Open systems: panoramic views of gene expression.

Since their development in the early 1990s, differential gene expression (DGE) technologies have been applied to a multitude of biological challenges, both for the purpose of basic biological research and as a valuable tool for the discovery and development of pharmaceuticals. In this review we survey a class of DGE technologies collectively referred to as 'open' architecture systems. These technologies are distinct from the 'closed' DGE technologies (quantitative PCR, chip technologies), in that no pre-existing biological or sequence information is necessary and they are applicable to any species. Examples of open systems include GeneCalling, SAGE, TOGA, READS, and their progenitor DGE technologies, differential display and cDNA representational difference analysis. We review these technologies and summarize a specific application using GeneCalling for novel gene discovery. Additionally, the significance of data management and experimental design in this new age of expression analysis is discussed.

Global expression analysis of extracellular matrix-integrin interactions in monocytes.

Central to immune and inflammatory responses are the integrin-mediated adhesive interactions of cells with their extracellular matrix (ECM)-rich environment. Using a comprehensive and quantitative mRNA profiling technique, we analyzed the effect of ECM-induced attachment on monocyte gene expression, its regulation by growth factors, and the integrin specificity of this event. Adhesion of cells to fibronectin resulted in increased expression of a large number of genes, which was strongly potentiated by the presence of growth factors. Adhesion activated both the NF-kappaB and Jak/STAT pathways of gene transcription and increased expression of genes involved in inflammatory and immune responses, revealing the importance of ECM-integrin interactions in these processes.

Dispelling the "mystery" of computational cognitive science.

H. Crowther-Heyck (1999) argued that early advocates of computational cognitive science, especially George Miller, aimed to bring about a revival of traditional mentalism, including the issues of consciousness and free will. He therefore found it inexplicable, and even "ironic," that they selected the computer as their main research tool because computers seem no more conscious and no more free than, for instance, the telephone switchboard that was one of the behaviorists' key metaphors. I argue, by contrast, that this misunderstands the main thrust of cognitive science, which was not to bring back all of traditional mentalism, but was rather only to give a rigorous account of intentionality. Once this is recognized, Crowther-Heyck's "mystery" of cognitive science is dispelled because, as is well known, computers use symbolic representations, and thus were seen by the early cognitive scientists as being prime mechanical models of intentional processes.

Binding of the 60-kDa Ro autoantigen to Y RNAs: evidence for recognition in the major groove of a conserved helix.

The 60-kDa Ro autoantigen is normally complexed with small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the Ro protein is also complexed with a large class of variant 5S rRNA precursors that are folded incorrectly. Using purified baculovirus-expressed protein, we show that the 60-kDa Ro protein binds directly to both Y RNAs and misfolded 5S rRNA precursors. To understand how the protein recognizes these two distinct classes of RNAs, we investigated the features of Y RNA sequence and structure that are necessary for protein recognition. We identified a truncated Y RNA that is stably bound by the 60-kDa Ro protein. Within this 39-nt RNA is a conserved helix that is proposed to be the binding site for the Ro protein. Mutagenesis of this minimal Y RNA revealed that binding by the 60-kDa Ro protein requires specific base pairs within the conserved helix, a singly bulged nucleotide that disrupts the helix, and a three-nucleotide bulge on the opposing strand. Chemical probing experiments using diethyl pyrocarbonate demonstrated that, in the presence of the two bulges, the major groove of the conserved helix is accessible to protein side chains. These data are consistent with a model in which the Ro protein recognizes specific base pairs in the conserved helix by binding in the major groove of the RNA. Furthermore, experiments in which dimethyl sulfate was used to probe a naked and protein-bound Y RNA revealed that a structural alteration occurs in the RNA upon Ro protein binding.

The thoroughly modern Aristotle: was he really a functionalist?

In recent years a debate has developed over whether Aristotle's theory of the psuche is properly characterized as having been "functionalist" in the sense that contemporary computational cognitive scientists claim to be adherents of that position. It is argued here that there are indeed some similarities between Aristotle's theory and that of contemporary functionalists but that the differences between them make it misleading, at best, for functionalists to look to Aristotle for ancient support. In particular, it is argued that Aristotle would not have--indeed, specifically did not--support the claim, central to functionalism, that the mind can in principle be transported from one body to another simply by instantiating in the new body some set of organizational properties that were instantiated in the old.

Two separate mechanisms for ligand-independent activation of the estrogen receptor.

Transient transfection experiments in which three different estrogen response element-containing reporter genes were cotransfected into HeLa cells, together with constitutively expressed estrogen receptor (ER) constructs, demonstrate that activation of the transcription of the reporter genes by epidermal growth factor (EGF) and by cholera toxin with 3-isobutyl-1-methyl-xanthine, which elevate cellular cAMP, is dependent upon the presence of functional ER. Cotransfection of the reporter genes with truncated versions of the ER shows that the two non-ligand activators of ER require different regions of the receptor to produce their effects on transcription. EGF acts primarily by means of transactivation domain AF-1, whereas cAMP acts via transactivation domain AF-2 of the ER. A point mutation that removes a major site of inducible phosphorylation within the AF-1 domain of the ER abolishes the response to EGF, but the response to estradiol and cAMP is retained. Specific inhibition of cAMP-activated protein kinase (protein kinase A) prevents the response to elevated cAMP but does not affect EGF or estradiol responses. Overexpression of the protein kinase A catalytic subunit in HeLa cells results in an amplified response to estradiol, similar to that induced by cholera toxin with 3-isobutyl-1-methyl-xanthine. Comparable experiments performed using COS-1 cells produce similar results but also reveal cell type- and promoter-specific aspects of the activation mechanisms. Apparently, the ER may be activated by three different signal molecules, estradiol, EGF, and cAMP, each using a mechanism that is distinguishable from that of the others.

Interaction between estradiol and growth factors in the regulation of specific gene expression in MCF-7 human breast cancer cells.

The response of two endogenous, estrogen-induced genes, LIV-1 and pS2, to growth factor stimulation of MCF-7 cells was examined. Epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and insulin-like growth factor-1 (IGF-1) were each able to induce an increase in the two mRNAs in the absence of estradiol, and their effects were additive to that of an optimally inducing concentration (10(-8) M) of the hormone. Induction by EGF and TGF alpha, but not by IGF-1, were also additive to induction by a saturating concentration (2 microg/ml) of insulin. TGFbeta, an antimitogenic growth factor for MCF-7 cells, did not induce LIV-1 or pS2 mRNA but inhibited induction by estradiol. Increases in mRNA were shown to reflect increases in specific gene transcription. Induction by growth factors, but not by estradiol, was dependent upon protein synthesis. Induction by both growth factors and estradiol was inhibited by the pure antiestrogen, ICI 164384 (ICI), and by the mixed agonist/antagonist, tamoxifen. Despite differences in patterns of expression in vivo and in vitro, both LIV-1 and pS2 appeared to be responsive to growth factors via a mechanism distinct from that of estradiol but requiring the estrogen receptor.

Interaction between estradiol and cAMP in the regulation of specific gene expression.

The mRNA levels of LIV-1 and pS2, two estrogen-responsive genes, are increased by the agents, cholera toxin (CT) plus 3-isobutyl-l-methylxanthine (IBMX), which cause an increase in cAMP in MCF-7 human breast cancer cells. The simultaneous addition of estradiol and CT/IBMX results in a synergistic induction of the two mRNAs. The changes in mRNA reflect changes in transcription of the two genes. Interestingly, the addition of CT/IBMX to estradiol not only causes a greater increase in transcription rate but the increase is longer-lasting that seen with the hormone alone. Stimulation of mRNA levels by CT/IBMX, but not by estradiol, was prevented by cycloheximide. Stimulation by both estradiol and by CT/IBMX was prevented by the antiestrogen, ICI 164387. Transcription of LIV-1 and pS2 genes is by both estradiol and cAMP, via separate mechanisms both requiring the estrogen receptor.

Insulin/IGF-1 modulation of the expression of two estrogen-induced genes in MCF-7 cells.

Estrogen responses of human breast cancer cell lines have frequently been shown to be promoted by insulin. We have examined the action of insulin, and its interaction with estradiol, in regulating the expression of the estrogen-induced genes, LIV-1 and pS2. Both hormones cause increases in mRNA levels of the two genes but do so by distinct mechanisms. The concentration of insulin required to produce this effect suggests that it is acting via its ability to bind to the IGF-1 receptor. Both insulin and estradiol exert their effects at the level of transcription. Induction by insulin is dependent upon continued protein synthesis whereas induction by estradiol is not. Induction by both insulin and estradiol is prevented by the pure antiestrogen. ICI 164384, indicating the requirement for an activatable estrogen receptor. Insulin does not stimulate LIV-1 expression via the androgen receptor. These results demonstrate that both estradiol and insulin can stimulate the transcription of these estrogen-inducible genes, by separate mechanisms both of which involve the estrogen receptor.

Oestrogen-induced genes, pLIV-1 and pS2, respond divergently to other steroid hormones in MCF-7 cells.

The level of oestrogen-responsive gene expression in breast tumours has been proposed as a predictor of the response of the tumour to endocrine (anti-oestrogen) therapy. We demonstrate that different oestrogen-responsive genes may differ in their responses to other hormones. pLIV-1 and pS2 are two oestrogen-regulated genes that are expressed in the MCF-7 human breast cancer cell line. We show that pLIV-1 mRNA, but not pS2 mRNA, is also induced, to a lesser extent, by progesterone, 5 alpha-dihydrotestosterone and dexamethasone. For pLIV-1, combinations of these hormones with oestradiol and with the pure anti-oestrogen, ICI 164384, indicate that the mechanism of its response to these other steroid hormones is clearly separable from its response to oestrogen. Such behaviour in breast tumours in vivo could explain the lack of absolute correlation between marker gene expression and anti-oestrogen sensitivity and between the expression of individual marker genes.

Differential expression of oestrogen regulated genes in breast cancer.

Pathological endpoints such as tumour size, lymph node status and vascular invasion remain the most useful guides in selecting treatment strategies for breast cancer. There is a need, however, to further investigate the molecular mechanisms that determine the properties of an individual tumour e.g., hormone responsiveness and probability of metastasis. While numerous prognostic factors have now been identified few have contributed to defining clinical response to therapy. Oestrogen-regulated genes are likely to be important since they not only define a functional oestrogen receptor, but alterations in their expression might provide insights into the mechanisms involved in tumour progression and loss of endocrine sensitivity. Recently an oestrogen responsive gene, pLIV1, has been isolated and shown to be expressed in ER+ disease where it appears to predict nodal involvement. The present paper describes aspects of its regulation and discusses the potential role of this and other genes in the development of endocrine resistance.

All that glitters: a review of psychological research on the aesthetics of the golden section.

Since at least the time of the Ancient Greeks, scholars have argued about whether the golden section-a number approximately equal to 0.618-holds the key to the secret of beauty. Empirical investigations of the aesthetic properties of the golden section date back to the very origins of scientific psychology itself, the first duties being conducted by Fechner in the 1860s. In this paper historical and contemporary issues are reviewed with regard to the alleged aesthetic properties of the golden section. In the introductory section the most important mathematical occurrences of the golden section are described. As well, brief reference is made to research on natural occurrences of the golden section, and to ancient and medieval knowledge and application of the golden section, primarily in art and architecture. Two major sections then discuss and critically examine empirical studies of the putative aesthetic properties of the golden section dating from the mid-19th century up to the 1950s, and the empirical work of the last three decades, respectively. It is concluded that there seems to be, in fact, real psychological effects associated with the golden section, but that they are relatively sensitive to careless methodological practices.

Tissue-specific expression of the large ATP synthase gene cluster in spinach plastids.

Plastids present in different tissues may vary morphologically and functionally, despite the fact that all plastids within the same plant contain identical genomes. This is achieved by regulation of expression of the plastid genome by tissue-specific factors, the mechanisms of which are not fully understood. The proton translocating ATP synthase/ATPase is a multisubunit complex composed of nine subunits, six encoded in the plastid and three in the nucleus. We have investigated the tissue-specific expression of the large ATP synthase gene cluster in spinach (Spinacia oleracea). This gene cluster encodes four of the six plastid-encoded ATP synthase genes. Transcript abundance, transcriptional activity, and transcript stability were investigated relative to gene dosage in root plastids and in stem, leaf, and flower chloroplasts. All three of these factors display significant tissue-specific variation. It was intriguing to discover that, although transcript abundance normalized to gene dosage varies in each tissue, transcript abundance as a proportion of the entire plastid RNA population in each tissue is not significantly different. Thus it appears that in these tissues the variation in transcription and stability of transcripts derived from the large ATP synthase gene cluster balances to yield an equivalent proportion of these transcripts in the plastid RNA population. Expression of this gene cluster in photosynthetic as well as non-photosynthetic tissues may facilitate the plasticity of structure and function which is characteristic of plastids.

Oestrogen-regulated genes in breast cancer: association of pLIV1 with lymph node involvement.

In order to isolate markers of oestrogen responsiveness in breast cancer, we have cloned a number of oestrogen-regulated genes. Two of these, pLIV1 and pLIV2 (pS2), have been shown to be predominantly expressed in oestrogen receptor (ER)+ tumours. In this study, we examined their expression in relation to various clinical and histopathological features of breast cancer, and showed that pLIV1, but not pS2, is significantly associated with lymph node involvement (P < 0.01), while pS2 is more frequently observed in premenopausal patients (P < 0.05). Subdivision of the pLIV1 data by ER and nodal status of the tumour identified a highly significant association between pLIV1 expression and lymph node involvement in ER-positive disease, with 15/24 (63%) ER+ pLIV1+ tumours showing nodal involvement. Conversely, 20/23 (87%) ER+ pLIV1- patients were lymph node-negative (P < 0.001). Subdivision of the pS2 data by ER status did not reach significance. The application of pLIV1 as a marker of lymph node involvement was further exemplified in small tumours (< < 2 cm), where 11/12 (92%) lymph node-positive patients expressed pLIV1, while 17/22 (77%) node-negative patients were pLIV1 negative (P < 0.001). Similarly, pLIV1 expression identified lymph node involvement in moderately differentiated tumours (P < 0.01), but was independent of vascular invasion. pLIV1 may, therefore, represent a candidate gene for metastatic spread in ER+ breast cancer.

Induction of germ-cell alkaline phosphatase by butyrate and cyclic AMP in BeWo choriocarcinoma cells.

BeWo choriocarcinoma cells synthesize two alkaline phosphatase isoenzymes: germ-cell alkaline phosphatase and tissue-unspecific alkaline phosphatase. We have made use of the differential heat-stabilities of these two isoenzymes to study the induction of germ-cell alkaline phosphatase by sodium butyrate and cyclic AMP (cAMP). Sodium butyrate causes a large induction of germ-cell alkaline phosphatase activity (approx. 35-fold after 96 h) after an initial lag period of 12-24 h. We showed that butyrate increases germ-cell alkaline phosphatase mRNA. Dibutyryl cAMP also induces germ cell alkaline phosphatase (approx. 2.5-fold after 96 h). When optimal concentrations of butyrate and dibutyryl cAMP were added simultaneously to cells, they caused a synergistic induction of activity. This suggested that these compounds use separate mechanisms to induce germ-cell alkaline phosphatase activity and that it is the cAMP moiety of dibutyryl cAMP that induces enzyme activity. This was confirmed by the use of two additional cAMP analogues, 8-(4-chlorophenylthio) cAMP and 8-bromo cAMP, and of two compounds, 3-methyl-1-isobutylxanthine and cholera toxin, which raise the endogenous concentration of cAMP. All four compounds caused a 2-fold increase in enzyme activity. Treatment of cells with 8-(4-chlorophenylthio) cAMP, 8-bromo cAMP and cholera toxin increased germ-cell alkaline phosphatase mRNA between 2- and 7-fold. These data suggest that this alkaline phosphatase isoenzyme is regulated at the level of its mRNA by cAMP, in a manner distinct from that of butyrate.

Placental alkaline phosphatase activity is inversely related to cell growth rate in HeLaS3 cervical cancer cells.

Placental alkaline phosphatase is an inducible enzyme, expressed in HeLaS3 cells, which has been shown to possess protein phosphotyrosine phosphatase activity. Since phosphotyrosine levels are known to increase in actively dividing cells we sought an inverse correlation between PLAP activity and growth rate in HeLaS3 cells. We found that PLAP inducers, Na-butyrate, dexamethasone, bromodeoxyuridine and dibutyryl cAMP caused a dose-dependent reduction in growth rate. Mimosine, an agent that blocks the cell cycle in G1, caused an increase in PLAP activity whilst the mitogen EGF caused a corresponding decrease in PLAP activity. PLAP activity may therefore be related to cell proliferation rate.