recommended protocol (Invitrogen). Gene specific primer pairs were designed using custom scripts and Primer 3 [RNA was obtained from H9 cells before and after induction of differentiation using a 30-day embryoid body protocol. Undifferentiated H9 cells maintained for 7 days on matrigel  in media conditioned by mouse embryonic fibroblasts and supplemented with 4 ng/ml fibroblast growth factor (bFGF-2) were harvested for embryoid body formation. Briefly, the cells were incubated with TrypLE (Invitrogen) for 10 minutes at 37°C and then collected by scraping. Resultant cell aggregates were subsequently cultured in non-adherent dishes using KOSR-based media without FGF2, for 15 to 30 days. At appropriate time-points RNA was extracted into Trizol. cDNA was synthesized from 2.0 ug of DNase I (DNA-Primer 3  to ampliPrimer 3  using a The following additional data are available with the online version of this paper. Additional data file Summary of mouse specific tag types identified.Click here for fileGenomic mappings for 268,515 unique tag sequences found in nine independent human embryonic stem cell lines.Click here for fileTag counts are expressed for each GO category for the top 1,000 by tag count.Click here for fileStatistically significant differentially expressed LongSAGE tags found between embryonic stem cells and terminally differentiated tissues.Click here for fileThe 4,337 genes found in common across 8 undifferentiated human embryonic stem cell lines.Click here for fileThe 20,047 LongSAGE tags exclusively expressed in embryonic stem cell lines.Click here for fileThe 634 LongSAGE tags exclusively expressed in ESCs that uniquely map to the human genome at least 2 kb away from an annotated transcript.Click here for fileThe 301 LongSAGE tags exclusively expressed in ESCs that uniquely map to species conserved regions of the human genome at least 2 kb away from an annotated transcript.Click here for fileThe 52 ESC specific transcripts identified by 5' RACE.Click here for fileRACE and qPCR primer sequences used in this study.Click here for file


With a cultivation area of 1.75 Mio ha and sugar yield of 16.7 Mio tons in 2006, sugar beet is a crop of great economic importance in Europe. The productivity of sugar beet is determined significantly by seed vigour and field emergence potential; however, little is known about the molecular mechanisms underlying these traits. Both traits exhibit large variations within sugar beet germplasm that have been difficult to ascribe to either environmental or genetic causes. Among potential targets for trait improvement, an enhancement of stress tolerance is considered because of the high negative influence of environmental stresses on trait parameters. Extending our knowledge of genetic and molecular determinants of sugar beet germination, stress response and adaptation mechanisms would facilitate the detection of new targets for breeding crop with an enhanced field emergence potential.To gain insight into the sugar beet germination we initiated an analysis of gene expression in a well emerging sugar beet hybrid showing high germination potential under various environmental conditions. A total of 2,784 ESTs representing 2,251 'unigenes' was generated from dry mature and germinating seeds. Analysis of the temporal expression of these genes during germination under non-stress conditions uncovered drastic transcriptional changes accompanying a shift from quiescent to metabolically active stages of the plant life cycle. Assay of germination under stressful conditions revealed 157 genes showing significantly different expression patterns in response to stress. As deduced from transcriptome data, stress adaptation mechanisms included an alteration in reserve mobilization pathways, an accumulation of the osmoprotectant glycine betaine, late embryogenesis abundant proteins and detoxification enzymes. The observed transcriptional changes are supposed to be regulated by ABA-dependent signal transduction pathway.This study provides an important step toward the understanding of main events and metabolic pathways during germination in sugar beet. The reported alterations of gene expression in response to stress shed light on sugar beet stress adaptation mechanisms. Some of the identified stress-responsive genes provide a new potential source for improvement of sugar beet stress tolerance during germination and field emergence. Beta vulgaris L.) is a major crop for sugar production in countries with a temperate climate. The agronomic productivity of sugar beet, as a direct seeded crop, is determined significantly by the uniformity of seedling emergence in the field . Sequences were subjected to clustering and assembly analysis with known sugar beet ESTs available at the GeneBank. Obtained consensus sequences were annotated against public nucleotide and protein databases and referred to different functional categories using Pedant-Pro™ Sequence Analysis Suite version 3 . Additionally, Blast searches were performed against the current set of Arabidopsis thaliana proteins (TAIR database). Gene Ontology (GO) and TAGGIT [A. thaliana hits detected with threshold levels of less than 1e-20.Expressed Sequence Tags (ESTs) were obtained by commercial sequencing of the selected cDNA clones . cDNA clones derived from dry seeds were sequenced either from 3'-ends using the M13 forward primer (5'-CCAGGGTTTTCCCAGTCACG) or from 5'-ends using the T7 primer (5'-TAATACGACTCACTATAGGG). cDNA clones obtained from the germinating seeds were sequenced from 5'-ends using the M13 reverse primer (5'-CGGATAACAATTTCACACAGG). EST sequences are integrated at GabiPD database d TAGGIT  functionTo compare our data with a recently published set of sugar beet seed proteins  tBlastn PCR amplification was carried out from individual bacterial clones in 96-well plates using the primers 5'-GCAGGTACCGGTCCGGAATTCCCGGG and 5'-CCCAGTCACGACGTTGTAAAACGACGGCCA for cDNAs inserted into the vector pSPORT1 and the primers 5'-GTTGTAAAACGACGGCCAGTGAG and 5'-GCTATGACCATGATTACGCCAAGC for inserts ligated into the vector pBluescript II SK (+). The PCR program included an initial denaturation step, 3 min at 94°C, followed by 30 cycles of 20 sec at 94°C, 1 min at 60°C, 2 min at 72°C and a final extension step of 7 min at 72°C. Amplification of cDNAs isolated previously from sugar beet leaf, root and inflorescence libraries  and kind33P-labeled cDNA was performed as described by Sreenivasulu et al. [Array hybridizations were performed at least twice per each time point using different biological replicates of plant material. The synthesis of u et al. . Hybridi2 = 0.99–1.00). Furthermore a high correlation between signal intensities representing biological replicates was observed (correlation coefficients r2 = 0.97–0.99). It was noted that low-intensity spots demonstrated a greater variability than middle and high intensity spots. To reduce the number of false positives, the spots showing normalized intensities below four averaged background levels over all analyzed time-course stages were removed from further consideration.The image data were evaluated by the AIDA program package . Signal intensities were normalized with the total amount of radioactivity bound to the arrays. Empty spots were used for background calculation with the 'weighted background dots' function in the AIDA Image Analyzer software. The intensities of double spots representing the same cDNA were averaged. Macroarray hybridization quality tests made by comparison of technical replicates for the stages T0 and T50 revealed a high reproducibility of results . For RNA dot blot analysis 5 μg of RNAs were loaded directly on membranes using a minifold dot-blot system . The membranes were hybridized with 32P-labelled probes prepared according to Megaprime DNA Labeling System protocol (Amersham Bioscience). For RNA loading control the membranes were stripped and hybridized again with 18S rDNA probe.For Northern analysis 10 μg of glyoxal denatured total RNAs isolated from dry and germinating sugar beet seeds were fractionated in 1% agarose gels and transferred onto Hybond NSugar beet ESTs were obtained by single-pass partial sequencing of selected cDNA clones of four representative cDNA libraries developed from dry mature seeds and seeds germinated under standard conditions. In total 2,784 ESTs were generated including 1,170 from dry seeds and 1,614 from germinated seeds. To estimate the number of genes represented by the entire EST data set, the sequences were assembled into contigs. With 1,917 singletons and 334 contigs consisting of 871 ESTs, the calculated gene number was 2,251. The contigs contain 2 to 12 ESTs with the largest contig of 12 ESTs showing similarity to a late embryogenesis abundant (LEA) protein and the second largest one of 10 ESTs similar to the 60S ribosomal protein L41.Arabidopsis thaliana proteins available at 'The Arabidopsis Information Resource' . Gene ontology (GO) classification was done for 1,576 (70%) of sugar beet sequences using the best A. thaliana hits detected with threshold levels of less than 1e-20 (data not shown). Carrera et al. [A. thaliana gene lists in relation to previously reported functions involved in embryo maturation, dormancy and germination. As only 21% of the A. thaliana hits comprising 14% of the total 'unigene' set could be annotated using the TAGGIT approach it was not further followed. Instead, sugar beet genes were manually annotated of using the combined information obtained by the different approaches.In order to obtain a better sequence annotation consensus sequences were generated for each contig. The consensus and singleton sequences making together the set of 2,251 'unigenes' were searched (BlastX) against a non-redundant protein database. As a result 1,670 sequences (74%) could be annotated to known or hypothetical plant proteins under the applied threshold value of 1e-20. In addition, the 'unigenes' were searched against the current set of a et al.  reportedRecently Catusse et al.  performe2O, 15°C) sugar beet seeds started to germinate after one day of imbibition , passed the time point T50 corresponding to 50% of germinated seeds at day 2 (T50 = 49 h) and reached a maximum germination value after day 4 (Tmax = 105 h)  analysed revealed a high correlation of the expression profiles among the different contig members. The occurrence of two dissimilar transcription patterns observed in the remaining 11 contigs suggests an inefficient resolution of genes representing gene family members. It should be noted that contigs were constructed based on partial, mostly 5'-end, sequences of the genes. Previously, it was shown that 5' sequence information is superior regarding gene discovery while sequence information obtained from the 3'-end is more useful for separation of individual members of gene families . Thus, tOut of the 1,602 genes analysed, 1,088 (67.9%) showed at least a 2-fold change in transcript level in the course of germination, 361 genes (22.5%) revealed a more than 5-fold change and 202 genes (12.6%) a more than 10-fold change. A given gene was declared to be differentially expressed when it satisfied two criteria: (1) at least a 2-fold change in mRNA level during germination and (2) statistical significance under the applied threshold cut-off q < 0.01 (1% FDR) ,32. The For 266 transcripts of cluster k1 and 301 transcripts of cluster k2 sequence analyses retrieved annotations with expected values below 1e-20. To compare the clusters of down- and upregulated genes the differentially regulated transcripts were assigned to different functional categories , abundant both in dry and germinating seeds 41 genes) and abundant in germinating seeds (33 genes)  encode ribosomal proteins and eukaryotic elongation factors. This finding emphasizes a central role of protein biosynthesis during both seed maturation and germination and is consistent with previous studies ,39. AmonAs expected, dry mature seeds contain a large amount of mRNAs coding for SSPs and LEAs, the proteins predominantly synthesized prior to seed desiccation. Germinating seeds are characterized by a more diverse spectrum of abundant mRNA species Fig. . These iArabidopsis [Several genes encoding enzymes involved in methionine biosynthesis and the corresponding regeneration pathways, are found to be actively transcribed either in germinating (methionine synthase and adenosylhomocysteinase) or both in dry and germinating (S-adenosyl-L-methionine synthase) seeds. This finding is in agreement with a previous report on a high representation of ESTs related to one-carbon metabolism in 4-day old sugar beet seedlings . Methionbidopsis .FG344171, FG345664 and FG345425), whose mRNA levels were low in dry seeds and upregulated 25 to 85 fold during germination, were assumed to be associated with SSP breakdown occurring inside protein storage vacuoles , with a low transcript level in dry seeds, showed an enormous upregulation  during germination and revealed the highest absolute mRNA level  among all the genes involved in reserve mobilization. Different time-dependent expression patterns were observed for two β-amylase genes, both characterised by medium mRNA level in dry seeds. One gene was 5-fold upregulated during germination whereas the second one was 3-fold downregulated. Amylase activity produces maltose, which is subsequently converted to glucose by the enzyme α-glucosidase. In contrast to medium to highly expressed amylase genes, the only gene with homology to α-glucosidase revealed a low mRNA level both in dry and germinating seeds. Given that alpha-glucosidases are encoded by several genes belonging to a large glycoside hydrolase gene family, we suppose that the specific family members involved in starch catabolism during germination were not represented on our array.As deduced from EST analysis, starch in sugar beet seeds is degraded predominantly through the hydrolytic pathway , osmotic (200 mM mannitol) and liquid excess 60 ml) stresses applied at a reduced temperature (10°C) delayed germination of sugar beet seeds significantly  and decreased the maximum germination rate from 99% to 92% compared to the standard conditions Fig. . The fir0 ml streThe striking finding is an altered expression of genes related to catabolism of starch and lipid storage reserves. Under the multistress conditions a stronger induction of genes involved in hydrolytic starch degradation (α- and β-amylases) and a weaker up-regulation of genes involved in lipid degradation  were observed Table , Fig. 7.'Multistress' germination results in increased mRNA levels of several genes encoding seed specific proteins  and transporters suggesting an essential role of these genes in osmotic stress adaptation and protection. Our finding of an enhanced expression of a gene encoding choline monooxygenase (CMO) indicates the activation of an additional pathway maintaining the osmotic potential of the cell, the accumulation of the osmoprotectant glycine betaine (GlyBet). The activation of this pathway is also supported by the enhanced expression of a downstream gene encoding betaine aldehyde dehydrogenase (BADH) (1.7-fold).Reduced mRNA levels of several genes encoding cytoplasmic ribosomal proteins, translation initiation factors, histones and tubulin under stress conditions point out a negative influence of stress on protein biosynthesis, cell division and growth and are in agreement with literature data .As expected, a considerable number of genes assigned to the stress response category, and especially those encoding ROS scavenging enzymes, showed an altered expression kinetic during germination under stress conditions, although not all of the genes demonstrated enhanced transcript amounts. The observed alterations of gene expression patterns are triggered by different signal transduction pathways and are governed by a hierarchical network of TFs. Two genes showing similarities to known protein kinases and three genes encoding putative TFs reveal enhanced transcript levels under stress conditions  is derived from a dry mature seed specific cDNA library. To our knowledge no ESTs isolated directly from dry mature sugar beet seeds have been reported so far, although some of the publicly available ESTs, especially derived from immature inflorescences , field-hDue to the applied cDNA library normalization technique based on several repetitive cycles of sequencing and colony hybridization (see Methods), the proportion of unique ESTs in the set was enriched up to 82%. For comparison de los Reyes et al.  have repArabidopsis thaliana seeds with a similar number of transcripts  detected in 24 h-imbibed seeds [Germination is a key developmental stage in plant life cycle. Upon imbibition, the dry quiescent seed resumes metabolic activity, and this metabolic switch is accompanied by a huge shift in gene expression. In the current study transcriptome profiles of dry mature and germinating sugar beet seeds were analysed using dedicated cDNA macroarrays representing 2, 251 unigenes. Recently it has been shown that as many as 12,470 mRNA species were quantitatively detected in dry ed seeds . TherefoMore than half of the analysed sugar beet genes revealed 2-fold changes of mRNA levels during germination. An assay of the genes demonstrates a considerable overrepresentation of transcripts encoding SSPs, LEAs, TFs, pre-mRNA splicing factors and RNA helicases in the cluster of downregulated genes. SSP genes  are known to be strongly expressed during seed maturation and reserve accumulation while LEA genes become activated later and play a protective role during seed desiccation ,43. BothA. thaliana showed that the mRNA profile of the imbibed seeds differed from that of dry seeds already 6 h after seed imbibition [Nine putative TFs belonging to six TF families  were found to be downregulated during germination. Members of these TF families were implicated in controlling many aspects of seed maturation, such as accumulation of storage compounds, cotyledon identity, acquisition of desiccation tolerance, dormancy, seed mass and seed yield -48, in mA. thaliana and B. vulgaris, the sugar beet transcripts detectable in dry  and germinating  seeds were annotated against A. thaliana genes and the obtained hits were compared with the mRNAs described by Nakabayashi et al. [A. thaliana seeds, correspondingly. The high proportion of functionally orthologous genes transcribed in Arabidopsis and sugar beet seeds might reflect a general conservation of the mechanisms regulating seed maturation and germination in dicotyledonous species.A high amount of the genes involved in energy production pathways, storage reserve mobilization, reinitiation of cell cycle, overall metabolism and activation of respiration was detected in the cluster of upregulated transcripts, thus confirming data reported for other species ,6,9. To sensu stricto and, with other seed reserves, are involved in the heterotrophic nutrition of young seedlings [Amount and efficient mobilization of seed storage reserves are important parameters determining early seedling vigour and field emergence. Sugar beet seeds contain starch, proteins and lipids as storage reserves. A previous study of sugar beet germination and early growth has shown that lipids are the major metabolic substrate during germination eedlings . To examThe presented transcriptome data for seed storage reserve mobilization Table , Fig. 5 2O2 solutions or in water [Drought or wetness, salt stress and non-optimal temperatures are critical environmental factors that limit seedling emergence in the field. Whereas some gene expression studies state that various stresses, like cold and drought, share a common set of signal transduction pathways , there ain water .We found that genes related to storage reserve catabolism are specifically affected by stress conditions during sugar beet germination: the mRNA levels of genes involved in starch degradation were increased, whereas the mRNA levels of genes related to lipid catabolism were decreased Table , Fig. 7.Given that the stress germination was conducted on pleated filter paper moistened with 60 ml of a NaCl/mannitol solution instead of 30 ml of deionized water, the observed gene expression changes might be related to hypoxia generated by an excess of liquid functioning as a diffusion barrier for oxygen. Indeed, some of the observed mRNA alterations point out changes characteristic for oxygen deprivation. It is known that under anaerobic conditions, the rate of glucose metabolism increases accompanied by enhanced fermentation. Similarly, in our study an increased expression of several glycolytic genes and a slight enhancement in the amount of alcohol dehydrogenase transcripts (1.5-fold) were observed. In germinating but still heterotrophic seeds the growing demand for glucose can only be satisfied by an enhanced degradation of seed reserves. In hypoxic conditions starch degradation might be favoured compared to the mobilization of lipids because the fatty acid β-oxidation pathway requires molecular oxygen as electron acceptor.versus germination on pleated paper moistened with a 100 mM NaCl/200 mM mannitol solution) and/or the different genetic background of the analysed sugar beet hybrids. Apparently, the reaction of sugar beet to stress depends remarkably on genotype, and different hybrids may use distinct strategies to overcome stress injuries.Alterations in expression of genes involved in starch and lipid catabolism during sugar beet germination under stress conditions have been reported previously. De los Reyes et al.  examinedMany genes respond to abiotic stresses at the transcriptional level, and these genes might be classified into two major groups. One group encodes products that directly protect plant cell against stresses (stress adaptation), whereas products of the other group regulate gene expression and signal transduction in stress responses (stress regulation) . Genes bThe majority of the transcriptional changes are in agreement with data published previously. Thus, the increased amount of transcripts encoding ROS detoxifying enzymes have been widely described in the literature as being important for stress tolerance ,35. LEA A. thaliana under stress caused by salicylic acid. These data are also in agreement with an existence of a small developmental window during which the seed can still recruit late maturation programs [Accumulation of LEA, seed maturation and oleosin transcripts proposes that a stress germination results in an increased transcription of genes that are normally accumulated during seed maturation and are under ABA control. Since ABA is a potent inhibitor of seed germination it would also account for the delay in sugar beet germination observed under multistress conditions. Similar results have been reported by Rajjou et al.  who deteprograms ,57.The enhanced expression of transporter genes in response to osmotic stress has been found for different plant species and reflects a necessary readjustment of cell water balance . AccumulAt least three putative TFs belonging to AP2-EREBP, MYB and CCCH-type zinc finger families of TFs show enhanced gene expression during germination of sugar beet seeds under multistress conditions  being activated later. Interestingly, the subsequent assay of germination under multistress conditions revealed significant alterations in the expression of genes involved in seed storage reserve mobilization, and suggested that in the analysed hybrid stress caused a transcriptionally regulated metabolic shift resulting in a favoured degradation of starch and reduced utilization of lipids. The approach also succeeded in the identification of other stress-responsive genes including those encoding TFs, protein kinases, LEA proteins, ROS detoxifying and GlyBet synthesizing enzymes, which have potential to contribute towards improvement of seed and seedling stress tolerance and, subsequently, field emergence potential in sugar beet.The new developed EST collection from sugar beet seeds and seedlings overlaps substantially with a recently published set of sugar beet seed-specific proteins and provides a useful resource to investigate the molecular bases of seed development, maturation and germination. The conducted gene expression profiling experiments enabled us to compare the transcriptional changes during sugar beet germination with the corresponding processes in the model species EP conducted RNA isolation, cDNA library development from germinating seeds, macroarray hybridizations, data analysis and drafted the manuscript. AW developed cDNA library from dry seeds, made preliminary gene expression experiments and provided several protocols. JM made germination assays, calculated germination parameters and prepared seed samples. AM provided bioinformatics support including contig assembling and consensus sequence annotation. UF and PW coordinated the project. All authors read and approved the final manuscript.Table A1: List of genes expressed differentially during germination under standard conditions.Click here for fileTable A2: List of genes expressed abundantly in dry and germinating sugar beet seeds.Click here for fileTable A3: List of genes showing significantly different transcript levels during germination under stress conditions.Click here for file


By means of immunohistochemistry, we previously demonstrated that the MAP kinase pathway is virtually inactive in Merkel cell carcinoma (MCC). Similarly to MCC Applying an siRNA approach RKIP expression was knocked down in UISO cells and a possible influence on MAP kinase pathway activity was assessed by Western blot analysis using phospho-specific antibodies. Moreover, a possible effect of RKIP knock down in UISO cells on proliferation as well as chemosensitivity to cisplatin were examined applying the MTS assay.Surprisingly the absence of phosphorylation of the MAP kinases ERK1 and ERK 2 even following growth factor stimulation was not affected by the RKIP knock down indicating that RKIP is not essential for blocking the MAP kinase pathway in the MCC cell line UISO. Moreover, proliferation as well as chemosensitivity towards cisplatin were not altered upon knock down of RKIP. Merkel cell carcinoma (MCC) is a highly aggressive skin cancer of neuroendocrine origin . The tumin situ the absence of MAP kinase activation is accompanied by high level expression of RKIP [A signal transduction pathway that is activated in many tumor entities is the MAP kinase pathway, a major regulator of cell growth, differentiation and survival. It comprises the three consecutive kinases Raf, MEK (MAP kinase kinase) and ERK (p42/p44 MAP kinases) which are controlled by the small G-Protein Ras . RecentlThe MCC cell line UISO, which has been established from a primary Merkel cell carcinoma of a 46 year old woman , was gro5 UISO cells were seeded in 24 well plates the day prior to transfection. Two different transfection reagents were used. The siRNA were transfected at 80 nM  or 10 nM  concentration according to the manufacturer's protocols.An Alexa Fluor 488 labelled scrambled siRNA as negative control (AAT TCT CCG AAC GTG TCA CGT) as well as an siRNA targeting RKIP (AAG GTG GCG TCC TTC CGT AAA) [® AQueous One Solution Cell Proliferation assay, Promega Corporation, Madison, WI, USA). To this end, 10 μl of CellTiter 96® AQueous One Solution Reagent containing a tetrazolium compound (MTS) were added to each well and the cells were incubated for approximately 90 min at 37°C. Metabolically active, viable cells convert MTS into a colored formazan product that was measured in a spectrophotometric microplate reader  at 493 nm.24 hours following siRNA transfection the cells were harvested with Trypsin/EDTA and were seeded with an equivalent of 2000 cells/well corresponding to the initial cell number in 96 well plates. For measurement of chemosensitivity cisplatin was added at this time point. To extend the period of high siRNA levels in the cells a second transfection using HiPerFect (Quiagen) was performed on day 3 following the first transfection. Another 3 days later proliferation and cell viability were assessed by the MTS assay . The antibodies used were the monoclonal antibodies α-Phospho-p44/42 MAP kinase (Thr202/Tyr204) , α-β-tubulin  and the polyclonal antibody α-RKIP .in situ, in particular the complete lack of ERK 1/2 phosphorylation associated with high expression of RKIP [The MCC cell line UISO displays the same MAP kinase pathway phenotype as it is observed for Merkel cell carcinoma  of RKIP . Remarka of RKIP ,8. ConseRas genes are the most frequently mutated oncogenes detected in human cancer [The importance of the Ras/Raf/MEK/ERK signalling pathway for carcinogenesis is reflected by the facts that n cancer ,10 and tn cancer . In MCC,n cancer ,12,13.in situ as well as in the MCC cell line UISO MAP kinase pathway activity as measured by ERK phosphorylation is not detectable at all . Since activation of the MAP kinase pathway in UISO cells induces apoptosis [In contrast, in MCC poptosis , demonstpoptosis . RKIP ispoptosis . Therefopoptosis . Consequpoptosis . Togethepoptosis , these fRKIP has been reported to be involved in the regulation of proliferation and apoptosis -18. HoweIn summary our data suggest that RKIP expression in the cell line UISO is not critical for inactivity of the MAPK pathway, proliferation properties and sensitivity of these MCC cells towards apoptosis inducing agents.


Promyelocytic Leukemia (PML) protein can interact with a multitude of cellular factors and has been implicated in the regulation of various processes, including protein sequestration, cell cycle regulation and DNA damage responses. Previous studies reported that misfolded proteins or proteins containing polyglutamine tracts form aggregates with PML, chaperones, and components of the proteasome, supporting a role for PML in misfolded protein degradation.In the current study, we have identified a reactive oxygen species (ROS) dependent aggregation of PML, small ubiquitin-like modifier 1 (SUMO-1), heat shock protein 70 (HSP70) and 20S proteasomes in human cell lines that have been transiently transfected with vectors expressing the puromycin resistance gene, puromycin n-acetyl transferase (pac). Immunofluorescent studies demonstrated that PML, SUMO-1, HSP70 and 20S proteasomes aggregated to form nuclear inclusions in multiple cell lines transfected with vectors expressing puromycin (puro) resistance in regions distinct from nucleoli. This effect does not occur in cells transfected with identical vectors expressing other antibiotic resistance genes or with vectors from which the pac sequence has been deleted. Furthermore, ROS scavengers were shown to ablate the effect of puro vectors on protein aggregation in transfected cells demonstrating a dependency of this effect on the redox state of transfected cells.Taken together we propose that puromycin vectors may elicit an unexpected misfolded protein response, associated with the formation of nuclear aggresome like structures in human cell lines. This effect has broad implications for cellular behavior and experimental design. The human promyelocytic leukemia PML gene was initially identified as the fusion partner of the retinoic acid receptor α gene in the T15;17) chromosomal translocation in acute promyelocytic leukemia 5;17 chro. Thus faNewly synthesized proteins must fold correctly to become functional molecules. Many events including errors in transcription, mRNA processing and translation lead to failure in achieving correct protein conformation . FurtherIn the current study we have identified a reactive oxygen species (ROS) dependent aggregation of proteins (including PML), proteasomes and chaperones into nuclear inclusions in cells lines that have been transfected with vectors expressing the puromycin resistance gene. This response is strikingly similar to previously described misfolded protein responses and has broad implications for cellular behavior and experimental design.During the course of our studies we observed an apparent relocalization of PML nuclear bodies (NBs) in cell lines transfected with shRNA vectors targeting specific E3 ligases (MDM2 and CHIP). Surprisingly, we also observed this effect in cells transfected with control vectors expressing non targeting shRNA sequences and those without DNA inserts (empty vectors). To examine this further, U2OS cells were transiently transfected with empty vectors using Fugene transfection reagent and immunostained for PML 24 h and 48 h after transfection. PML localized to discrete foci (11 ± 5 foci per cell) dispersed throughout the nuclei of non transfected and mock transfected cells Fig . These fWe next aimed to quantify the percentage of transfected cells in which PML aggregation occurred. To this end, pEGFP Neo empty vector was cotransfected with pBABE Puro into U2OS cells, and the cells immunostained for PML 48 h after transfection. Transfected cells were identified by EGFP expression. PML localization was not altered in cells transfected with pEGFP Neo alone Fig. . In contPML aggregates similar to that observed in Figs Next, we wished to address why this effect was specific to certain vectors but not others. The empty vectors that caused PML, HSP70, SUMO-1, and 20S proteasome aggregation  are retroviral vectors that express the puromycin resistance gene. The vectors that did not cause this effect  are non-retroviral vectors that express the neomycin resistance gene. We questioned whether this effect could be related to either retroviral genetic sequences or expression of the puromycin resistance gene, puromycin n-acetyl transferase (pac). In order to investigate this, we used multiple pBABE vectors which differed only in the selection marker expressed . U2OS cells were transfected with these pBABE vectors and immunostained 48 h later for HSP70 and PML. While pBABE Puro caused PML and HSP70 coaggregation in transfected cells  we did not detect any aggregation (n = 3 separate experiments). Cell lines are often maintained in culture over long periods and are subjected to frequent passaging and multiple freeze-thaw cycles. Notably, ROS levels have been reported to increase in cells maintained in culture for prolonged periods, and multiple studies have linked elevated ROS levels with protein aggregation and misfolding ,18,19. WFinally, we examined whether transfected cells maintain protein aggregation after undergoing puromycin selection. For this, U2OS cells were transfected with pBABE puro or left non-transfected. Four days later, cells were placed in media containing puromycin (0–15 μg/mL). Non-transfected cells were killed at all doses of puromcyin 2 days after treatment. Transfected cells were grown for two weeks in puromycin in order to select for stable, puro-resistant cells. Stable cells were seeded on coverslips in the presence or absence of puromycin and stained 3 days later for PML and HSP70. Analysis of transfected cells grown in the absence of puromycin demonstrated these cells do not display PML and/or HSP70 aggregation Fig . SimilarIn the current study, we have described a ROS sensitive aggregation of PML, HSP70, SUMO-1 and 20S proteasomes into nuclear inclusions in cells transfected with vectors expressing the selection marker gene, puromycin n-acetyl transferase (pac). Moreover, aggregation of HSP70 and proteasomes was also observed in the cytoplasm of transfected cells. Cytoplasmic inclusions containing HSP70 and proteasomes have been previously described and are associated with aggregates of misfolded proteins . MisfoldOur results demonstrate that protein aggregation in cells transfected with empty puro vectors depends on the presence of the puromycin-resistance (pac) gene. It is presently unclear why this effect is specific to pac and why similar aggregation does not occur upon transfection with vectors expressing other selection markers. One possible reason could be that puromycin n-acetyl transferase protein (PAC) is more aggregation prone than other antibiotic resistance proteins. We propose that PAC protein may colocalize with both nuclear and cytosolic aggregates and that the presence of proteasomes and chaperones may represent the cells response to aggregated PAC protein. However, in the absence of commercially available antibodies specific to PAC, we were unable to confirm this hypothesis. The composition and the primary structure of a polypeptide determine to a large extent its propensity to aggregate. We have used two recently developed bioinformatics software programs as a preliminary approach to compare the protein sequences of neomycin, puromycin and hygromycin resistance gene products for aggregation propensity (data not shown). The software Aggrescan predicts aggregation hotspots in protein sequences . Using t2O2, the major cellular source of ROS, to water and oxygen. H2O2 readily passes through cell membranes allowing extracellular catalase (in cell medium) to drain H2O2 from cells thereby protecting against extracellular and intracellular H2O2 [2O2 and other ROS [During the course of this study, we observed the effect of puro vectors on protein aggregation in multiple cell types but not in low passage/newer cells (<10 passages). Many studies have described the generation of reactive oxygen species (ROS) in cultured cells and higher levels of ROS are associated with protein aggregation ,18,19,33lar H2O2 . Both ROlar H2O2 . It is plar H2O2 ,36. Manyther ROS -39. Cellther ROS ,41. Furtther ROS . We suggThis study has broad implications for cell experimentation. Many studies are based on malignant cell lines that are often maintained in culture and/or frozen over long periods. Cell lines are generally considered to be more robust than primary cells which often undergo stress during cell culture; however, our study shows that cell lines can also be susceptible to oxidative stress during prolonged culture. Our study clearly shows an alteration in cell phenotype/behavior in a ROS dependent manner. The aggregation of critical multifunctional proteins and complexes such as HSP70, PML and proteasomes into nuclear inclusions clearly may affect multiple cellular processes and may lead to inaccurate conclusions in experimental studies. Cell transfection itself is a critical component of cell biology studies. Typically DNA vectors are used without consideration for the effects of resistance proteins in transfected cells. It is surprising that the puromycin resistance gene can cause these effects in cells however it is worth noting that this gene expresses a foreign protein in mammalian cells. We suggest caution when using puromycin-based DNA vectors, as these vectors may elicit an unexpected misfolded-protein response that includes PML, HSP70, SUMO-1, and proteasome coaggregation, and that could alter the outcome or interpretation of experiments.HeLa, MCF-7 and U2OS cells were obtained from ATCC and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 μg/mL). PBABE Puro, pBABE Hygro, pBABE Neo, pBABE GFP, pBABE hcRed and pSUPER plasmids were obtained from Addgene . PLKO.1 Puro plasmid, N-Acetyl Cysteine, Bovine Catalase and puromycin were obtained from Sigma Aldrich . Fugene-6 transfection reagent was obtained from Roche (Nutley NJ).Fugene transfection reagent was equilibrated in serum free DMEM prior to mixing with vector DNA (2 μg). Fugene/DNA was incubated at room temperature prior to being added dropwise to cells. To establish stable cell lines, media was refreshed on transfected cells 4 days later with media containing puromycin . Transfected cells were treated with puromycin, refreshed every 3 days, for > 2 weeks.Immunofluorescent staining was performed as previously described . BrieflyPrimers were designed for flanking regions of the pac sequence in the pBABE Puro plasmid. Deletion of the pac gene was performed using the Quikchange XL mutagenesis kit  exactly as the manufacturer described and the resulting plasmid was renamed pBABE Puro del. Deletion of the pac sequence was confirmed by DNA sequencing of the pBABE Puro del plasmid.2DCFDA  for 30 min at 37°C in dark. Cells were next washed with ice-cold PBS. Fluorescence was quantified by flow cytometry using a BD FACSCanto . Data was analyzed using FlowJo 8.8. Geometric Mean fluorescence was used as average fluorescence value for each population.Cells were grown to 70% confluency in 35 mm dishes. Cells were trypsinized and incubated with 25 μM Carboxy HDM conceived, designed and performed the experimental work for this study and wrote this manuscript. HS performed the ROS detection experiments Figure . CGM par


Human papillomaviruses (HPVs) are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious life cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture. Here, we used chemical biology approaches to reveal that cell cycle progression through mitosis is critical for HPV infection. Using infectious HPV16 particles containing the intact viral genome, G1-synchronized human keratinocytes as hosts, and early viral gene expression as a readout for infection, we learned that the recipient cell must enter M phase (mitosis) for HPV infection to take place. Late M phase inhibitors had no effect on infection, whereas G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented infection. We conclude that host cells need to pass through early prophase for successful onset of transcription of the HPV encapsidated genes. These findings provide one reason why HPVs initially establish infections in the basal compartment of stratified epithelia. Only this compartment of the epithelium contains cells progressing through the cell cycle, and therefore it is only in these cells that HPVs can establish their infection. By defining a major condition for cell susceptibility to HPV infection, these results also have potentially important implications for HPV control. Human papillomaviruses (HPV), which comprise more than 100 genotypes, are the most prevalent sexually transmitted infection and are associated with multiple human cancers including all cervical cancers, many other anogenital cancers, and 25% of head and neck cancers. The HPV life cycle is closely linked to epithelial differentiation of skin keratinocytes, with initial infection occurring only in the undifferentiated proliferating basal compartment of the epithelium and progeny virus production only in the terminally differentiated suprabasal compartment. So far, little is known about how host cells restrict the HPV life cycle to specific stages of skin cell development. Here, by identifying small molecule inhibitors of HPV infection, we discovered that cell cycle progression through mitosis is critical for the establishment of HPV infection. In addition, our further chemical genetic dissection of this process showed that early steps of mitosis are required for HPV infection and early gene expression. Our findings provide one reason why HPV only infects undifferentiated proliferating cells and provide new leads for the development of preventive and therapeutic strategies against HPV infection. Papillomaviruses (HPV) are small, non-enveloped, double stranded DNA viruses that infect the cutaneous and/or mucosal epithelium in many vertebrates The HPV life cycle is tightly linked to the differentiation of its host epithelial cells The tight link between keratinocyte differentiation and the HPV life cycle has made large scale production of mature infectious HPV particles difficult, greatly restricting studies of the mechanisms of natural HPV infection To define the pathways and mechanisms involved in these early steps of HPV infection, we tested approximately 5,000 bioactive compounds with known mechanisms of action for effects on the entry of HPV capsids containing reporter genes or the full HPV genome, and identified a subset of cell cycle inhibitors that completely blocked wild type HPV infection. Our further studies showed that cell cycle progression through early stages of mitosis is critical for successful HPV infection. These findings reveal new insights into the mechanism by which HPV infects cells and provide one reason why HPV infects only undifferentiated, proliferating cells. These results also provide new leads for developing preventive and therapeutic strategies against HPV infection.hpvSEAP). High activity effectors identified in this assay were retested in a second infectivity assay using HPV pseudovirions containing renilla luciferase driven by the natural HPV16 promoter (hpv16wpA-RL) to exclude false positive compounds that may selectively affect the SV40 promoter or alkaline phosphatase (data not shown). This screening assay was performed in human kidney 293T cells owing to the high signal to noise ration, and resulting high z scores obtained in this cell line. Secondary screens performed in HaCaT cells, an immortalized line of human keratinocytes, which are natural host cells for HPV infection, confirmed the validity of the hits, as indicated below.To identify mechanistic pathways and informative modulators of HPV infection, we tested nearly 5,000 compounds in known bioactive molecule libraries from Prestwick  and LOPAC , using HPV pseudovirions containing an SV40 promoter-driven secreted alkaline phosphatase (SEAP) reporter gene  is critical for early HPV infection, we synchronized HaCaT cells with aphidicolin or etoposide and then tightly controlled subsequent cell cycle progression. In the first set of experiments  breakdown, which is triggered by a signal cascade that involves polo I like kinase 1 (plk1) and cyclin B1-associated CDK1 Another notable cellular event in early mitosis is the reorganization of cytoplasmic microtubules to support mitotic spindle formation, chromosome segregation, and cell division In addition, restructuring of PML oncogenic bodies (PODs) and chromatin in early mitosis could be necessary for establishing HPV infection in the nucleus. PODs, also known as ND10, are subnuclear bodies implicated in multiple cellular functions including transcription, DNA repair, viral defense, stress, cell cycle regulation, proteolysis and apoptosis Many genes such as polo-like kinases and aurora kinases are exclusively expressed in early mitosis to certify completion of DNA replication, overcome mitotic checkpoint, and initiate mitosis in vivo models such as mouse and non-human primate. In addition, real-time particle imaging studies could be employed to identify what specific steps in infection are blocked upon cell cycle arrest; however, the ability to interpret accurately such imaging studies rests on the ability to distinguish particles leading to infectious events from the vast majority of particles that give rise to abortive or non-infections events. To date no imaging method exists able to make such a distinction at the individual particle level.HPV is thought to be able to infect the proliferating basal layer of stratified epithelium only when wounds allow HPV to penetrate the physical barrier of upper skin layers. Wounding also can provide HPV particles access to laminin 5 or heparin sulfate moieties, both components of the extra cellular matrix (ECM) component of the epithelial basement membrane for which HPV particles have high affinity, thereby localizing the virus particles to basal surface of epithelial cells it needs to infect in vivo mouse model of HPV infection in mouse reproductive organs The recent development of HPV vaccines offers an extremely important avenue for control of HPV infections and their associated cancers pEF399, containing the complete W12E HPV16 genome Human embryonic kidney cell line 293T from ATCC, and its enhanced SV40 T antigen-expressing daughter cell line 293TT HPV virions and pseudovirions were prepared as described previously Known Bioactive Library (KBA01) consists of 3 commercially available collections totaling 4,160 compounds. KBA01 consists of 880 high purity compounds of known safety and bioavailability in humans of which over 85% are marketed drugs from Prestwick Chemical. The Prestwick compounds cover several therapeutic areas including neuropsychiatry, cardiology, immunology, inflammation, analgesia, etc. Also included in the KBA01 library is 2000 diverse FDA approved drugs and natural products from Spectrum Chemical Collection From Microscource Discovery Systems, Inc. and 1280 compounds from the LOPAC of Sigma representing marketed drugs, failed development candidates and “gold standards” that have well-characterized activities. These compounds are the results of lead optimization efforts and have been rationally designed by structure-activity relationship studies. The NCI03 library consists of 235 natural products that were selected from the NCI open repository on the basis of structural diversity and availability of compound. Etoposide, Aphidicolin, Monastrol, 5-Fluorouracil, CGP-74514A were purchased from Sigma.hpvSEAP at 50 vge /cell for 48 hrs. Culture supernatant 2.5 µl was used for virus infectivity assay using a Phospha-Light a chemiluminescent alkaline phosphatase assay (Applied Biosystems) and cell lysate was used for cell viability assay using CellTiter-Glo Luminescent cell viability assay (Promega). Biomek FX (Beckman Coulter) was used for automated liquid handling and a Victor 3-V plate reader (Perkin Elmer) was used for measuring luminescence. Screening was performed in the Small Molecule Screening Facility at the University of Wisconsin Comprehensive Cancer Center.293TT cells in 384-well plates were treated with 3.3 µM of library compounds for 4 hrs and inoculated with hpvSEAP) was measured using a Phospha-Light a chemiluminescent alkaline phosphatase assay (Applied Biosystems) and the activity of pHPV16wpA-RL-encapsidating pseudovirions (hpv16wpA-RL) was assayed using Renilla Luciferase Assay System (Promega). Infectivity of wild type HPV16 virions was examined by quantitative reverse transcriptase PCR (qRT-PCR) by amplifying viral mRNA signals from HPV16-treated 293TT cells. 293TT cells were inoculated with 50–100 vge/cell of packaged virus and incubated for 48 h at 37°C. Total RNA was isolated using the RNeasy total RNA purification kit (Qiagen), treated with RQ DNaseI (Promega) to remove possible DNA contaminants, purified again on RNeasy columns to remove DNaseI, and quantified by spectrophotometer. cDNA was synthesized from 20 µg of total RNA with oligo (dT) using a SuperScript cDNA synthesis kit (Invitrogen), and qPCR was performed with QuantiTect SYBR Green PCR Kit (Qiagen). Oligonucleotide primers  incorporation method. Briefly, HaCaT cells were harvest, homogenized, fixed with cold 70% ethanol for 30 min at −20°C, and incubated with PI staining solution  for 30 min at room temperature. Stained cells were filtered through 35 µm-pore nylon mesh cell filtering caps  and analyzed by flow cytometry using Becton Dickinson FACSCalibur (488 nm laser excitation).Figure S1Selected cell cycle inhibitors abrogate HPV infection. HPV16 early gene (A) and β-actin expression (B) was measured by quantitative RT-PCR. 293T cells were inoculated with wild type HPV16 after 4 hr pre-treatment with 3 µM of each compound or with 1∶100 dilution of neutralizing antibody (H16.7E). Total RNA was extracted after 48 hrs, and expression levels of HPV16 RNA transcripts were measured by qRT-PCR using E7, E2, and E5 sequence specific primers . The exp(0.12 MB PDF)Click here for additional data file.Figure S2Cell cycle progression through M phase is critical for HPV infection. 293T cells were synchronized with aphidicolin  and etoposide (set 3) 4 hrs before wild type HPV16 inoculation. Cell cycle progression was released in set 1 and kept blocked in sets 2 and 3 using aphidicolin and etoposide, respectively. In set 4, cell cycle progressed through S phase but arrested at G2 phase by switching the compounds in medium from aphidicolin to etoposide. In set 5, cell cycle was released for 20 hrs to progress through M phase and arrest at G2 phase by etoposide addition. Wild type HPV16 gene expression was measured from total RNA extracts as indicated in (0.06 MB PDF)Click here for additional data file.Figure S3Quantitative PCR primers of HPV16 early genes. (A) Oligonucleotide primers were designed using the Primer3 primer design program. (B) The HPV genomic position of each primer is indicated by an arrow.(0.01 MB PDF)Click here for additional data file.