Significant Differences In Transcript Levels Research Articles

Analysis of gene expression related to polyamine concentration and dimorphism induced in ornithine decarboxylase (odc) and spermidine synthase (spd) Ustilago maydis mutants

Polyamines are ubiquitous small organic cations, and their roles as regulators of several cellular processes are widely recognized. They are implicated in the key stages of the fungal life cycle. Ustilago maydis is a phytopathogenic fungus, the causal agent of common smut of maize and a model system to understand dimorphism and virulence. U. maydis grows in yeast form at pH 7 and it can develop its mycelial form in vitro at pH 3. Δodc mutants that are unable to synthesize polyamines, grow as yeast at pH 3 with a low putrescine concentration, and to complete its dimorphic transition high putrescine concentration is require. Δspd mutants require spermidine to grow and cannot form mycelium at pH 3. In this work, the increased expression of the mating genes, mfa1 and mfa2, on Δodc mutants, was related to high putrescine concentration. Global gene expression analysis comparisons of Δodc and Δspd U. maydis mutants indicated that 2,959 genes were differentially expressed in the presence of exogenous putrescine at pH 7 and 475 genes at pH 3. While, in Δspd mutant, the expression of 1,426 genes was affected by exogenous spermine concentration at pH 7 and 11 genes at pH 3. Additionally, we identified 28 transcriptional modules with correlated expression during seven tested conditions: mutant genotype, morphology (yeast, and mycelium), pH, and putrescine or spermidine concentration. Furthermore, significant differences in transcript levels were noted for genes in modules relating to pH and genotype genes involved in ribosome biogenesis, mitochondrial oxidative phosphorylation, N-glycan synthesis, and Glycosylphosphatidylinositol (GPI)-anchor. In summary, our results offer a valuable tool for the identification of potential factors involved in phenomena related to polyamines and dimorphism.

Biomolecular evaluation of three contrasting rice cultivars (Oryza sativa L.) in salt stress response at seedling stage.

Salt contamination of soils due to climate change faces a severe environmental issue that affects crop production today. However, the response mechanism in plants to salt stress is not fully understood. The present study investigated molecular and biochemical changes under salt stress in rice seedlings of three rice cultivars, i.e., AGPPS114 (salt-tolerant), OM6967 (moderately tolerance), VD20 (salt-sensitive). Increasing salt concentration leads to a reduction in shoot/root length but different levels among the cultivars. In contrast, reactive oxygen species (ROS) accumulation and lipid peroxidation increased progressively with increasing salt concentration and time course treatment. However, at 250 ?M of NaCl, these parameters were more adversely affected in VD20 than AGPPS114 and OM6967. Using ICP showed that Na+ accumulation in rice root increased gradually with increasing NaCl concentrations in all cultivars under salt treatment but was low in salt-sensitive cultivar VD20 compared to other cultivars. Antioxidant enzyme activity analysis indicated catalase (CAT) and superoxide dismutase (SOD) were induced during salt treatment in all cultivars. The results also showed greater proline and glycine betaine accumulation in the AGPPS114 than OM6976 and VD20. qPCR indicated a significant difference in transcript levels of the Na+-transporter gene OsSOS1, OsNHX1 and OsHKT1s in AGPPS114 and OM6967 cultivars compared to VD20 cultivar. In summary, the active regulation of genes related to Na+ transport at the transcription level and with high glycine betaine and proline accumulation levels may be involved in salt tolerance mechanisms and thus might be useful for selecting tolerant plants.

Infection by Diverse HIV-1 Subtypes Leads to Different Elevations in HERV-K Transcriptional Levels in Human T Cell Lines.

Human endogenous retroviruses (HERVs) make up ~8% of the human genome, and for millions of years, they have been subject to strict biological regulation. Many HERVs do not participate in normal physiological activities in the body. However, in some pathological conditions, they can be abnormally activated. For example, HIV infection can cause abnormal activation of HERVs, and under different infection conditions, HERV expression may be different. We observed significant differences in HERV-K transcription levels among HIV-1 subtype-infected individuals. The transcriptional levels in the HERV-K gag region were significantly increased in HIV-1 B subtype-infected patients, while the transcriptional levels in the HERV-K pol region were significantly increased in CRF01_AE and CRF07_BC subtype-infected patients. In vitro, the transcriptional levels of HEVR-K were increased 5-fold and 15-fold in MT2 cells transfected with two different HIV-1 strains (B and CRF01_AE, respectively). However, there was no significant difference in transcriptional levels among regions of HERV-K. When MT2 cells were infected with different subtypes of HIV-1 Tat proteins (B, CRF01_AE), which is constructed by lentiviruses, and the transcription levels of HERV-K were increased 4-fold and 2-fold, respectively. Thus, different subtypes of HIV-1 have different effects on HERV-K transcription levels, which may be caused by many factors, not only Tat protein.

Transcriptome and methylome analysis reveals effects of ripening on and off the vine on flavor quality of tomato fruit

Abstract Current commercial harvest procedures for tomatoes usually involve harvesting the fruit at an early ripening stage with high firmness suitable for storage and transportation followed by ethylene treatment to accelerate fruit ripening. However, harvesting at an immature stage results in reduced flavor quality. Here, we provide major insights into the effect of ethylene-induced ripening of immature fruit on consumer liking, flavor-related compounds, transcriptomes and DNA methylation. Reduced flavor quality of off-vine ripened fruit treated with ethylene is associated with significantly higher content of malic acid, a lower sugar/acid ratio, as well as changes in major volatiles. Altered levels of flavor-related compounds are associated with significant changes in expression of genes encoding key biosynthetic enzymes for malic acid and specific volatiles. Transcripts involved in ethylene formation and signaling are induced by exogenous ethylene treatment. Meanwhile, significant difference in transcript levels of genes related to auxin response are observed between fruit ripened on and off the vine. Differences in transcript levels of volatile synthesis-related genes, including LOXC, BCAT1 and AADC1A, are accompanied by major changes in DNA methylation status of their transcriptional promoter regions. Our analysis provides molecular insights into tomato fruit flavor loss caused by artificial ripening using postharvest ethylene treatment.

Environmentally relevant concentrations of carbamazepine induce liver histopathological changes and a gender-specific response in hepatic proteome of Chinese rare minnows (Gobiocypris rarus)

To assess hepatotoxicity and to determine the underlying mechanisms of carbamazepine (CBZ) toxicity in fish, histopathology and the liver proteome were examined after Chinese rare minnow (Gobiocypris rarus) were exposed to 1, 10, and 100 μg/L CBZ for 28 days. Histopathological changes included disruption of spatial structure, pyknotic nuclei, cellular vacuolization and deformation of cell nuclei, in addition to marked swelling of hepatocytes in all treatment groups. Protein analysis revealed that there were gender-specific responses in rare minnow following exposure, and there were 47 proteins in females and 22 proteins in males identified as differentially abundant following CBZ treatments. Pathway analysis revealed that cellular processes affected by CBZ included apoptosis, cell differentiation, cell proliferation, and the respiratory chain, indicating impaired energy homeostasis. Noteworthy was that 15 proteins identified as different in abundance were associated with carcinogenicity. Relative mRNA levels for select transcripts were consistent with the changes of proteins N-myc downstream regulated gene (NDRG), Tropomyosin 2-Beta (TPM2) and annexin A4 (ANXA4). Protein pyruvate kinase, liver and RBC (PKLR) were increased at 1 and 100 μg/L CBZ without significant difference in transcript levels. These findings characterize molecular responses and histological changes in the liver that generate new insights into CBZ hepatotoxicity in Chinese rare minnow.

Differential gene expression pattern in biopsies with renal allograft pyelonephritis and allograft rejection

Differentiating acute pyelonephritis (APN) from acute rejection (AR) in renal allograft biopsies can sometimes be difficult because of overlapping clinical and histologic features, lack of positive urine cultures,and variable response to antibiotics. We wanted to study differential gene expression between AR and APN using biopsy tissue. Thirty-three biopsies were analyzed using NanoString multiplex platform and PCR (6 transplant baseline biopsies, 8 AR, 15 APN [8 culture positive, 7 culture negative], and 4 native pyelonephritis [NP]). Additional 22 biopsies were tested by PCR to validate the results. CXCL9, CXCL10, CXCL11, and IDO1 were the top differentially expressed genes, upregulated in AR. Lactoferrin (LTF) and CXCL1 were higher in APN and NP. No statistically significant difference in transcript levels was seen between culture-positive and culture-negative APN biopsies. Comparing the overall mRNA signature using Ingenuity pathway analysis, interferon-gamma emerged as the dominant upstream regulator in AR and allograft APN, but not in NP (which clustered separately). Our study suggests that chemokine pathways in graft APN may differ from NP and in fact resemble AR, due to a component of alloreactivity, resulting in variable response to antibiotic treatment. Therefore, cautious addition of steroids might help in resistant cases of graft APN.

Comparative Analysis of Extremely Thermophilic Caldicellulosiruptor Species Reveals Common and Unique Cellular Strategies for Plant Biomass Utilization.

Microbiological, genomic and transcriptomic analyses were used to examine three species from the bacterial genus Caldicellulosiruptor with respect to their capacity to convert the carbohydrate content of lignocellulosic biomass at 70°C to simple sugars, acetate, lactate, CO2, and H2. Caldicellulosiruptor bescii, C. kronotskyensis, and C. saccharolyticus solubilized 38%, 36%, and 29% (by weight) of unpretreated switchgrass (Panicum virgatum) (5 g/liter), respectively, which was about half of the amount of crystalline cellulose (Avicel; 5 g/liter) that was solubilized under the same conditions. The lower yields with C. saccharolyticus, not appreciably greater than the thermal control for switchgrass, were unexpected, given that its genome encodes the same glycoside hydrolase 9 (GH9)-GH48 multidomain cellulase (CelA) found in the other two species. However, the genome of C. saccharolyticus lacks two other cellulases with GH48 domains, which could be responsible for its lower levels of solubilization. Transcriptomes for growth of each species comparing cellulose to switchgrass showed that many carbohydrate ABC transporters and multidomain extracellular glycoside hydrolases were differentially regulated, reflecting the heterogeneity of lignocellulose. However, significant differences in transcription levels for conserved genes among the three species were noted, indicating unexpectedly diverse regulatory strategies for deconstruction for these closely related bacteria. Genes encoding the Che-type chemotaxis system and flagellum biosynthesis were upregulated in C. kronotskyensis and C. bescii during growth on cellulose, implicating motility in substrate utilization. The results here show that capacity for plant biomass deconstruction varies across Caldicellulosiruptor species and depends in a complex way on GH genome inventory, substrate composition, and gene regulation.

Identification and fine-mapping of a new resistance gene, Xa40, conferring resistance to bacterial blight races in rice (Oryza sativa L.).

A new bacterial blight resistance gene has been identified through fine-mapping, which confers high levels of resistance to all Korean Xanthomonas oryzae pv. oryzae (Xoo) races, including the new Xoo race K3a. Rice bacterial leaf blight (BB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo) is a serious constraint to rice production in Asia and Africa. The japonica advanced backcross breeding lines derived from the indica line IR65482-7-216-1-2 in the background of cultivar Junam are resistant to all Korean BB races, including K3a. To identify the gene(s) involved in resistance to Korean Xoo races, the association of genotypic and phenotypic variations was examined in two F2 populations derived from the crosses between 11325 (IR83261-3-7-23-6-2-1-1-2-1-2)/Anmi and 11325/Ilpum. The segregation ratios of F2 individuals from the crosses of 11325/Anmi and 11325/Ilpum were 578 resistant:209 susceptible and 555 resistant:241 susceptible, respectively, which is consistent with the expected allelic frequency of a 3:1 ratio. Genetic analysis using graphical mapping indicated that resistance (R) was controlled by a new resistance gene linked with the flanking markers RM27320 and ID55.WA18-5 within an approximately 80-kb region between 28.14 and 28.22 Mbp on chromosome 11. The eight candidate genes functionally predicted were included in the target region. Examination of the candidate genes by RT-PCR analysis only corroborated with the significant difference in transcript levels of the WAK3 gene in the presence or absence of pathogen infection. Allelism tests performed with other known BB R-genes revealed that the allele was distinct from others having a similar chromosomal location.

Evaluation of the estrogenic and oxidative stress effects of the UV filter 3-benzophenone in zebrafish (Danio rerio) eleuthero-embryos

Personal care products have been detected in superficial waters, representing an environmental risk to the biota. Some studies indicated that 3-benzophenone (3BP) alters hormones, inducing vitellogenesis and having adverse effects on fish reproduction. Other studies have reported generation of free radicals and changes in antioxidant enzymes. Therefore, the aim of the present study was to test acute exposure to 3BP at concentrations within and beyond that found environmentally to provide important toxicological information regarding this chemical. We evaluated the effect of 3BP on vitellogenin 1 (VTG1) gene expression and the transcription of the enzymes catalase (CAT), superoxide dismutase (SOD) or glutathione peroxidase (GPx), which are involved in cellular redox balance. Zebrafish eluthero-embryos (168hpf) were exposed to 1,10, 100, 1000µg/L 3BP, in addition to a negative control and a 0.1% ethanol control for 48h. The results of our study indicated a positive significant correlation between exposure concentrations and VTG1 expression (r=0.986, p=0.0028) but only 1000µg/L 3BP produced a significant increase from control. Acute exposure showed no significant differences in transcription levels of CAT, SOD or GPx at the tested conditions. Nevertheless, a trend toward increase in GPx expression was observed as a positive significant correlation (r=0.928, p=0.017) was noted.

Bagging Treatment Influences Production of C6 Aldehydes and Biosynthesis-Related Gene Expression in Peach Fruit Skin

Bagging is a useful method to improve fruit quality by altering its exposure to light, whereas its effect on fruit volatiles production is inconsistent, and the genes responsible for the observed changes remain unknown. In the present study, single-layer yellow paper bags were used to study the effects of bagging treatment on the formation of C6 aldehydes in peach fruit (Prunus persica L. Batsch, cv. Yulu) over two succeeding seasons. Higher concentrations of n-hexanal and (E)-2-hexenal, which are characteristic aroma volatiles of peach fruit, were induced by bagging treatment. After bagging treatment, peach fruit had significantly higher LOX and HPL enzyme activities, accompanying increased contents of C6 aldehydes. The gene expression data obtained through real-time PCR showed that no consistent significant differences in transcript levels of LOX genes were observed over the two seasons, but significantly up-regulated expression was found for PpHPL1 after bagging treatment In addition, bagging-treated fruit produced more (E)-2-hexenal and had higher expression levels of PpHPL1 during postharvest ripening at room temperature. The regulatory role of the LOX-HPL pathway on the biosynthesis of n-hexanal and (E)-2-hexenal in response to bagging treatment during peach fruit development is discussed in the text.

Excisions of a defective transposable CACTA element (Tetu1) generate new alleles of a CYCLOIDEA-like gene of Helianthus annuus

tubular ray flower (turf) is a sunflower mutant that caught attention because it bears actinomorphic ray flowers, due to the presence of an active, although non-autonomous CACTA transposon (Tetu1) in the TCP domain of a CYCLOIDEA-like gene, HaCYC2c, a major regulator of sunflower floral symmetry. Here, we analyzed its excision rates in F3 population deriving from independent crosses of turf with common sunflower accessions. Our results suggest that the excision rate, ranging from 1.21 to 6.29%, depends on genetic background; moreover, the absence of somatic sectors in inflorescences of revertant individuals analyzed (182) and genetic analyses suggests a tight developmental control of Tetu1 excision, likely restricted to germinal cells. We individuate events of Tetu1 excision through molecular analysis that restore the wild type (WT) HaCYC2c allele, but even transposon excisions during which footprints are left. All mutations we detected occurred at the TCP basic motif and cause a change in ray flower phenotype. In particular, we selected five mutants with a one-to-four amino acid change that influence the capacity of reproductive organ development and ray flower corolla shaping (MUT-1, -2, -3, -4, -5). Revertant alleles not affecting turf phenotype (i.e. reading frame mutations) have also been identified (MUT-6). In all mutants, Real-time quantitative PCR (qPCR) experiments revealed variations of the steady state level of HaCYC2c mRNA. MUT-1 and MUT-4 showed a significant HaCYC2c down-regulation with respect to WT. A large variation within the biological replicates of MUT-2, MUT-3 and MUT-5 was detected and not significant differences in transcription levels between mutants and WT were observed. We detected low steady state level of HaCYC2c mRNA both in turf as in MUT-6. A three dimensional (3D) structure prediction tool let us predict an incorrect folding of the TCP protein already after a single amino acid deletion. This in turn is detectable as the restore of traits that are not peculiar of WT ray flowers, such as male fertility. Our analysis of an active TE sheds light on the TCP motif of the HaCYC2c gene and suggests that Tetu1 may be useful to obtain new natural mutants and for transposon tagging in different inbred lines of sunflower.

A TrkB–STAT3–miR-204-5p regulatory circuitry controls proliferation and invasion of endometrial carcinoma cells

BackgroundWe previously identified TrkB as an oncogene involved in promoting metastasis in endometrial carcinoma (EC). Here, we sought to delineate the effect of changes in TrkB expression on the global profile of microRNAs (miRNAs) in EC cells and further investigated the correlation between the expression of certain miRNA and TrkB in the clinicopathologic characteristics of EC patients.Methods and resultsUsing quantitative reverse transcription-PCR (qRT-PCR), we found that expression of TrkB mRNA has no significant difference in transcript levels between normal endometrium and EC cells captured by laser capture microdissection, while immunohistochemistry results demonstrated a markedly higher expression of TrkB protein in EC tissues. The microRNA array showed that ectopic overexpression and knockdown of TrkB expression caused global changes in miRNA expression in EC cells. qRT-PCR results showed that elevated TrkB repressed miR-204-5p expression in EC cells. Furthermore, immunoblotting assays revealed that TrkB overexpression in IshikawaTrkB cells noticeably increased JAK2 and STAT3 phosphorylation, which, however, was aborted by TrkB knockdown in HEC-1BshTrkB cells. Moreover, ChIP assays showed that phospho-STAT3 could directly bind to STAT3-binding sites near the TRPM3 promoter region upstream of miR-204-5p. Interestingly, using bioinformatics analysis and luciferase assays, we identified TrkB was a novel target of miR-204-5p. Functionally, the MTT assays, clonogenic and Transwell assays showed that miR-204-5p significantly suppressed the clonogenic growth, migration and invasion of EC cells. Furthermore, miR-204-5p also inhibited the growth of tumor xenografts bearing human EC cells. Importantly, we found lower miR-204-5p expression was associated with advanced FIGO stages, lymph node metastasis and probably a lower chance for survival in EC patients.ConclusionsThis study uncovers a new regulatory loop involving TrkB/miR-204-5p that is critical to the tumorigenesis of EC and proposes that reestablishment of miR-204-5p expression could be explored as a potential new therapeutic target for this disease.

Intraspecific sequence variation and differential expression in starch synthase genes of Arabidopsis thaliana

BackgroundNatural accessions of Arabidopsis thaliana are a well-known system to measure levels of intraspecific genetic variation. Leaf starch content correlates negatively with biomass. Starch is synthesized by the coordinated action of many (iso)enzymes. Quantitatively dominant is the repetitive transfer of glucosyl residues to the non-reducing ends of α-glucans as mediated by starch synthases. In the genome of A. thaliana, there are five classes of starch synthases, designated as soluble starch synthases (SSI, SSII, SSIII, and SSIV) and granule-bound synthase (GBSS). Each class is represented by a single gene. The five genes are homologous in functional domains due to their common origin, but have evolved individual features as well. Here, we analyze the extent of genetic variation in these fundamental protein classes as well as possible functional implications on transcript and protein levels.FindingsIntraspecific sequence variation of the five starch synthases was determined by sequencing the entire loci including promoter regions from 30 worldwide distributed accessions of A. thaliana. In all genes, a considerable number of nucleotide polymorphisms was observed, both in non-coding and coding regions, and several amino acid substitutions were identified in functional domains. Furthermore, promoters possess numerous polymorphisms in potentially regulatory cis-acting regions. By realtime experiments performed with selected accessions, we demonstrate that DNA sequence divergence correlates with significant differences in transcript levels.ConclusionsExcept for AtSSII, all starch synthase classes clustered into two or three groups of haplotypes, respectively. Significant difference in transcript levels among haplotype clusters in AtSSIV provides evidence for cis-regulation. By contrast, no such correlation was found for AtSSI, AtSSII, AtSSIII, and AtGBSS, suggesting trans-regulation. The expression data presented here point to a regulation by common trans-regulatory transcription factors which ensures a coordinated action of the products of these four genes during starch granule biosynthesis. The apparent cis-regulation of AtSSIV might be related to its role in the initiation of de novo biosynthesis of granules.

Transcription profiling of immune genes during parasite infection in susceptible and resistant strains of the flour beetles (Tribolium castaneum).

The flour beetle, Tribolium castaneum, is an intermediate host for the tapeworm Hymenolepis diminuta and has become an important genetic model to explore immune responses to parasite infection in insect hosts. The present study examined the immune responses to tapeworm infection in resistant (TIW1) and susceptible (cSM) strains of the red flour beetle, T. castaneum, using real-time quantitative reverse transcription PCR on 29 immunity-related genes that exhibit antimicrobial properties. Thirteen of the 29 genes showed constitutive differences in expression between the two strains. Fourteen to fifteen of the 29 genes exhibited significant differences in transcription levels when beetles were challenged with tapeworm parasite in the resistant and susceptible strains. Nine genes (GNBP3, cSPH2, lysozyme4, defensin1, PGRP-SA, defensin2, coleoptericin1, attacin2 and serpin29) in cSM and 13 genes (lysozyme2, proPO1, GNBP3, cSPH2, lysozyme4, defensin1, PGRP-SA, defensin2, coleoptericin1, attacin2, proPO2/3, PGRP-LE and PGRP-SB) in TIW1 were up-regulated by infections or showed parasite infection-induced expression. Seven genes (attacin2, coleoptericin1, defensin1, defensin2, lysozyme2, PGRP-SA and PGRP-SB) were more than 10 folds higher in the resistant TIW1 strain than in the susceptible cSM strain after exposure to tapeworm parasites. This study demonstrated the effects of genetic background, the transcription profile to parasite infection, and identified the immunity-related genes that were significantly regulated by the infection of tapeworms in Tribolium beetles.

GacS-Dependent Regulation of Polyhydroxyalkanoate Synthesis in Pseudomonas putida CA-3

To date, limited reports are available on the regulatory systems exerting control over bacterial synthesis of the biodegradable polyester group known as polyhydroxyalkanoates (PHAs). In this study, we performed random mini-Tn5 mutagenesis of the Pseudomonas putida CA-3 genome and screened transconjugants on nitrogen-limited medium for reduced PHA accumulation phenotypes. Disruption of a GacS sensor kinase in one such mutant was found to eliminate medium-chain-length PHA production in Pseudomonas putida CA-3. Recombinant expression of wild-type gacS from a pBBRgacS vector fully restored PHA accumulation capacity in the mutant strain. PCR-based screening of the P. putida CA-3 genome identified gene homologues of the GacS/GacA-rsm small RNA (sRNA) regulatory cascade with 96% similarity to published P. putida genomes. However, reverse transcription-PCR (RT-PCR) analyses revealed active transcription of the rsmY and rsmZ sRNAs in gacS-disrupted P. putida CA-3, which is atypical of the commonly reported Gac/Rsm regulatory cascade. Quantitative real-time RT-PCR analyses of the phaC1 synthase responsible for polymer formation in P. putida CA-3 indicated no statistically significant difference in transcript levels between the wild-type and gacS-disrupted strains. Subsequently, SDS-PAGE protein analyses of these strains identified posttranscriptional control of phaC1 synthase as a key aspect in the regulation of PHA synthesis by P. putida CA-3.

Genomic and physiological responses to strong selective pressure during late organogenesis: few gene expression changes found despite striking morphological differences

BackgroundAdaptations to a new environment, such as a polluted one, often involve large modifications of the existing phenotypes. Changes in gene expression and regulation during critical developmental stages may explain these phenotypic changes. Embryos from a population of the teleost fish, Fundulus heteroclitus, inhabiting a clean estuary do not survive when exposed to sediment extract from a site highly contaminated with polycyclic aromatic hydrocarbons (PAHs) while embryos derived from a population inhabiting a PAH polluted estuary are remarkably resistant to the polluted sediment extract. We exposed embryos from these two populations to surrogate model PAHs and analyzed changes in gene expression, morphology, and cardiac physiology in order to better understand sensitivity and adaptive resistance mechanisms mediating PAH exposure during development.ResultsThe synergistic effects of two model PAHs, an aryl hydrocarbon receptor (AHR) agonist (β-naphthoflavone) and a cytochrome P4501A (CYP1A) inhibitor (α-naphthoflavone), caused significant developmental delays, impaired cardiac function, severe morphological alterations and failure to hatch, leading to the deaths of reference embryos; resistant embryos were mostly unaffected. Unexpectedly, patterns of gene expression among normal and moderately deformed embryos were similar, and only severely deformed embryos showed a contrasting pattern of gene expression. Given the drastic morphological differences between reference and resistant embryos, a surprisingly low percentage of genes, 2.24% of 6,754 analyzed, show statistically significant differences in transcript levels during late organogenesis between the two embryo populations.ConclusionsOur study demonstrates important contrasts in responses between reference and resistant natural embryo populations to synergistic effects of surrogate model PAHs that may be important in adaptive mechanisms mediating PAH effects during fish embryo development. These results suggest that statistically significant changes in gene expression of relatively few genes contribute to the phenotypic changes and large morphological differences exhibited by reference and resistant populations upon exposure to PAH pollutants. By correlating cardiac physiology and morphology with changes in gene expression patterns of reference and resistant embryos, we provide additional evidence for acquired resistance among embryos whose parents live at heavily contaminated sites.

Involvement of hexokinase1 in plant growth promotion as mediated by Burkholderia phytofirmans.

Potato plantlets inoculated with strain PsJN of the bacterium Burkholderia phytofirmans exhibit consistent and significant increases in plant growth under in vitro conditions, when compared with uninoculated plants. The greatest influence on the degree and type of growth enhancement that develops has been shown to be mediated by the sugar concentration in the agar media. Bacterial growth promotion has been suggested in other studies to be regulated by the sugar sensor enzyme hexokinase1, the role of which is activation of glucose phosphorylation. In this present study, we examined the co-relationship between root and stem development in potato plants treated with PsJN and the activity of hexokinase1. Plants grown in the presence of 1.5% and 3% sucrose showed increased levels of hexokinase1 activity only in the roots of inoculated plants, suggesting that the increased enzyme levels may be associated with root growth. Analysis for mRNA using reverse transcriptase did not reveal any significant differences in transcription levels of the gene between inoculated and uninoculated plants. When PsJN-inoculated plants were grown in 1.5% and 3% concentrations of glucose and fructose, stem height and mass, leaf number, root mass, and overall biomass increased. No growth promotion occurred when PsJN-inoculated plants were grown in 3% maltose. Subsequently, a hexokinase1 activity assay showed that PsJN-induced growth of potato plants was found to only occur when plants were grown in the presence of sugars that are recognized by the plant hexokinase1. The results suggest that PsJN may enhance sugar uptake in plants by direct or indirect stimulation of hexokinase1 activity in roots and this results in enhanced overall plant growth.

Abstract 1532: The identification of osteoblastic and osteolytic factors in prostate cancer bone metastases

Abstract Prostate Cancer (PCa) metastasizes to the bone in approximately 90% of patients who die of PCa. Growth of tumor cells in the bone can lead to replacement of bone marrow, spinal cord compression, severe bone pain, cachexia and death. The bone response to a metastasis can manifest as a spectrum of osteoblastic to osteoblastic/osteolytic (mixed) or osteolytic responses in the same patient at different biopsy sites. A number of proteins have been proposed to promote the osteoblastic and osteolytic responses in PCa bone metastasis. Our objective was to determine whether known bone forming and degrading factors are associated with the osteoblastic or osteolytic response in pre-clinical models and clinical specimens of PCa bone metastasis. Additionally, we wanted to identify novel proteins that may be associated with the osteoblastic and osteolytic response in PCa bone metastases. Using specimens obtained at rapid autopsy at the University of Washington, we compared the gene expression profile of osteoblastic and osteolytic metastases (n=14) from 13 patients and validated our findings by qRT-PCR. We also interrogated the same factors at the protein level in 18 osteoblastic and 18 osteolytic PCa bone metastases from 27 patients by immunohistochemistry (IHC). Additionally, we are currently interrogating the levels of these factors in 6 novel LuCaP xenograft models of PCa that promote an osteoblastic response and 4 that promote an osteolytic response in the tibia of immune compromised mice by IHC. Using gene expression arrays, validated by qRTPCR, we identified a putative osteoblastic factor EMID1 (p=0.01) and two putative osteolytic factors, MMP12 (p=0.03) and sFRP1 (p=0.02). By IHC, MMP12 expression approached significance (p=0.07), whereas sFRP1 was significantly higher in the osteolytic samples (p=0.02). Interestingly, we did not observed significant differences in transcript levels between osteoblastic and osteolytic metastases for a number of proposed osteoblastic and osteolytic factors including BMP2 and 7, tachykinin 1, endothelin 1, osteoprotegerin, and sclerostin. Furthermore, none of the proposed osteoblastic and osteolytic factors we interrogated by IHC showed a significant difference in protein expression between osteoblastic and osteolytic PCa bone metastases. This is the first detailed analysis of PCa osteoblastic and osteolytic factors in human specimens and animal models of PCa in the bone. Our data suggest that many of the factors important in bone remodeling may not be central to the bone response in PCa bone metastases. Additionally we have identified three factors that may have a role in bone remodeling in PCa bone metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1532. doi:1538-7445.AM2012-1532

P1-05-05: Prognostic Utility of Histone Modifier Gene Expression Profiles in Human Breast Cancer.

Abstract Background: Gene expression is stringently controlled under physiological conditions by epigenetic mechanisms, including the specific methylation of cytosine residues within CpG dinucleotides and orchestrated adjustments in the histone dependent organisation of chromatin. The organisation of DNA within the chromatin template depends upon highly conserved histone proteins, the properties of which continue to exceed the simplistic packaging role originally assigned to them. Histone modifier enzymes impart a dynamic histone code and specific permutations have significant implications for chromatin topology and the functional configuration of promoters. This study follows our initial report describing potential tumour suppressor function associated with the histone methyltransferase SETD2 in human breast cancer. The objective was to evaluate the expression profiles of sixteen additional histone modifier genes in women with primary operable breast cancer within a well annotated cohort with extended follow-up. Methods: Primary breast cancer tissues (n= 127) and adjacent benign/normal tissues (n=33) underwent RNA extraction and reverse transcription. The transcript levels of histone modifier genes were evaluated using real-time quantitative PCR, these included: histone acetyltransferases (CREBBP), Class 1 (HDAC1 and HDAC2), II (HDAC5) and III (SIRT1) histone deacetylases and histone methyltransferases (SUV39H1 and SUV39H2) amongst others. Transcript levels were analysed against a range of clinico-pathological variables, including: tumour size, grade, nodal involvement, histological subtype, receptor status, TNM stage, Nottingham Prognostic Index, disease free and overall survival over a 10 year follow-up period. Results: Transcript levels of the histone modifier genes in breast cancer tissues differed significantly from non-malignant samples (HDAC5, HDAC1, KDM4A and KDM6A). Amongst breast cancers, significant differences in transcript levels were associated with established pathological parameters and prognostic indices: tumour grade (KAT5, HDAC1, KDM4A, SUV39H1 and KDM6A), receptor status (KAT5, SMYD3 and KDM1A), histological type (KAT5, KDM5C, MYST1, KDM4A and MLL), TNM stage (SUV39H1, KAT2B, KDM1A, KDM4A, KDM5C, MYST1, HDAC5 and KAT5), Nottingham Prognostic Index (KDM5C, MLL, MYST1 and SMYD3), disease free survival (SUV39H1, SMYD3, HDAC5, KDM6A, HDAC1, KDM1A, KDM4A, MYST1, KDM5C, KAT5 and MLL) and overall survival (MYST1). Interestingly, significant correlations were also identified between the differential expression profiles of particular histone modifying genes. Conclusion: The expression profiles of histone modifier genes differ significantly between breast cancer tissues and normal/benign samples. Particular expression profiles in breast cancer are significantly associated with established pathological parameters, prognostic indices, disease free and overall survival. The biological significance and clinical relevance of altered expression of specific histone modifier genes and particular permutations of misexpression remain to be fully elucidated and further study is warranted. Epigenetic signatures derived from histone modifier genes may offer utility as biomarkers and histone modifier enzymes have potential for targeted therapeutic strategies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-05-05.

Construction of a chassis for hydrogen production: physiological and molecular characterization of a Synechocystis sp. PCC 6803 mutant lacking a functional bidirectional hydrogenase

Cyanobacteria are photosynthetic prokaryotes that are promising 'low-cost' microbial cell factories due to their simple nutritional requirements and metabolic plasticity, and the availability of tools for their genetic manipulation. The unicellular non-nitrogen-fixing Synechocystis sp. PCC 6803 is the best studied cyanobacterial strain and its genome was the first to be sequenced. The vast amount of physiological and molecular data available, together with a relatively small genome, makes Synechocystis suitable for computational metabolic modelling and to be used as a photoautotrophic chassis in synthetic biology applications. To prepare it for the introduction of a synthetic hydrogen producing device, a Synechocystis sp. PCC 6803 deletion mutant lacking an active bidirectional hydrogenase (ΔhoxYH) was produced and characterized at different levels: physiological, proteomic and transcriptional. The results showed that, under conditions favouring hydrogenase activity, 17 of the 210 identified proteins had significant differential fold changes in comparisons of the mutant with the wild-type. Most of these proteins are related to the redox and energy state of the cell. Transcriptional studies revealed that only six genes encoding those proteins exhibited significant differences in transcript levels. Moreover, the mutant exhibits similar growth behaviour compared with the wild-type, reflecting Synechocystis plasticity and metabolic adaptability. Overall, this study reveals that the Synechocystis ΔhoxYH mutant is robust and can be used as a photoautotrophic chassis for the integration of synthetic constructs, i.e. molecular constructs assembled from well characterized biological and/or synthetic parts (e.g. promoters, regulators, coding regions, terminators) designed for a specific purpose.