CausalTAB: the PSI-MITAB 2.8 updated format for signalling data representation and dissemination.

MOTIVATION: Combining multiple layers of information underlying biological complexity into a structured framework represent a challenge in systems biology. A key task is the formalization of such information in models describing how biological entities interact to mediate the response to external and internal signals. Several databases with signalling information, focus on capturing, organizing and displaying signalling interactions by representing them as binary, causal relationships between biological entities. The curation efforts that build these individual databases demand a concerted effort to ensure interoperability among resources. RESULTS: Aware of the enormous benefits of standardization efforts in the molecular interaction research field, representatives of the signalling network community agreed to extend the PSI-MI controlled vocabulary to include additional terms representing aspects of causal interactions. Here, we present a common standard for the representation and dissemination of signalling information: the PSI Causal Interaction tabular format (CausalTAB) which is an extension of the existing PSI-MI tab-delimited format, now designated PSI-MITAB 2.8. We define the new term 'causal interaction', and related child terms, which are children of the PSI-MI 'molecular interaction' term. The new vocabulary terms in this extended PSI-MI format will enable systems biologists to model large-scale signalling networks more precisely and with higher coverage than before. AVAILABILITY AND IMPLEMENTATION: PSI-MITAB 2.8 format and the new reference implementation of PSICQUIC are available online (https://psicquic.github.io/ and https://psicquic.github.io/MITAB28Format.html). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Identification of novel neuroendocrine-specific tumour genes.

Neuroendocrine tumours (NETs) comprise a heterogenous group of malignancies with an often unpredictable course, and with limited treatment options. Thus, new diagnostic, prognostic, and therapeutic markers are needed. To shed new lights into the biology of NETs, we have by cDNA transcript profiling, sought to identify genes that are either up- or downregulated in NE as compared with non-NE tumour cells. A panel of six NET and four non-NET cell lines were examined, and out of 12 743 genes examined, we studied in detail the 200 most significantly differentially expressed genes in the comparison. In addition to potential new diagnostic markers (NEFM, CLDN4, PEROX2), the results point to genes that may be involved in the tumorigenesis (BEX1, TMEPAI, FOSL1, RAB32), and in the processes of invasion, progression and metastasis (MME, STAT3, DCBLD2) of NETs. Verification by real time qRT-PCR showed a high degree of consistency to the microarray results. Furthermore, the protein expression of some of the genes were examined. The results of our study has opened a window to new areas of research, by uncovering new candidate genes and proteins to be further investigated in the search for new prognostic, predictive, and therapeutic markers in NETs.

Gene expression analysis and clinical diagnosis.

BACKGROUND: A new basis for diagnostic tests is being provided by the vast amount of data on gene expression that are now becoming available through large-scale measurement of mRNA abundance. The insights gained from these resources are most likely going to provide both a better basic understanding of disease mechanisms, and to identify molecular markers for more precise diagnoses and for prediction of prognosis and treatment response. METHODS: Some quantitative RT-PCR assays are utilized today for diagnosis of both malignant and non-malignant disease, but the use of gene expression measurements in clinical medicine can be expected to increase dramatically. CONCLUSIONS: There are important technical issues that must be adequately solved in order to obtain robust assays, such as standardized protocols with appropriate quality controls that ensure reliable data for the specific samples being analysed and good inter-laboratory reproducibility.

Identification of novel growth factor-responsive genes in neuroendocrine gastrointestinal tumour cells.

Targeting growth-regulatory pathways is a promising approach in cancer treatment. A prerequisite to the development of such therapies is characterisation of tumour growth regulation in the particular tumour cell type of interest. In order to gain insight into molecular mechanisms underlying proliferative responses in neuroendocrine (NE) gastrointestinal (GI) tumours, we investigated gene expression in human carcinoid BON cells after exposure to gastrin, hepatocyte growth factor (HGF), pituitary adenylate cyclase-activating polypeptide or epidermal growth factor. We particularly focused on gastrin- and HGF-induced gene expression, and identified 95 gastrin- and 101 HGF-responsive genes. The majority of these genes are known mediators of processes central in tumour biology, and a number of them have been associated with poor prognosis and metastasis in cancer patients. Furthermore, we identified 12 genes that were regulated by all four factors, indicating that they may be universally regulated during NE GI tumour cell proliferation. Our findings provide useful hypotheses for further studies aimed to search for new therapeutic targets as well as tumour markers in NE GI tumours.

Molecular mechanisms involved in gastrin-mediated regulation of cAMP-responsive promoter elements.

In the present study, we explore the role of cAMP-responsive (CRE) promoter elements in gastrin-mediated gene activation. By using the minimal CRE promoter reporter plasmid, pCRELuc, we show that gastrin can activate CRE. This activation is blocked by H-89 and GF 109203x, which inhibit protein kinases A and C, respectively. Moreover, Ca(2+)-activated pathways seem to be involved, because the calmodulin inhibitor W-7 reduced gastrin-mediated activation of pCRELuc. Deletion of CRE from the c-fos promoter rendered this promoter completely unresponsive to gastrin, indicating that CRE plays a central role in c-fos transactivation. Interestingly, gastrin-induced expression of the inducible cAMP early repressor (ICER), a gene that is known to be regulated by CRE promoter elements, was not reduced by H-89, W-7, or GF 109203x. Furthermore, bandshift analyses indicated that the region of the ICER promoter containing the CRE-like elements CARE 3-4 binds transcription factors that are not members of the CRE-binding protein-CRE modulator protein-activating transcription factor, or CREB/CREM/ATF-1, family. Our results underline the significance of the CRE promoter element in gastrin-mediated gene regulation and indicate that a variety of signaling mechanisms are involved, depending on the CRE promoter context.

[Measurement of gene activity by DNA microarrays]. / Måling av genaktivitet med DNA-mikromatriser.

BACKGROUND: DNA microarray is a tool that can be used to measure in one single analysis simultaneous changes in the activity of tens of thousands of genes. MATERIAL AND METHODS: The method is based upon advanced robotic techniques; High-density arrays of DNA probes are placed on a solid surface; this is followed by hybridisation with a fluorescence labelled sample and analysis of fluorescence signals. RESULTS: The analysis create huge data sets which have to be transformed into formats that can be interpreted and correlated with existing knowledge. This means that bioinformatics is an integrated part of microarray analysis. INTERPRETATION: DNA microarray may be used to examine complex physiological and pathological conditions and will most likely be very important in functional studies addressing the structural knowledge of genes obtained through the Human Genome Project. Dedicated microchips are already being tested in the diagnosis of malignant and premalignant diseases and being used to characterize HIV viruses with respect to choice of therapy.

[New knowledge derived from measurement of gene expression with the DNA microarray method]. / Ny kunnskap fra måling av genuttrykk gjennom DNA-mikromatrise.

BACKGROUND: The cDNA microarray method offers the first possibility of obtaining a global understanding of biological processes in living organisms, by simultaneous read-outs of tens of thousands of mRNAs. Initial experiments suggest that genes with similar function have similar expression patterns. MATERIAL AND METHODS: Understanding this level of biological complexity will, however, require completely new approaches to data analysis. Computer science methods, such as data mining and knowledge discovery, can synthesize interpretable if-then rules that model the relation between gene expressions and functions and use the rules to classify unknown genes. The huge body of existing biological and medical knowledge makes it necessary to develop methods for extracting knowledge from such repositories. RESULTS: Models of relations between gene expressions and gene functions in a data set from a publicly available source are synthesized semiautomatically and applied to classify unknown genes. Encouraging results have been achieved. The method is applied in the analysis of data from our microarray system which has recently become operational. INTERPRETATION: The principles are of general importance and will be used to evaluate a wide range of complex data sets like decision support in clinical medicine, for situations in which physicians need to handle a large volume of data for each patient.

A literature network of human genes for high-throughput analysis of gene expression.

We have carried out automated extraction of explicit and implicit biomedical knowledge from publicly available gene and text databases to create a gene-to-gene co-citation network for 13,712 named human genes by automated analysis of titles and abstracts in over 10 million MEDLINE records. The associations between genes have been annotated by linking genes to terms from the medical subject heading (MeSH) index and terms from the gene ontology (GO) database. The extracted database and accompanying web tools for gene-expression analysis have collectively been named 'PubGene'. We validated the extracted networks by three large-scale experiments showing that co-occurrence reflects biologically meaningful relationships, thus providing an approach to extract and structure known biology. We validated the applicability of the tools by analyzing two publicly available microarray data sets.

Predicting gene function from gene expressions and ontologies.

We introduce a methodology for inducing predictive rule models for functional classification of gene expressions from microarray hybridisation experiments. The basic learning method is the rough set framework for rule induction. The methodology is different from the commonly used unsupervised clustering approaches in that it exploits background knowledge of gene function in a supervised manner. Genes are annotated using Ashburner's Gene Ontology and the functional classes used for learning are mined from these annotations. From the original expression data, we extract a set of biologically meaningful features that are used for learning. A rule model is induced from the data described in terms of these features. Its predictive quality is fine-turned via cross-validation on subsets of the known genes prior to classification of unknown genes. The predictive and descriptive quality of such a rule model is demonstrated on the fibroblast serum response data previously analysed by Iyer et. al. Our analysis shows that the rules are capable of representing the complex relationship between gene expressions and function, and that it is possible to put forward high quality hypotheses about the function of unknown genes.

Regulation of inducible cAMP early repressor expression by gastrin and cholecystokinin in the pancreatic cell line AR42J.

The CREM gene encodes both activators and repressors of cAMP-induced transcription. Inducible cAMP early repressor (ICER) isoforms are generated upon activation of an alternative, intronic promoter within the CREM gene. ICER is proposed to down-regulate both its own expression and the expression of other genes that contain cAMP-responsive elements such as a number of growth factors. Thus, ICER has been postulated to play a role in proliferation and differentiation. Here we show that ICER gene expression is induced by gastrin, cholecystokinin (CCK), and epidermal growth factor in AR42J cells. The time course of gastrin- and CCK-mediated ICER induction is rapid and transient, similar to forskolin- and phorbol 12-myristate 13-acetate-induced ICER expression. The specific CCK-B receptor antagonist L740,093 blocks the gastrin but not the CCK response, indicating that both the CCK-B and the CCK-A receptor can mediate ICER gene activation. Noteworthy, CREB is constitutively phosphorylated at Ser-133 in AR42J cells, and ICER induction proceeds in the absence of increased CREB Ser(P)-133. Gastrin-mediated ICER induction was not reduced in the presence of the protein kinase A inhibitor H-89, indicating a protein kinase A-independent mechanism. This is the first report on ICER inducibility via G(q)/G(11) protein-coupled receptors.

Selective inhibitors of cytosolic or secretory phospholipase A2 block TNF-induced activation of transcription factor nuclear factor-kappa B and expression of ICAM-1.

TNF signaling mechanisms involved in activation of transcription factor NF-kappaB were studied in the human keratinocyte cell line HaCaT. We show that TNF-induced activation of NF-kappaB was inhibited by the well-known selective inhibitors of cytosolic phospholipase A2 (cPLA2): the trifluoromethyl ketone analogue of arachidonic acid (AACOCF3) and methyl arachidonyl fluorophosphate. The trifluoromethyl ketone analogue of eicosapentaenoic acid (EPACOCF3) also suppressed TNF-induced NF-kappaB activation and inhibited in vitro cPLA2 enzyme activity with a similar potency as AACOCF3. The arachidonyl methyl ketone analogue (AACOCH3) and the eicosapentanoyl analogue (EPACHOHCF3), which both failed to inhibit cPLA2 enzyme activity in vitro, had no effect on TNF-induced NF-kappaB activation. TNF-induced NF-kappaB activation was also strongly reduced in cells stimulated in the presence of the secretory PLA2 (sPLA2) inhibitors 12-epi-scalaradial and LY311727. Addition of excess arachidonic acid suppressed the inhibitory effect of 12-epi-scalaradial and LY311727. Moreover, both methyl arachidonyl fluorophosphate and 12-epi-scalaradial blocked TNF-mediated enhancement of expression of ICAM-1. Activation of NF-kappaB by IL-1beta was markedly less sensitive to both cPLA2 and sPLA2 inhibitors. The results indicate that both cPLA2 and sPLA2 may be involved in the TNF signal transduction pathway leading to nuclear translocation of NF-kappaB and to NF-kappaB-activated gene expression in HaCaT cells.

A non-competitive P55 TNF receptor antibody enhances the specific activity of lymphotoxin-alpha.

In the present study the authors elucidated the involvement of the two TNF receptors (TNFR) in discriminating TNF and lymphotoxin-alpha (LT-alpha) effects in human SW480-beta Gal and KYM-1 cell lines. A non-competitive p55 TNFR monoclonal antibody (MoAb) 44E strongly enhanced LT-alpha-mediated gene regulation and cytotoxicity up to the level of the responses caused by TNF. TNF-induced biological responses were only weakly influenced by 44E. 44E did not affect both binding and the rate of dissociation of the cytokines. The combination of the two p55 TNFR MoAb 44E and Htr5 elicited strong TNF responses, while none of them were agonistic alone. When the p75 TNFR was blocked with Utr1, LT-alpha was still less potent than TNF in mediating CMV promoter activation and cytotoxicity. However, the addition of 44E in the presence of Utr1 merged the LT-alpha dose-response curves with those obtained with TNF plus Utr1. Using antagonistic TNFR MoAb, the authors further showed that TNF functions through both TNFR types while LT-alpha mediates its effects largely via the p55 TNFR. These data suggest that LT-alpha is less potent than TNF due to its lower ability to properly trigger the p55 TNFR and because of its lack of signalling through the p75 TNFR.

Four mutations in the porphobilinogen deaminase gene in patients with acute intermittent porphyria.

We have detected four different mutations in the porphobilinogen deaminase (PBGD) gene in acute intermittent porphyria (AIP) families from England, Norway, and Sweden. A splicing mutation in the first position of intron 8 (Int8 + 1) was found in a family from England and a missense mutation in exon 12 (Glu250) was detected in a Norwegian family. Two mutations were identified in Swedish families, one splicing mutation in the first position of intron 3 (Int3 + 1) and one missense mutation in exon 8 (Pro119).

Tumor necrosis factor induces lipopolysaccharide tolerance in a human adenocarcinoma cell line mainly through the TNF p55 receptor.

This study demonstrates that lipopolysaccharide (LPS) mediates induction of transcription factor NF kappa B and activation of the cytomegalovirus (CMV) promoter-enhancer in the SW480 cell line. These cells do not express a functional membrane CD14. The LPS response in SW480 cells was weaker and markedly slower than the tumor necrosis factor (TNF) response. Pretreatment with TNF for 72 h inhibited both TNF, tumor necrosis factor receptor (TNFR) p55, TNFR p75, and LPS-mediated activation of nuclear factor -kappa B (NF kappa B), whereas pretreatment with LPS only inhibited the LPS response. TNFR p55 antibody pretreatment resulted in marked inhibition of the LPS response, while pretreatment with TNFR p75 antiserum only had a weak inhibitory effect. Flowcytometric analysis showed that LPS binding as well as expression of TNFR p55 and TNFR p75 were not affected by LPS or TNF pretreatment, indicating that the observed inhibition is not due to reduction of specific binding sites at the cell surface. The results suggest that LPS signaling in SW480 cells involves intracellular components which may be depleted or inactivated via TNFR p55, indicating that the LPS and TNFR p55 pathways overlap. We propose that TNFR p55 can mediate activation of NF kappa B and cytomegalovirus promoter-enhancer in SW480 cells via two distinct mechanisms, one which is activated only via TNFR p55 and leads to rapid activation of NF kappa B, and another which is overlapping with the LPS pathway.

Immunohistologic detection of non-pancreatic phospholipase A2 (type II) in human placenta and its possible involvement in normal parturition at term.

Placenta is one of the richest sources of non-pancreatic phospholipase A2 (npPLA2) (type II) in the human body, and the enzyme is the key enzyme releasing unsaturated fatty acids from membrane phospholipids. Prostaglandins (PGs) play a critical role in the initiation and progression of parturition. Cytokines are presumed to play a central role in the initiation of normal labor at term, and cytokines are also found to regulate both synthesis and secretion of npPLA2 enzyme. In an attempt to resolve the physiologic function of the npPLA2 enzyme in placental tissue, immunohistologic localization and enzymatic activity measurements of npPLA2 enzyme were performed. NpPLA2 protein was detected in vascular smooth muscle, endothelial cells and connective tissue cells in placenta and umbilical cord, and the protein was weakly stained in trophoblast cells in placenta. Enzymatic activity was not increased in sera from mother nor child compared to sera from healthy individuals, but the activity in amniotic fluid was considerably higher. Our findings support the candidacy for npPLA2 in enzymatic arachidonic acid release from foetal membranes.

Tumor necrosis factor receptor p75 mediates cell-specific activation of nuclear factor kappa B and induction of human cytomegalovirus enhancer.

The functional role of human tumor necrosis factor receptor (TNFR) p75 was studied by the use of TNFR p75-specific agonistic antibodies. Human SW480T adenocarcinoma cells, stably transfected with a reporter construct containing beta-galactosidase under the control of human cytomegalovirus immediate early enhancer, were stimulated with anti-TNFR p75 polyclonal antiserum or monoclonal antibodies followed by measurement of beta-galactosidase activity and analysis by electrophoretic mobility shift assays. It was found that cross-linking of TNFR p75 led to strong induction of the human cytomegalovirus enhancer as well as activation of nuclear factor-kappa B (NF-kappa B). Stimulation of TNFR p75 also mediated activation of NF-kappa B in human KYM-1 rhabdomyosarcoma cells but not in other cell types such as U937 and HL-60 monocytic cells or in Eahy 926 endothelial cells. NF-kappa B activation induced by TNFR p75 was delayed approximately 15 min compared with NF-kappa B activation induced by TNFR p55, indicating that the two TNFRs activate NF-kappa B through different signaling pathways. The data presented in this study identify intracellular responses mediated by TNFR p75 which have not been reported previously and suggest that TNFR p75-induced activation of NF-kappa B is strictly cell type-specific.

Elevated expression of human nonpancreatic phospholipase A2 in psoriatic tissue.

In involved psoriatic tissue, which is characterized by chronic inflammation in both epidermis and dermis, elevated levels of arachidonic acid and eicosanoids have been measured. This implies that a phospholipase A2 (PLA2) may be involved in the pathogenesis of psoriasis. The PLA2's are a group of enzymes that release unsaturated fatty acids from the sn2-position of membrane phospholipids. Once released, the fatty acids are converted by various enzymes into biologically very important signaling molecules. Release of arachidonate initiates the arachidonate cascade, leading to the synthesis of eicosanoids such as prostaglandins, thromboxanes, leukotrienes, and lipoxines. Eicosanoids are important in a variety of physiological processes and play a central role in inflammatory mediators, such as lyso-PAF (a precursor for PAF) and other lysophospholipids, may also be formed through the action of a PLA2. We report for the first time the detection of transcripts of nonpancreatic phospholipase A2 (npPLA2, type II) and cytosolic (c) PLA2 in human skin, and overexpression of npPLA2 in involved skin from patients with psoriasis (plaque psoriasis and pustular psoriasis). Limited amounts of npPLA2 enzyme are detected immunologically in the uppermost layers of epidermis from healthy persons. Both involved and uninvolved psoriatic epidermis contain higher levels of npPLA2 than normal skin. Positive cells in dermis showed significantly higher levels of npPLA2 than epidermal cells. In dermis from healthy persons, only weak staining of a few cells could be detected. The two PLA2 enzymes detected in psoriatic skin (cytosolic and nonpancreatic) may both be involved in eicosanoid overproduction in psoriatic tissue, and the npPLA2 may also be involved in potentiating cell activation, especially T cells.

Genetic carrier detection in Norwegian families with acute intermittent porphyria.

Early detection of carriers of acute intermittent porphyria (AIP) is of great value as an assistance for correct diagnosis and prevention of attacks. In order to complement traditional biochemical methods, restriction fragment length polymorphism (RFLP) studies as well as analysis for a previously identified point mutation were included in a study of three Norwegian AIP families. Several asymptomatic carriers could be identified, and the study thus demonstrates the usefulness of the combination of biochemical and genetic analysis.

The release of soluble p55 TNF receptor from U937 cells studied by a new p55 immunoassay.

A highly specific and sensitive immunoassay for soluble p55 tumor necrosis factor receptor (TNFR) has been established. The immunoassay was based on a newly developed monoclonal antibody (IV4E) recognizing a non-TNF-binding site of the p55 TNFR. The IV4E antibody immunoprecipitated a 55 kDa TNF binding protein from HL-60 cells. No binding of IV4E to the p75 TNFR could be detected. Bound TNFR to IV4E was detected with digoxigenin (DIG) labeled TNF. This assay could detect down to 300 pg/ml of soluble p55, which represents an 8-10-fold increase in sensitivity compared to earlier developed immunoassays. The assay was specific for soluble p55 TNFR present in serum and cell culture supernatants, since addition of excess unlabeled TNF together with DIG labeled TNF inhibited the signal. TNF concentrations up to 10 ng/ml in the TNFR sample did not affect the assay, indicating that TNFRs can be measured in samples containing TNF. The new immunoassay was used to study the mechanisms underlying the release of soluble p55 TNFR from U937 cells stimulated with TPA. The TPA induced release of soluble p55 TNFR from U937 cells occurred in two phases. First, a rapid increase of soluble p55 was observed after the addition of TPA. Later, the release of p55 occurred at a slower rate, and this release was inhibited by known inhibitors of protein synthesis and intracellular transport. Addition of TPA increased the p55 mRNA expression in U937 cells. The results suggest that TPA induces both release and new synthesis of p55 in U937 cells.

Trace amounts of ganglioside GM1 in human milk inhibit enterotoxins from Vibrio cholerae and Escherichia coli.

Gangliosides were isolated from human milk fat and purified by silica gel column chromatography and high performance liquid chromatography (HPLC). Low amounts of the ganglioside GM1, detected by high performance thin layer chromatography (HPTLC)-immunoassay, were found in all fractions with enterotoxin-inhibitory activity, while fractions without GM1 were inactive. It is concluded that GM1 is responsible for enterotoxin-inhibitory activity in the ganglioside fraction from human milk.