Biomedical subjects

F V Peale

Publications and source records attributed to F V Peale.

14 recordsLinked to original sources

PubMed2004

Expression of vascular endothelial growth factor, hypoxia inducible factor 1alpha, and carbonic anhydrase IX in human tumours.

AIMS: To measure vascular endothelial growth factor (VEGF-A) mRNA in a large, diverse cohort of tumours and to investigate whether VEGF-A expression is associated with markers of hypoxia, including hypoxia inducible factor 1alpha (HIF-1alpha) and carbonic anhydrase IX (CA9). METHODS: The expression of VEGF-A and CA9 was assessed in 5067 fresh frozen human tissue samples and 238 cell lines by DNA microarray analysis. In addition, tissue microarrays were constructed from 388 malignancies to investigate the expression of VEGF-A and HIF-1alpha by in situ hybridisation and immunohistochemistry, respectively. RESULTS: VEGF-A was significantly upregulated in primary malignancies of the breast, cervix, colon and rectum, oesophagus, head and neck, kidney, ovary, skin, urinary system, and white blood cells by DNA microarray analysis. However, VEGF-A expression only correlated with CA9 expression in renal tissues. In the tissue microarrays, HIF-1alpha positive cores showed a significant increase in VEGF-A expression in lung, ovary, soft tissue, and thyroid malignancies. CONCLUSIONS: The expression of VEGF-A is upregulated in a large proportion of human malignancies, and may be associated with markers of hypoxia. VEGF-A expression can be induced in the absence of hypoxia and hypoxia does not always provoke VEGF-A upregulation in tumours.

Antigens, Neoplasm↗

PubMed2003

Quantitative analysis of colorectal tissue microarrays by immunofluorescence and in situ hybridization.

The accuracy and reliability of in situ studies may be compromised by qualitative interpretations. Quantitation imposes a greater degree of objectivity, is more reproducible, and facilitates the clarity of definitions. The aim of this study was to validate the utility of laser imaging systems for the in situ quantitative analysis of gene expression in tissue microarrays. Immunofluorescence was employed to quantify the expression of the tumour suppressor p53, a marker of proliferation (Ki67), an endothelial cell marker (CD31), and the mismatch repair proteins human Mut L homologue 1 and human Mut S homologue 2 in an arrayed series of colorectal tissues (n = 110). Quantitative data on this panel of antigens were compared objectively with qualitative scoring of immunohistochemical chromogen deposition. In addition, the expression of vascular endothelial growth factor (VEGF)-A, placental growth factor, hepatocyte growth factor, and c-Met mRNA was quantified by phosphor image analysis of in situ hybridization reactions. The quantified data on p53, Ki67, and CD31 expression were significantly associated with the pathologist's score (p < or = 0.001). While hepatocyte growth factor and placental growth factor were not up-regulated, c-Met expression was increased up to 2.5-fold and the median VEGF-A expression was elevated 4-fold (p = 0.003) in this series of colorectal tumours. Laser imaging systems are therefore feasible for high-throughput, quantitative profiling of tissue microarrays.

Biomarkers, Tumor↗

PubMed2001

Detection of novel gene expression in paraffin-embedded tissues by isotopic in situ hybridization in tissue microarrays.

Correlating altered gene expression patterns with particular disease states is a critical step in understanding disease processes and developing treatment strategies. Many thousands of novel gene sequences have recently been annotated in public and private databases and are now available for analysis. Tissue-specific expression patterns of these sequences can be evaluated physically on DNA arrays and other high throughput assays, or virtually by bioinformatics mining of expressed sequence tag (EST) databases. As a secondary screening tool, in situ hybridisation (ISH) not only confirms tissue specificity, but also reveals what is often valuable information about cell-type expression patterns of nov16l sequences. Due to their availability and long-term stability at room temperature, formalin-fixed paraffin-embedded clinical specimens provide an invaluable resource for evaluating expression patterns of novel human genes. We describe a high-throughput approach for identifying and quantifying the expression of novel genes in paraffin-embedded human tissues using isotopic in situ hybridisation and tissue microarrays (TMA).

Blotting, Northern↗

PubMed2001

Dobutamine stress cine-MRI of cardiac function in the hearts of adult cardiomyocyte-specific VEGF knockout mice.

A mouse model of non-necrotic vascular deficiency in the adult heart was studied using cine-magnetic resonance imaging (MRI) and other techniques. The mice lacked cardiomyocyte-derived vascular endothelial growth factor (VEGF) following a targeted knockout in the ventricular cardiomyocytes. Quantitative endothelial labeling showed that the capillary density was significantly reduced in the hearts of knockout mice. Gene expression patterns suggested that they were hypoxic. Semiautomated MR image analysis was employed to obtain both global and regional measurements of left ventricular function at 10 or more time points through the cardiac cycle. MRI measurements showed a marked reduction in ejection fraction both at rest and under low- and high-dose dobutamine stress. Regional wall thickness, thickening, and displacement were all attenuated in the knockout mice. A prolonged high-dose dobutamine challenge was monitored by MRI. A maximal response was sustained for 90 minutes, suggesting that it did not depend on endogenous glycogen stores.

Animals↗

PubMed2001

Gene profiling techniques and their application in angiogenesis and vascular development.

The analysis of gene expression in specific tissues and physiological processes has evolved over the last 20 years from the painstaking identification of selected genes to the relatively efficient and open-ended surveying of potentially all genes expressed in a tissue. Current art for gene discovery includes the use of large-scale arrays of cDNA sequences or oligonucleotides, and molecular 'tagging' techniques such as GeneCalling and SAGE. Common to each of these techniques is a reliance on the increasingly comprehensive databases of human and mouse EST and full-length gene sequences. Early efforts to characterize candidate genes were limited by their narrow scope, while current efforts are confounded by the enormous volume of data returned. Sophisticated software tools are an integral part of the analysis, helping to organize information into coherent groups with temporal or functional similarity. These techniques, in conjunction with the continued analysis of human genetic syndromes, transgenic, and knockout mice, have driven genetic analysis of angiogenesis and vascular development from describing which individual genes are involved to defining the outlines of regulatory networks.

Animals↗

PubMed2000

FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family.

Bronchoalveolar lavage fluid from mice with experimentally induced allergic pulmonary inflammation contains a novel 9.4 kDa cysteine-rich secreted protein, FIZZ1 (found in inflammatory zone). Murine (m) FIZZ1 is the founding member of a new gene family including two other murine genes expressed, respectively, in intestinal crypt epithelium and white adipose tissue, and two related human genes. In control mice, FIZZ1 mRNA and protein expression occur at low levels in a subset of bronchial epithelial cells and in non-neuronal cells adjacent to neurovascular bundles in the peribronchial stroma, and in the wall of the large and small bowel. During allergic pulmonary inflammation, mFIZZ1 expression markedly increases in hypertrophic, hyperplastic bronchial epithelium and appears in type II alveolar pneumocytes. In vitro, recombinant mFIZZ1 inhibits the nerve growth factor (NGF)-mediated survival of rat embryonic day 14 dorsal root ganglion (DRG) neurons and NGF-induced CGRP gene expression in adult rat DRG neurons. In vivo, FIZZ1 may modulate the function of neurons innervating the bronchial tree, thereby altering the local tissue response to allergic pulmonary inflammation.

Amino Acid Sequence↗

PubMed1998

Multiplex display polymerase chain reaction amplifies and resolves related sequences sharing a single moderately conserved domain.

We present a technique, multiplex display polymerase chain reaction (MD-PCR), that amplifies and resolves coding sequences from messenger RNAs sharing only a single moderately conserved domain encoding eight or nine amino acids. The technique, a form of single-sided PCR, allows detection of known and novel genes in a family by using one degenerate primer complementary to a gene family-specific domain. A second common primer is complementary to an oligonucleotide ligated to a nearby restriction enzyme cleavage site. Uniquely, restriction enzyme digestion of single-stranded cDNA, a technique never previously performed to useful advantage, is used to increase the specificity and sensitivity of the technique. Up to several hundred bases of coding sequence are amplified simultaneously from many (potentially from all) genes in a specific family, yielding products of different sizes from different genes, and allowing amplified products to be resolved electrophoretically. Typically, more than 50% of the amplified sequences are from the targeted gene family and many of the amplified products are novel sequences. mRNAs representing less than 1 in 100,000 messages can be detected. The method allows the focused yet open-ended examination of genes in families known to be important in both normal cellular homeostasis and the etiology of many diseases.

Animals↗

PubMed1998

Expression of novel potassium channels in the chick basilar papilla.

Ionic currents are critical for the functioning of the inner ear auditory sensory epithelium. We set out to identify and molecularly clone the genes encoding the channels responsible for several currents in the chick basilar papilla. Here we describe an inward-rectifying K+ channel, cKir2.3, present in both hair cells and support cells in the apical end of the chick basilar papilla. The biophysical properties of the human ortholog, hKir2.3, are similar to those of an inward-rectifying channel found in the apical end of the chick basilar papilla, suggesting that this channel may contribute to the corresponding current. Additionally, we describe two new members of the Kv6 subfamily of putative regulatory voltage-gated K channels, cKv6.2 and cKv6.3. Both are expressed in hair cells in the apical end of the chick basilar papilla; cKv6.2 is also strongly expressed in support cells and in the brain.

Amino Acid Sequence↗

PubMed1998

Characterization of CMIX, a chicken homeobox gene related to the Xenopus gene mix.1.

Members of the TGFbeta, Wnt and FGF families act in concert to induce and pattern the mesoderm of gastrulating embryos. Downstream effectors for these growth factors include homeobox proteins, which also feed back to activate and repress upstream signaling pathways (e.g. Fainsod, A., Steinbeisser, H., De Robertis, E.M. 1994. On the function of BMP-4 in patterning the marginal zone of the Xenopus embryo. EMBO J. 13, 5015-5025; Carnac, G., Kodjabachian, L., Gurdon, J.B., Lemaire, P. 1996. The homeobox gene Siamois is a target of the Wnt dorsalization pathway and triggers organizer activity in the absence of mesoderm. Development 122, 3055-3065). As well as having interwoven upstream and downstream regulatory pathways Mix.1, siamois and goosecoid, all paired-type homeobox genes, may physically interact with each other as heterodimers to regulate dorsal-ventral polarity (Mead, P.E., Brivanlou, I.H., Kelley, C.M., Zon, L.I. 1996. BMP-4 responsive regulation of dorsal-ventral patterning by the homeobox protein Mix.1. Nature 382, 357-360). We report here a chicken paired-type homeobox gene, CMIX, with a homeodomain having 72% aa identity to its nearest homolog, Xenopus Mix.1. CMIX is expressed in the epiblast of the posterior marginal zone of early chick embryos, and later along the entire anterior-posterior axis of the primitive streak in cells of the medial ectoderm, in nascent mesoderm, but not in endoderm. Coincident with formation of prechordal mesoderm, CMIX mRNA levels rapidly decline throughout the embryo.

Amino Acid Sequence↗

PubMed1992

Cooperative estrogen receptor interaction with consensus or variant estrogen responsive elements in vitro.

Specific binding of estradiol-liganded, partially purified calf uterine estrogen receptor (ER) to a 38-base pair estrogen responsive element (ERE) consensus sequence, containing the inverted repeat 5'-GGTCAnnnTGACC-3', was measured in vitro. The ERE sites were inserted as single or multiple tandem copies in a plasmid vector [p GEM-7Zf(+)]. Results showed that one dimeric ER can interact with one ERE, and steric constraints do not inhibit binding of ER to adjacent EREs. Molybdate-stabilized monomeric (4S) ER did not bind to EREs. ER bound to single and tandem double EREs with Kd values of 0.24 and 0.23 nM, respectively. When the plasmid contained three or more tandem copies of the ERE, ER bound in a cooperative manner, as indicated by convex Scatchard plots and Hill coefficients greater than 1.5. To determine those characteristics of the consensus sequence that are important for maximal high-affinity ER binding, ten variant ERE oligomer sequences were synthesized and cloned into pGEM-7Zf(+) as single copies or as four copies in tandem. ER binding affinity was maximal for the consensus ERE and was reduced for variants containing one or two nucleotide changes in the inverted repeat. The number of nucleotides separating the inverted repeat in the ERE was critical for high-affinity ER binding. Certain sequence-variant EREs when cloned as single copies bound less ER compared to the consensus ERE, yet when cloned as four tandem copies, ER binding displayed cooperativity by Scatchard and Hill analyses. Results demonstrate that cooperative interactions noted in vivo by others are present when measured in vitro. Results strongly imply that the number, spacing, and nucleotide sequence of EREs could precisely control the amount of ER binding to estrogen-responsive genes.

Base Sequence↗

PubMed1990

A microtiter well assay for quantitative measurement of estrogen receptor binding to estrogen-responsive elements.

Reproducible, rapid measurement of estrogen receptor (ER) binding to DNA was accomplished in microtiter wells treated so that ER-DNA complexes or DNA bound in preference to free ER. Mixtures of 35S-labeled DNA and [3H]estrogen-charged ER ([3H]ER), incubated to equilibrium in microfuge tubes, were transferred to microtiter wells previously treated with histone followed by gelatin. After binding of the DNA or ER-DNA complex to the treated wells, free ER was removed by washing. Radioactivity retained in each well was measured by placing individual wells from snap-apart microtiter plates directly in scintillation fluid. Binding of DNA was saturable, and ER-DNA complex binding was complete within 2 h at 4 C. The use of 35S-labeled DNA and [3H]ER allowed stoichiometric determination of ER bound to DNA. The amount of ER specifically bound to a consensus estrogen-responsive element (ERE) containing the inverted repeat GGTCAgagTGACC was determined by comparing ER bound to plasmid containing or lacking the ERE. At saturating concentrations of ER, plasmids bearing one, two, and four EREs in tandem bound approximately one, two, and four dimeric ER molecules, respectively. Scatchard analysis of saturation binding data revealed a Kd of 0.15 nM for specific ER binding to a single ERE site. Thus, the assay detects ER retaining both DNA-binding and estrogen-binding functions. ER complexed with DNA in the well was also detected using a monoclonal antibody specific for the receptor. Simple modifications of this method would allow study of other DNA-protein interactions.

Animals↗

PubMed1989

Rapid purification of the estrogen receptor by sequence-specific DNA affinity chromatography.

Rapid purification of calf uterine estrogen receptor (ER) to near homogeneity has been accomplished by use of sequence-specific DNA affinity resin. Very high selectivity for the estrogen receptor is achieved through the use of DNA-Sepharose containing eight tandem copies of a consensus estrogen response element (ERE) DNA sequence. The highly purified ER prepared by this new scheme may be labeled economically with ligands of high specific activity. This purification scheme selects for intact receptors retaining function in both estrogen-binding and DNA-binding domains. Purified receptor has an electrophoretic mobility consistent with a molecular weight of 68,000, sediments as a 5S species on sucrose gradients, and reacts with antibody specific to the human estrogen receptor.

Animals↗

PubMed1988

Properties of a high-affinity DNA binding site for estrogen receptor.

It has been shown previously that a short sequence from the 5' regulatory region of the Xenopus laevis vitellogenin gene A2, when appropriately placed, can confer estrogen responsiveness to another gene. Using the Xenopus sequence and similar sequences from the 5' regulatory regions of other estrogen-responsive genes, we derived a consensus sequence 38 nucleotides long. The sequence contains an inverted repeat (5' C-A-G-G-T-C-A-G-A-G-T-G-A-C-C-T-G 3') and an A/T-rich region. Plasmids carrying a single copy of the sequence bound 3-fold-more partially purified estrogen receptor (ER) than did control plasmids when assayed by gel filtration. Maximum specificity for ER binding occurs at 100-150 mM ionic strength and pH 7.5-8.0. Plasmids carrying multiple copies of the sequence bound correspondingly more ER. The dissociation constant for ER bound to the sequence is 0.5 nM. This value is lower by a factor of about 400 than the dissociation constant for ER bound to an equivalent length of plasmid DNA. Portions of the consensus sequence were evaluated for binding efficiency. Plasmids containing the inverted repeat alone bound ER, though less efficiently than did plasmids containing the entire sequence. The A/T-rich region alone was ineffective in binding ER. Linearization of the plasmid DNA did not enhance specific binding efficiency for ER. This model system represents an effective tool for characterization of ER binding to DNA sequences involved in the regulation of gene expression.

Animals↗

PubMed1985

An analysis of the decrease in the assayed level of charged bovine estrogen receptor observed at physiological ionic strength.

Lower assayed levels of heifer uterine estrogen receptor (ER) occur at physiologic ionic strength when ER is separated from [3H]estradiol by Dextran-coated charcoal treatments, or by gel filtration on Sephadex or polyacrylamide resins. The assayed level of charged ER in buffers containing 150-200 mM ionic strength is approximately one-half that of ER levels assayed in buffers either at 0-50 or 400-450 mM ionic strength. Treatment of ER with trypsin or molybdate eliminates this observed reduction. Evidence is presented that the decrease results from a preferential adsorption of ER to the assay resins at 150-200 mM ionic strength. This adsorption is likely to be mediated by a hydrophobic region of the ER, which is removed by trypsin cleavage.

Acrylic Resins↗