Gene expression profiling of human hibernating myocardium: Increased expression of B-type natriuretic peptide and proenkephalin in hypocontractile vs normally-contracting regions of the heart

A greater understanding of the molecular basis of hibernating myocardium may assist in identifying those patients who would most benefit from revascularization. Paired heart biopsies were taken from hypocontractile and normally-contracting myocardium (identified by cardiovascular magnetic resonance) from 6 patients with chronic stable angina scheduled for bypass grafting. Gene expression profiles of hypocontractile and normally-contracting samples were compared using Affymetrix microarrays. The data for patients with confirmed hibernating myocardium were analysed separately and a different, though overlapping, set (up to 380) of genes was identified which may constitute a molecular fingerprint for hibernating myocardium. The expression of B-type natriuretic peptide (BNP) was increased in hypocontractile relative to normally-contracting myocardium. The expression of BNP correlated most closely with the expression of proenkephalin and follistatin 3, which may constitute additional heart failure markers. Our data illustrate differential gene expression in hypocontractile and/hibernating myocardium relative to normally-contracting myocardium within individual human hearts. Changes in expression of these genes, including increased relative expression of natriuretic and other factors, may constitute a molecular signature for hypocontractile and/or hibernating myocardium.

Temporal regulation of expression of immediate early and second phase transcripts by endothelin-1 in cardiomyocytes

Background: Endothelin-1 stimulates Gq protein-coupled receptors to promote proliferation in dividing cells or hypertrophy in terminally differentiated cardiomyocytes. In cardiomyocytes, endothelin-1 rapidly (within minutes) stimulates protein kinase signaling, including extracellular-signal regulated kinases 1/2 (ERK1/2; though not ERK5), with phenotypic/physiological changes developing from approximately 12 h. Hypertrophy is associated with changes in mRNA/protein expression, presumably consequent to protein kinase signaling, but the connections between early, transient signaling events and developed hypertrophy are unknown. Results: Using microarrays, we defined the early transcriptional responses of neonatal rat cardiomyocytes to endothelin-1 over 4 h, differentiating between immediate early gene (IEG) and second phase RNAs with cycloheximide. IEGs exhibited differential temporal and transient regulation, with expression of second phase RNAs within 1 h. Of transcripts upregulated at 30 minutes encoding established proteins, 28 were inhibited >50% by U0126 (which inhibits ERK1/2/5 signaling), with 9 inhibited 25-50%. Expression of only four transcripts was not inhibited. At 1 h, most RNAs (approximately 67%) were equally changed in total and polysomal RNA with approximately 17% of transcripts increased to a greater extent in polysomes. Thus, changes in expression of most protein-coding RNAs should be reflected in protein synthesis. However, approximately 16% of transcripts were essentially excluded from the polysomes, including some protein-coding mRNAs, presumably inefficiently translated. Conclusion: The phasic, temporal regulation of early transcriptional responses induced by endothelin-1 in cardiomyocytes indicates that, even in terminally differentiated cells, signals are propagated beyond the primary signaling pathways through transcriptional networks leading to phenotypic changes (that is, hypertrophy). Furthermore, ERK1/2 signaling plays a major role in this response.

A specific group of genes respond to cold dehydration stress in cut Alstroemeria flowers whereas ambient dehydration stress accelerates developmental senescence expression patterns

Petal development and senescence entails a normally irreversible process. It starts with petal expansion and pigment production, and ends with nutrient remobilization and ultimately cell death. In many species this is accompanied by petal abscission. Post-harvest stress is an important factor in limiting petal longevity in cut flowers and accelerates some of the processes of senescence such as petal wilting and abscission. However, some of the effects of moderate stress in young flowers are reversible with appropriate treatments. Transcriptomic studies have shown that distinct gene sets are expressed during petal development and senescence. Despite this, the overlap in gene expression between developmental and stress-induced senescence in petals has not been fully investigated in any species. Here a custom-made cDNA microarray from Alstroemeria petals was used to investigate the overlap in gene expression between developmental changes (bud to first sign of senescence) and typical post-harvest stress treatments. Young flowers were stressed by cold or ambient temperatures without water followed by a recovery and rehydration period. Stressed flowers were still at the bud stage after stress treatments. Microarray analysis showed that ambient dehydration stress accelerates many of the changes in gene expression patterns that would normally occur during developmental senescence. However, a higher proportion of gene expression changes in response to cold stress were specific to this stimulus and not senescence related. The expression of 21 transcription factors was characterized, showing that overlapping sets of regulatory genes are activated during developmental senescence and by different stresses.

Glycogen synthase kinases 3α and 3β in cardiac myocytes: regulation and consequences of their inhibition

Inhibition of glycogen synthase kinase 3β (GSK3β) as a consequence of its phosphorylation by protein kinase B/Akt (PKB/Akt) has been implicated in cardiac myocyte hypertrophy in response to endothelin-1 or phenylephrine. We examined the regulation of GSK3α (which we show to constitute a significant proportion of the myocyte GSK3 pool) and GSK3β in cardiac myocytes. Although endothelin increases phosphorylation of GSK3 and decreases its activity, the response is less than that induced by insulin (which does not promote cardiac myocyte hypertrophy). GSK3 phosphorylation induced by endothelin requires signalling through the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade and not the PKB/Akt pathway, whereas the reverse is true for insulin. Cardiac myocyte hypertrophy involves changes in morphology, and in gene and protein expression. The potent GSK3 inhibitor 1-azakenpaullone increases myocyte area as a consequence of increased cell length whereas phenylephrine increases both length and width. Azakenpaullone or insulin promotes AP1 transcription factor binding to an AP1 consensus oligonucleotide, but this was significantly less than that induced by endothelin and derived principally from increased binding of JunB protein, the expression of which was increased. Azakenpaullone promotes significant changes in gene expression (assessed by Affymetrix microarrays), but the overall response is less than with endothelin and there is little overlap between the genes identified. Thus, although GSK3 may contribute to cardiac myocyte hypertrophy in some respects (and presumably plays an important role in myocyte metabolism), it does not appear to contribute as significantly to the response induced by endothelin as has been maintained.

Pretranslational regulation of the expression of the lipoprotein lipase (EC 3.1, l.34) gene by dietary fatty acids in the rat

Although there have been a number of studies of effects of diet and hormones on lipoprotein lipase (EC 3.1.1.34; LPL) activity and levels of LPL mRNA (Raynolds et al. 1990), there have been no studies which have investigated effects of different dietary fatty acids on LPL gene expression. In the present study male Wistar Albino rats were pair-fed diets containing 50 g fat/kg of different fatty acid composition for 2 weeks. The diets fed were (1) a mixed oil (450 g saturated fatty acids, 420 g monounsaturated fatty acids, 130 g polyunsaturated fatty acids/kg; n 8), (2) maize oil (n 8), or (3) fish oil (n 8). Animals were killed, RNA was extracted from liver and perirenal and epididymal fat pads, and analysed by ‘Northern methodology’. Samples were hybridized to a human cDNA probe for LPL (Gotoda et al. 1989). Two transcripts were identified in epididymai and perirenal adipose tissue which were approximately 3·7 and 1·7 kb in size. The results suggested that (1) fish oil-fed animals had significantly greater production of LPL mRNA in epididymai adipose tissue compared with maize oil-fed animals (P < 0·05), (2) maize oil-fed animals had significantly greater production of LPL mRNA in perirenal fat compared with the other dietary groups (P < 0·05), (3) expression in the liver was not significant. Rats fed on a fish oil diet had significantly reduced plasma triacylglycerol concentrations compared with the mixed-oil group (P < 0·05), but there were no significant differences in plasma cholesterol. The differences in LPL could not be explained directly by the changes in plasma immunoreactive-insulin and glucose-dependent insulinotrophic polypeptide levels in the three groups.

Complex history of a chromosomal paralogy region: insights from amphioxus aromatic amino acid hydroxylase genes and insulin-related genes

Aromatic amino acid hydroxylase (AAAH) genes and insulin-like genes form part of an extensive paralogy region shared by human chromosomes 11 and 12, thought to have arisen by tetraploidy in early vertebrate evolution. Cloning of a complementary DNA (cDNA) for an amphioxus (Branchiostoma floridae) hydroxylase gene (AmphiPAH) allowed us to investigate the ancestry of the human chromosome 11/12 paralogy region. Molecular phylogenetic evidence reveals that AmphiPAH is orthologous to vertebrate phenylalanine (PAH) genes; the implication is that all three vertebrate AAAH genes arose early in metazoan evolution, predating vertebrates. In contrast, our phylogenetic analysis of amphioxus and vertebrate insulin-related gene sequences is consistent with duplication of these genes during early chordate ancestry. The conclusion is that two tightly linked gene families on human chromosomes 11 and 12 were not duplicated coincidentally. We rationalize this paradox by invoking gene loss in the AAAH gene family and conclude that paralogous genes shared by paralogous chromosomes need not have identical evolutionary histories.

Eight-plex iTRAQ analysis of variant metastatic human prostate cancer cells identifies candidate biomarkers of progression: an exploratory study.

BACKGROUND: Due to the heterogeneity in the biological behavior of prostate cancer, biomarkers that can reliably distinguish indolent from aggressive disease are urgently needed to inform treatment choices. METHODS: We employed 8-plex isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to profile the proteomes of two distinct panels of isogenic prostate cancer cells with varying growth and metastatic potentials, in order to identify novel biomarkers associated with progression. The LNCaP, LNCaP-Pro5, and LNCaP-LN3 panel of cells represent a model of androgen-responsive prostate cancer, while the PC-3, PC-3M, and PC-3M-LN4 panel represent a model of androgen-insensitive disease. RESULTS: Of the 245 unique proteins identified and quantified (>or=95% confidence; >or=2 peptides/protein), 17 showed significant differential expression (>or=+/-1.5), in at least one of the variant LNCaP cells relative to parental cells. Similarly, comparisons within the PC-3 panel identified 45 proteins to show significant differential expression in at least one of the variant PC-3 cells compared with parental cells. Differential expression of selected candidates was verified by Western blotting or immunocytochemistry, and corresponding mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Immunostaining of prostate tissue microarrays for ERp5, one of the candidates identified, showed a significant higher immunoexpression in pre-malignant lesions compared with non-malignant epithelium (P < 0.0001, Mann-Whitney U-test), and in high Gleason grade (4-5) versus low grade (2-3) cancers (P < 0.05). CONCLUSIONS: Our study provides proof of principle for the application of an 8-plex iTRAQ approach to uncover clinically relevant candidate biomarkers for prostate cancer progression.

Feedback regulation by Atf3 in the endothelin-1-responsive transcriptome of cardiomyocytes: Egr1 is a principal Atf3 target

Endothelin-1 promotes cardiomyocyte hypertrophy by inducing changes in gene expression. Immediate early genes including activating transcription factor 3 (Atf3), Egr1 and Ptgs2 are rapidly and transiently upregulated by endothelin-1 in cardiomyocytes. Atf3 regulates expression of downstream genes and is implicated in negative feedback regulation of other immediate early genes. To identify Atf3-regulated genes, we knocked down Atf3 expression in cardiomyocytes exposed to endothelin-1 and used microarrays to interrogate the transcriptomic effects. Of upregulated mRNAs, expression of 23 (including Egr1, Ptgs2) was enhanced and expression of 25 was inhibited by Atf3 knockdown. Using quantitative PCR, we determined that knockdown of Atf3 had little effect on upregulation of Egr1 mRNA over 30 min, but abolished the subsequent decline, causing sustained Egr1 mRNA expression and enhanced protein expression. This resulted from direct binding of Atf3 to the Egr1 promoter. Mathematical modelling established that Atf3 can suffice to suppress Egr1 expression. Given the widespread co-regulation of Atf3 with Egr1, we suggest that the Atf3-Egr1 negative feedback loop is of general significance. Loss of Atf3 caused abnormal cardiomyocyte growth, presumably resulting from dysregulation of target genes. Our data therefore identify Atf3 as a nexus in cardiomyocyte hypertrophy required to facilitate the full and proper growth response.

Profiling dehydrin gene sequence and physiological parameters in drought tolerant and susceptible spring wheat cultivars.

Physiological and yield traits such as stomatal conductance (mmol m-2s-1), Leaf relative water content (RWC %) and grain yield per plant were studied in a separate experiment. Results revealed that five out of sixteen cultivars viz. Anmol, Moomal, Sarsabz, Bhitai and Pavan, appeared to be relatively more drought tolerant. Based on morphophysiological results, studies were continued to look at these cultivars for drought tolerance at molecular level. Initially, four well recognized primers for dehydrin genes (DHNs) responsible for drought induction in T. durum L., T. aestivum L. and O. sativa L. were used for profiling gene sequence of sixteen wheat cultivars. The primers amplified the DHN genes variably like Primer WDHN13 (T. aestivum L.) amplified the DHN gene in only seven cultivars whereas primer TdDHN15 (T. durum L.) amplified all the sixteen cultivars with even different DNA banding patterns some showing second weaker DNA bands. Third primer TdDHN16 (T. durum L.) has shown entirely different PCR amplification prototype, specially showing two strong DNA bands while fourth primer RAB16C (O. sativa L.) failed to amplify DHN gene in any of the cultivars. Examination of DNA sequences revealed several interesting features. First, it identified the two exon/one intron structure of this gene (complete sequences were not shown), a feature not previously described in the two database cDNA sequences available from T. aestivum L. (gi|21850). Secondly, the analysis identified several single nucleotide polymorphisms (SNPs), positions in gene sequence. Although complete gene sequence was not obtained for all the cultivars, yet there were a total of 38 variable positions in exonic (coding region) sequence, from a total gene length of 453 nucleotides. Matrix of SNP shows these 37 positions with individual sequence at positions given for each of the 14 cultivars (sequence of two cultivars was not obtained) included in this analysis. It demonstrated a considerable diversity for this gene with only three cultivars i.e. TJ-83, Marvi and TD-1 being similar to the consensus sequence. All other cultivars showed a unique combination of SNPs. In order to prove a functional link between these polymorphisms and drought tolerance in wheat, it would be necessary to conduct a more detailed study involving directed mutation of this gene and DHN gene expression.

Optical genetic mapping defines regions of chromosomal variation in serovars of S. enterica subsp enterica of concern for human and animal health

Infections involving Salmonella enterica subsp. enterica serovars have serious animal and human health implications; causing gastroenteritis in humans and clinical symptoms, such as diarrhoea and abortion, in livestock. In this study an optical genetic mapping technique was used to screen 20 field isolate strains from four serovars implicated in disease outbreaks. The technique was able to distinguish between the serovars and the available sequenced strains and group them in agreement with similar data from microarrays and PFGE. The optical maps revealed variation in genome maps associated with antimicrobial resistance and prophage content in S. Typhimurium, and separated the S. Newport strains into two clear geographical lineages defined by the presence of prophage sequences. The technique was also able to detect novel insertions that may have had effects on the central metabolism of some strains. Overall optical mapping allowed a greater level of differentiation of genomic content and spatial information than more traditional typing methods.

Genome scale reconstruction of a Salmonella metabolic model comparison of similarity and differences with a commensal Escherichia coli strain

Salmonella are closely related to commensal Escherichia coli but have gained virulence factors enabling them to behave as enteric pathogens. Less well studied are the similarities and differences that exist between the metabolic properties of these organisms that may contribute toward niche adaptation of Salmonella pathogens. To address this, we have constructed a genome scale Salmonella metabolic model (iMA945). The model comprises 945 open reading frames or genes, 1964 reactions, and 1036 metabolites. There was significant overlap with genes present in E. coli MG1655 model iAF1260. In silico growth predictions were simulated using the model on different carbon, nitrogen, phosphorous, and sulfur sources. These were compared with substrate utilization data gathered from high throughput phenotyping microarrays revealing good agreement. Of the compounds tested, the majority were utilizable by both Salmonella and E. coli. Nevertheless a number of differences were identified both between Salmonella and E. coli and also within the Salmonella strains included. These differences provide valuable insight into differences between a commensal and a closely related pathogen and within different pathogenic strains opening new avenues for future explorations.

Proteomic analyses of a Listeria monocytogenes mutant lacking σB identify new components of the σB regulon and highlight a role for σB in the utilization of glycerol

In Listeria monocytogenes the alternative sigma factor σB plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the σB regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic ΔsigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was σB dependent; 17 of these proteins were found to require the presence of σB for full expression, while 21 were expressed at a higher level in the ΔsigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for σB in the general stress response and highlighted a probable role for σB in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of σB in glycerol metabolism. Five newly identified members of the σB regulon were shown to be under direct regulation of σB using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four σB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified σB-dependent genes, as well as proteomic analyses, σB was shown to play a major role in the stationary phase of growth in complex media.

Genistein protects prostate cells against hydrogen peroxide-induced DNA damage and induces expression of genes involved in the defence against oxidative stress

Prostate cancer is one of the most frequent cancer types in Western societies and predominately occurs in the elderly male. The strong age-related increase of prostate cancer is associated with a progressive accumulation of oxidative DNA damage which is presumably supported by a decline of the cellular antioxidative defence during ageing. Risk of developing prostate cancer is much lower in many Asian countries where soy food is an integral part of diet. Therefore, isoflavones from soy were suggested to have chemopreventive activities in prostate cells. Here, we have investigated the hypothesis that the soy-isoflavone genistein could protect DNA of LAPC-4 prostate cells from oxidative stress-related damage by enhancing the expression of antioxidative genes and proteins. A 24 h preincubation with genistein (1-30 microM) protected cells from hydrogen peroxide-induced DNA damage, as determined by the comet assay. Analysis of two cDNA macroarrays, each containing 96 genes of biotransformation and stress response, revealed a modulated expression of 3 genes at 1 microM and of 19 genes at 10 microM genistein. Real-time PCR confirmed the induction of three genes encoding products with antioxidant activities, namely glutathione reductase (2.7-fold), microsomal glutathione S-transferase 1 (1.9-fold) and metallothionein 1X (6.3-fold), at 1-30 microM genistein. 17Beta-estradiol, in contrast, decreased the expression of metallothionein 1X at 0.3 microM (2.0-fold), possibly pointing to an estrogen receptor-mediated regulation of this gene. Immunocytochemical staining revealed an induction of metallothionein proteins at 30 microM genistein, while their intracellular localization was unaffected. Metallothioneins were previously found to protect cells from hydrogen peroxide-induced DNA damage. Hence, our findings indicate that genistein protects prostate cells from oxidative stress-related DNA damage presumably by inducing the expression of antioxidative products, such as metallothioneins. Genistein, therefore, might counteract the age-related decline of important antioxidative defence systems which in turn maintain DNA integrity.

Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.

The early transcriptomic response to interleukin 1β and interleukin 33 in rat neonatal cardiomyocytes

In the heart, inflammatory cytokines including interleukin (IL) 1β are implicated in regulating adaptive and maladaptive changes, whereas IL33 negatively regulates cardiomyocyte hypertrophy and promotes cardioprotection. These agonists signal through a common co-receptor but, in cardiomyocytes, IL1β more potently activates mitogen-activated protein kinases and NFκB, pathways that regulate gene expression. We compared the effects of external application of IL1β and IL33 on the cardiomyocyte transcriptome. Neonatal rat cardiomyocytes were exposed to IL1β or IL33 (0.5, 1 or 2h). Transcriptomic profiles were determined using Affymetrix rat genome 230 2.0 microarrays and data were validated by quantitative PCR. IL1β induced significant changes in more RNAs than IL33 and, generally, to a greater degree. It also had a significantly greater effect in downregulating mRNAs and in regulating mRNAs associated with selected pathways. IL33 had a greater effect on a small, select group of specific transcripts. Thus, differences in intensity of intracellular signals can deliver qualitatively different responses. Quantitatively different responses in production of receptor agonists and transcription factors may contribute to qualitative differences at later times resulting in different phenotypic cellular responses.