Seasonal variations in the stable carbon isotopic signature of biogenic methane in a coastal sediment.

Systematic seasonal variations in the stable carbon isotopic signature of methane gas occur in the anoxic sediments of Cape Lookout Bight, a lagoonal basin on North Carolina's Outer Banks. Values for the carbon isotope ratio (delta 13C) of methane range from -57.3 per mil during summer to -68.5 per mil during winter in gas bubbles with an average methane content of 95%. The variations are hypothesized to result from changes in the pathways of microbial methane production and cycling of key substrates including acetate and hydrogen. The use of stable isotopic signatures to investigate the global methane cycle through mass balance calculations, involving various sediment and soil biogenic sources, appears to require seasonally averaged data from individual sites.

Serum regulation of the estrogen responsiveness of the human breast cancer cell line MCF-7.

Initially after receiving MCF-7 cells, we were able to confirm their estrogen responsiveness. We observed significant increases in thymidine incorporation, in thymidine kinase activity, and in cell numbers in response to 10(-8) M estradiol. Subsequently, however, the cells failed to show a response to estradiol. A growth response to estradiol could be restored by increasing the serum concentration in the medium. Cells grown in 15% serum (calf or human) responded to estradiol with increased rates of growth and thymidine incorporation and increased activities of thymidine kinase and DNA polymerase. We suggest that there is present in serum a "factor" which can influence the expression of a growth response to estradiol.

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.

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.

The role of four oestrogen-responsive genes, pLIV1, pS2, pSYD3 and pSYD8, in predicting responsiveness to endocrine therapy in primary breast cancer.

The expression of four oestrogen-responsive genes in 118 immunohistochemically defined primary breast tumours has been determined by northern analysis. While all the genes are induced by oestrogen in oestrogen receptor (ER)-positive cell lines, their behaviour is different in breast tumour biopsies. This behaviour can be divided into two groups; the first containing the genes, pLIV1 and pLIV2 (pS2), which both show a significant association with ER status (P = 0.001) and a corresponding inverse relationship with epidermal growth factor receptors (EGFR) (P = 0.01 and P = 0.08, respectively). In addition, the mRNA levels of both pLIV1 and pS2 are greater in ER-positive compared to ER-negative disease (P = 0.001). While a small number of ER-negative tumours were positive for either pLIV1 (12%) or pS2 (9%), we failed to observe co-expression of pLIV1 and pS2 in ER-negative disease. In ER-positive disease, four tumour populations were identified; ER+pLIV1-pS2-, ER+pLIV1-pS2+, ER+pLIV1+pS2- and ER+pLIV1+pS2+. Interestingly, the levels of pLIV1 and pS2 when co-expressed were significantly greater in ER+pLIV1+pS2+ tumours compared to either of the ER+pLIV1+pS2- (P = 0.03) or ER+pLIV1-pS2+ (P = 0.01) mixed phenotypes. Unlike pLIV1 and pS2, pSYD3 and pSYD8 belong to a group of genes which are expressed in the majority of tumours regardless of ER and EGFR status. However, lower pSYD8 mRNA levels were detected in moderately EGFR-positive disease (P = 0.06) while both pSYD3 positivity (P = 0.01) and mRNA levels (P = 0.001) were increased in highly proliferating tumours as shown by Ki67 immunostaining. These genes provide additional markers which, in conjunction with other parameters, may help to determine the likelihood of a given tumour to respond to endocrine therapy.

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.

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.

Polyamines and growth regulation of cultured human breast cancer cells by 17 beta-oestradiol.

The growth of ZR-75-1 cells, a line of human breast cancer cells in culture, is stimulated by oestradiol and inhibited by anti-oestrogens. Changes in growth rate caused by these agents are accompanied by changes in activity of ornithine decarboxylase, a rate-limiting enzyme for polyamine synthesis. Furthermore, the growth inhibition caused by tamoxifen, an anti-oestrogen, can be reversed by the addition of spermine, spermidine or putrescine to the cells. Insulin can also stimulate ZR-75-1 cell growth and this is again accompanied by an increase in ODC activity. The reduced cell growth rate observed when the cells become confluent is associated with a marked decrease in ornithine decarboxylase activity. Experiments performed with DFMO, a specific and irreversible inhibitor of ODC, show that this compound can prevent the stimulation of growth by oestradiol and that this may be overcome by the addition of putrescine to the cells. It would appear that increased ODC activity and polyamine synthesis are necessary components of the stimulation of breast cancer cell growth by oestradiol but that other growth regulatory stimuli also may act via this enzyme.

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.

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.

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.

Effects of oestrogen on the expression of a 4.4 kb mRNA in the ZR-75-1 human breast cancer cell line.

A cDNA library has been constructed from the poly(A)+ mRNA of oestrogen-stimulated ZR-75-1 human breast cancer cells. Screening by differential hybridization has identified eight clones which are stimulated between 4- and 16-fold by oestrogen. Two clones (pLIV-1) that are stimulated 4-fold, hybridize to three different mRNA species. A further five recombinants encode for a mRNA 600 bp long which is induced greater than 16-fold and have been shown to cross-hybridize to the oestrogen-responsive clone, pS2, isolated from the MCF-7 breast cancer cell line. Oestradiol was shown to be without detectable effect upon the expression of mRNA for dihydrofolate reductase, which is reported to be oestrogen regulated in MCF-7 cells. Actin gene expression is also unresponsive to oestradiol in ZR-75-1 cells. These results suggest that pLIV-1 represents a previously unidentified mRNA that may be involved in the oestrogen-regulated growth of ZR-75-1 human breast cancer cells.

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.

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.

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.