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PubMed1990

Species and regional differences in the expression of cell-type specific elements at the human and rat tyrosine hydroxylase gene loci.

The expression of the catecholamine biosynthetic enzyme, tyrosine hydroxylase (TH), is confined to several different types of neuroendocrine cells. Using a transient assay system, we examined more than 10 kb of the human TH gene and 6.5 kb of 5' flanking sequences of the rat TH gene for DNA elements that confer cell-type specific expression. Surprisingly, these elements do not appear to be conserved in position or sequence across species. When plasmids containing DNA sequences - 749 bp from the transcription start site of the rat gene were introduced into PC12 cells, up to sixfold higher levels of expression were observed as compared to the same fragments introduced into HepG2 cells or LAN-1 cells. In contrast to the rat gene, analogous fragments of the human 5' promoter failed to confer cell-type specific expression. However, when plasmids containing a truncated thymidine kinase promoter and either orientation of a 760 by 3' human TH gene fragment were introduced into PC12 and LAN-1 cells, we observed a six- and 3.5-fold increase, respectively, over that observed for HepG2 cells. Subsequent deletion of this fragment led to significant activation of transcription in PC12 and HepG2 cell lines. These data indicate the presence of multiple elements contributing to the cell-type specific expression of tyrosine hydroxylase genes.

Adrenal Gland Neoplasms↗
PubMed1992

Nuclear factors that bind two regions important to transcriptional activity of the simian immunodeficiency virus long terminal repeat.

Previous studies identified two regions in the U3 region of a molecular clone of simian immunodeficiency virus, SIVmac142, that are important to transcriptional activity under conditions of induction as well as basal-level expression (B. Renjifo, N. A. Speck, S. Winandy, N. Hopkins, and Y. Li, J. Virol. 64:3130-3134, 1990). One region includes the NF-kappa B binding site, while the other lies just 5' of this site between nucleotides -162 and -114 (the -162 to -114 region). The fact that the NF-kappa B site mutation attenuated transcriptional activity in uninduced T cells and fibroblasts where activated NF-kappa B would not be present suggested that a factor(s) other than NF-kappa B could be acting through this site. In this study, we have identified a factor which binds to a cis element overlapping the NF-kappa B site. This factor, which we call simian factor 3 (SF3), would play a role in regulation under conditions of basal level expression, whereas under conditions of induction, NF-kappa B would act via this region. SF3 may also bind to an element in the -162 to -114 region. In addition, we have identified two other factors that bind the -162 to -114 region. One, which we designated SF1, is a ubiquitous basal factor, and the other, SF2, is a T-cell-predominant phorbol myristate acetate-inducible factor. Through identification of nuclear factors that interact with the U3 region of the SIVmac142 long terminal repeat, we can gain insight into how this virus is transcriptionally regulated under conditions of basal-level expression as well as conditions of T-cell activation.

Animals↗
PubMed2002

An enhanced molecular marker based genetic map of perennial ryegrass (Lolium perenne) reveals comparative relationships with other Poaceae genomes.

A molecular-marker linkage map has been constructed for perennial ryegrass (Lolium perenne L.) using a one-way pseudo-testcross population based on the mating of a multiple heterozygous individual with a doubled haploid genotype. RFLP, AFLP, isoenzyme, and EST data from four collaborating laboratories within the International Lolium Genome Initiative were combined to produce an integrated genetic map containing 240 loci covering 811 cM on seven linkage groups. The map contained 124 codominant markers, of which 109 were heterologous anchor RFLP probes from wheat, barley, oat, and rice, allowing comparative relationships between perennial ryegrass and other Poaceae species to be inferred. The genetic maps of perennial ryegrass and the Triticeae cereals are highly conserved in terms of synteny and colinearity. This observation was supported by the general agreement of the syntenic relationships between perennial ryegrass, oat, and rice and those between the Triticeae and these species. A lower level of synteny and colinearity was observed between perennial ryegrass and oat compared with the Triticeae, despite the closer taxonomic affinity between these species. It is proposed that the linkage groups of perennial ryegrass be numbered in accordance with these syntenic relationships, to correspond to the homoeologous groups of the Triticeae cereals.

Chromosome Mapping↗
PubMed2002

Lymphomas and high-grade astrocytomas: comparison of water diffusibility and histologic characteristics.

PURPOSE: To determine if water diffusivity within lymphomas and high-grade astrocytomas correlates with cellularity. MATERIALS AND METHODS: Echo-planar diffusion-weighted magnetic resonance (MR) images obtained in 11 patients with brain lymphomas (19 lesions) and in 17 patients with astrocytomas (19 lesions) were retrospectively reviewed. Regions of interest were drawn on apparent diffusion coefficient (ADC) maps in enhancing tumor. ADC values were normalized by dividing ADC values of tumors by those of normal-appearing regions and expressing the quotient as a ratio. Histologic samples from 11 patients with astrocytomas (11 lesions) and seven patients with lymphoma (seven lesions) were reviewed. Cellularity was measured by calculating the percentage of nuclear area and the percentage of cytoplasmic area and expressing the results as the nuclear-to-cytoplasmic (N/C) ratio. The ADC and N/C ratios of both tumor types were compared by using a two-tailed t test. RESULTS: Mean ADC ratio of lymphomas was 1.15 (SD, 0.33; standard error of the mean [SEM], 0.10), and that of high-grade astrocytomas was 1.68 (SD, 0.48; SEM, 0.11) (P <.01). Mean N/C ratio of lymphoma was 1.45 (SD, 0.94; SEM, 0.36), and that of high-grade astrocytomas was 0.24 (SD, 0.18; SEM, 0.05) (P <.01). CONCLUSION: Measurements of water diffusivity and cellularity suggest that higher cellularity contributes to more restricted diffusion.

Adult↗
PubMed1993

Carotid baroreflex responsiveness to lower body positive pressure-induced increases in central venous pressure.

Lower body positive pressure (LBPP) was applied at 4 and 30 Torr to increase central venous pressure (CVP). CVP increased with LBPP (r = 0.55, P < 0.01), and the increases were significant at 4 and 30 Torr (7.4 and 7.8 mmHg) from the control (6.0 mmHg). During LBPP cardiac output increased, which was significantly related to the increase in CVP (r = 0.63, P < 0.01). The carotid baroreflex was elicited by trains of neck pressure and suction from 40 to -65 Torr. The carotid-cardiac and carotid-vasomotor baroreflex responses were assessed by determining the maximal gains of heart rate (HR) interval and intraradial mean arterial pressure (MAP) to changes in carotid sinus pressure using a logistic model. The carotid-cardiac and carotid-vasomotor baroreflex gains were negatively related to LBPP, and at 30 Torr, both gains (5.6 ms/mmHg and -0.14 mmHg/mmHg) were significantly smaller than the control (12.4 ms/mmHg and -0.24 mmHg/mmHg). The decreases in carotid-cardiac and carotid-vasomotor baroreflex sensitivity during LBPP were primarily associated with the increases in CVP (r = -0.52, P < 0.01, and r = -0.74, P < 0.01, respectively). These data suggest that the increases in CVP and/or central blood volume diminish the sensitivity of the carotid baroreflex control of HR and MAP by enhancing the tonic inhibitory influence from the cardiopulmonary baroreceptors.

Adult↗
PubMed1994

Altered host range phenotype of the transformation-defective Ad12 mutant CS-1 is due to deletions in the E1 region.

Although it grows well in bulk infection, human adenovirus type 12 (Ad12) does not plaque efficiently in Vero cells of simian origin. After long-term passage of the virus or after transfection of Ad12 DNA into these cells, however, transformation-defective, host-range mutants giving high plaque yields in Vero cells were isolated. The original mutants have deletions in both E1a and E1b as well as additions of viral sequences at the right terminus of the genome. We have constructed a recombinant virus (Ad12d169) carrying both E1 alterations of the original mutant CS-1 on the Ad12 wild-type background. Another mutant (Ad12mut2) has additional sequences at the right terminus and an intact E1 region. In plaque assays mutant virus Ad12d169 carrying E1a deletions has an about thousandfold higher efficiency in Vero cells than Ad12wt and Ad12mut2, mapping the enhanced replication in Vero cells to the deletions in E1. Mechanical models for the influence of the E1 deletions, for example by up-regulation of the E2-encoded DNA-binding protein, are discussed to explain the efficient replication of mutant adenoviruses in Vero cells under plaque assay conditions.

Adenovirus E1 Proteins↗
PubMed2001

Auditory evoked response data reduction by PCA: development of variables sensitive to reading disability.

Long latency auditory evoked responses (AER) were formed on 232 healthy normal and learning impaired subjects to tone pairs of 50 msec inter-stimulus interval (TALAER) and also to the words "tight" and "tyke" (TTAER). Both evoked potential (EP) type have been used to demonstrate differences between good readers (WIAT Basic Reading score > 115, N = 42) and poor readers (Reading score < 85, N = 42). A largely automated, hands off approach was used to reduce artifact contamination, to develop canonical measures for discriminating good from poor readers, and to predict reading scores across the entire population including intermediate (average) readers. Eye and muscle artifact were diminished by multiple regression. Substantial EP data reduction was enabled by an unrestricted use of Principal Components Analysis (PCA). For each EP type, 40 factors encompassed 70-80% of initial variance, a meaningful data reduction of about 90:1. Factor interpretation was enhanced by mapping of the factor loadings. By discriminant analysis, resulting factors predicted reading group membership with over 80% jackknifed and also split--half replication accuracy. By multiple regression, they produced a canonical variate correlating significantly (p < 0.001) with the Basic Reading score (r = 0.39). The TTAER factors were more useful than the TALAER factors. The relevance of rapid auditory processing and phonemic discrimination measurements to dyslexia is discussed.

Brain Mapping↗
PubMed1999

Fine-scale transgenic mapping of the MyoD core enhancer: MyoD is regulated by distinct but overlapping mechanisms in myotomal and non-myotomal muscle lineages.

Skeletal muscle lineage determination is regulated by the myogenic regulatory genes, MyoD and Myf-5. Previously, we identified a 258 bp core enhancer element 20 kb 5' of the MyoD gene that regulates MyoD gene activation in mouse embryos. To elucidate the cis control mechanisms that regulate MyoD transcription, we have mutagenized the entire core enhancer using linker-scanner mutagenesis, and have tested the transcriptional activity of enhancer mutants using lacZ reporter gene expression in transgenic mouse embryos. In total, 83 stable transgenic lines representing 17 linker-scanner mutations were analyzed in midgestational mouse embryos. Eight linker-scanner mutations resulted in a partial or complete loss of enhancer activity, demonstrating that MyoD is primarily under positive transcriptional control. Six of these mutations reduced or abolished transgene expression in all skeletal muscle lineages, indicating that activation of MyoD expression in trunk, limb and head musculature is regulated, in part, by shared transcriptional mechanisms. Interestingly, however, two adjacent linker-scanner mutations (LS-14 and LS-15) resulted in a dramatic reduction in transgene expression specifically in myotomes at 11.5 days. At later stages, transgene expression was absent or greatly reduced in myotomally derived muscles including epaxial muscles (deep back muscles) and hypaxial muscles of the body wall (intercostal muscles, abdominal wall musculature). In contrast, head muscles, as well as muscles of the body derived from migrating muscle progenitor cells (e.g. limb, diaphragm), were unaffected by these mutations. In Pax-3-mutant mice, LS-14 and LS-15 transgene expression was eliminated in the body, but was unaffected in the head, yielding an identical expression pattern to the endogenous MyoD gene in mice mutant for both Myf-5 and Pax-3. These data support the hypothesis that LS-14 and LS-15 define the core enhancer targets for Myf-5-dependent activation of MyoD in myotomal muscles.

Alternative Splicing↗
PubMed1995

Body surface Laplacian electrocardiographic mapping--a review.

It is of great importance and significance to be able to noninvasively map spatially distributed cardiac electrical activity from body surface electrical recordings. The standard electrocardiographic monitoring techniques provide little spatial information regarding cardiac electrical activity. Recently, a new approach-body surface Laplacian electrocardiographic mapping-has been aggressively pursued to provide high-resolution spatial mapping of cardiac electrical activity. The fundamental innovation is the measurement of the Laplacian electrocardiogram distribution over the body surface. The body surface Laplacian electrocardiographic maps have been shown to provide enhanced ability to map multiple spatially separate cardiac bioelectric sources. This article reviews the theoretical and experimental aspects of this emerging mapping technique. First of all, the paper briefly reviews the historical development of body surface mapping and inverse solutions for mapping the distributed cardiac electrical activity. Then the paper reviews the theoretical basis of body surface Laplacian mapping and the biophysical interpretation of body surface Laplacian signals, as well as technical consideration of the Laplacian recording and instrumentation. Investigations of body surface Laplacian maps in computer models and a physical tank model, as well as physiological studies are also reviewed.

Animals↗
PubMed2001

Characterization of the rat GAD67 gene promoter reveals elements important for basal transcription and glucose responsiveness.

GAD65 and GAD67 are two isoforms of the enzyme glutamic acid decarboxylase which catalyze the production of GABA from glutamate, primarily in the brain. However, GAD and GABA also prevail in the retina, testes and islets of Langerhans. The main function of GABA is in neurotransmission, and it is involved in paracrine signalling in islets, but has also been suggested to play a role as a trophic factor in synaptogenesis and to be an important metabolite feeding into the tricarboxylic acid cycle via the GABA-shunt. Both GAD isoforms are subject to regulation, e.g. by synaptic activity. GAD65 is regulated at the level of enzyme activity by association and dissociation from its cofactor, PLP, whereas GAD67 is controlled at the level of its mRNA. To study this process in further detail, we have isolated and characterized the 5'-flanking region of the rat GAD67 gene. We report the transcriptional initiation sites and promoter sequences important for expression in islet beta-cells and C6 glioma cells, and demonstrate that the GAD67 promoter harbors elements that are responsive to glucose in primary islet cells.

5' Flanking Region↗
PubMed2002

Antivasculature effects of doxorubicin-containing liposomes in an intracranial rat brain tumor model.

Increased neovascularization and vascular hyperpermeability are integral processes in tumors, and various therapeutic strategies seek to reverse the angiogenic phenotype. Long-circulating liposomes extravasate in tumors such as the rat 9L gliosarcoma and accumulate in perivascular areas. Under such conditions, liposome-encapsulated doxorubicin (DOX) provides approximately 30% increase in life span, but free DOX is no more beneficial than a saline control. However, the relationship between drug deposition and therapeutic effect is understood poorly. In the present work, magnetic resonance (MR) and functional MR (fMR) imaging were used for noninvasive, serial evaluation of intracranial 9L tumor responses to repetitive doses of free DOX or DOX in sterically stabilized long-circulating liposomes (SSL-DOX). After multiple doses of SSL-DOX, MR imaging revealed the induction of intratumor hemorrhage in 63-75% of rats (n = 8). No hemorrhage was observed by MR imaging after a single dose of SSL-DOX, in normal brain regions in animals treated with free DOX (n = 3) or in saline controls (n = 9). Histological sections from rats sacrificed immediately after MR imaging verified the putative hemorrhagic regions and revealed necrotic and apoptotic tumor cells surrounding the area of the hemorrhage. fMR maps were obtained by comparing paired images acquired during air and Carbogen (7% CO2 and 93% oxygen) breathing. These blood oxygenation level-dependent fMR maps showed enhanced image intensity after both single and multiple doses of SSL-DOX, which suggested increased and progressive vascular permeabilization. The results suggest that the breakdown of tumor vasculature induced by SSL-DOX may arise from the perivascular accumulation of liposomes in tumor and cytotoxic effects on tumor vascular endothelium.

Angiogenesis Inhibitors↗
PubMed1990

Transcriptional activation of the human papillomavirus-16 P97 promoter by an 88-nucleotide enhancer containing distinct cell-dependent and AP-1-responsive modules.

The P97 promoter upstream of the oncogenic early genes of human papillomavirus (HPV)-16 is active in keratinocytes and in cervical carcinoma cells due to a 5' keratinocyte-dependent cis enhancer. In this study, we have mapped the main enhancer activity to an 88-nucleotide (nt) fragment composed of multiple cis elements. A 63-nt promoter-proximal enhancer core was sufficient for P97 activation in a human keratinocytic cell line, HaCaT, and in cervical carcinoma cells. Although the enhancer functioned poorly in hepatoma cells or in fibroblasts, nuclear extracts from different cells protected similar cis elements from DNase I digestion. Two protected half-palindromic NF-I/CTF sites within the 63-nt core were necessary for its function; one represents a "cytokeratin element" (CK), a previously described 8-nt sequence shared with cytokeratin gene promoters. Both sites formed complexes of the same apparent size and relative binding affinity with NF-I/CTF-like factor(s) present in all cells tested. Although cell-dependent P97 activation could be determined by similar, yet distinct NF-I/CTF-like proteins, adjacent cis elements in the enhancer core were also required for function, and may thus interact with additional transcription factors. A 25-nt distal module with two AP-1 sites increased enhancer activity and cooperated with cis elements of the proximal core. Each AP-1 site as well as a third AP-1 site near the promoter bound c-Jun and Jun/Fos in vitro, and was activated by c-Jun and c-Fos in transfections. In addition to cell type-dependent activation, HPV-16 P97 transcription may therefore respond to growth factors and oncogene products via the AP-1 pathway.

Base Sequence↗
PubMed1991

Modulation by physostigmine of head-up tilt- and bilateral carotid occlusion-induced baroreflexes in rats.

In anesthetized normotensive rats, 45 degrees head-up tilt induced a transient marked drop followed by a long-lasting slight drop in mean arterial pressure (MAP), and with cessation of tilt there is a transient pressor response. Heart rate (HR) did not change appreciably by tilting. Bilateral occlusion of common carotid arteries (BCO) induced pressor responses with tachycardia. Physostigmine (5 and 15 micrograms i.c.v.) attenuated the tilt-induced decreases in MAP. Neither the HR during tilt nor the restoration-related transient pressor response was altered by physostigmine. Acetylcholine (ACh, 5 micrograms i.c.v.) exhibited a similar attenuation of tilt-induced changes in MAP. Physostigmine enhanced BCO-induced pressor responses whereas not affecting BCO-induced HR responses. The time for onset of these changes in arterial pressure was slower than that of physostigmine-induced pressor response. Depletion of brain ACh with hemicholinium-3 (20 micrograms i.c.v.) or its derivative A-5 (20 micrograms i.c.v.) or blockade of muscarinic-receptors with atropine (1 microgram i.c.v.) blocked physostigmine-elicited modulation of both tilt- and BCO-induced reflexes. These results suggest involvement of central cholinergic systems for modulation not only of BCO-induced but also tilt-induced cardiovascular reflexes. In particular, ACh appears to enhance these reflexes by acting within the central nervous system.

Animals↗
PubMed1995

Difference images calculated from ictal and interictal technetium-99m-HMPAO SPECT scans of epilepsy.

UNLABELLED: Image processing techniques were applied to SPECT brain images to aid in the localization of epileptic foci. METHODS: Ictal and interictal cerebral perfusion SPECT images were acquired from 12 epilepsy patients (6 temporal, 6 extratemporal) after injection of 20 mCi 99mTc-HMPAO. Each ictal scan was registered to the same patient's interictal scan. Normalization of the three-dimensional data was applied to account for global percent brain uptake and total injected activity. After registration, normalization and subtraction of the SPECT images and functional difference images were computed. Difference images were calculated, which give a quantitative measure of perfusion alterations during ictus. The resulting difference images were also registered with each patient's MRI scan which permits localization of perfusion changes onto anatomical structures. RESULTS: Areas in the brain where significant perfusion increases occur correlate with areas confirmed to be seizure foci. Four of the six patients with known temporal lobe seizure foci exhibited significant perfusion increases on the difference images. These areas demonstrate a percent increase of perfusion larger than 40%. For the extratemporal seizure patients, four of the four confirmed seizure sites were diagnosed with difference images. Results on the remaining two patients were inconclusive. CONCLUSION: When compared to side-by-side visual interpretation of the ictal and interictal SPECT images, registration of SPECT and MR images together with calculated difference maps greatly enhances the ability to localize epileptic seizure foci. This offers the potential to locate epileptic seizure foci using a noninvasive, inexpensive imaging procedure and data processing algorithm.

Adolescent↗
PubMed1996

NG-nitro-L-arginine methyl ester attenuates the maintenance and expression of methamphetamine-induced behavioral sensitization and enhancement of striatal dopamine release.

We examined the roles of nitric oxide (NO) in methamphetamine (MAP)-induced behavioral sensitization and enhancement of striatal dopamine (DA) release using both in vivo and in vitro methods. Repeated administration of MAP produced augmentation of MAP-induced locomotor activity after 3-day withdrawal of MAP and an enhancement of MAP-evoked DA release from striatal slices after 6-day withdrawal. When the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L- NAME) was administered only during the period of MAP withdrawal, the behavioral sensitization and enhancement of DA release were attenuated significantly. In contrast, NG-nitro-D- arginine methyl ester, an inactive isomer of L-NAME, exhibited no such effect. When L-NAME was administered acutely before the challenge injection of MAP, behavioral sensitization was also attenuated only when the dose of L-NAME was high. Coadministration of L-NAME with MAP did not block the development of sensitization to MAP. We also examined whether MAP-induced behavioral sensitization and enhancement of DA release could be observed in vivo in a microdialysis experiment. Challenge injection of MAP caused marked enhancement of DA release in MAP-sensitized rats compared with saline-treated controls corresponding to robust augmentation of locomotor activity. When L-NAME was injected during the MAP withdrawal period, the enhancement of DA release and locomotor activity induced by challenge injection of MAP were attenuated. These results suggest that NO production plays a role in the maintenance (expression) of MAP-induced behavioral sensitization and enhancement of DA release but not in the development of these effects.

Animals↗
PubMed1994

A myeloid-lineage-specific enhancer upstream of the mouse myeloperoxidase (MPO) gene.

The myeloperoxidase (MPO) gene is selectively expressed during haemopoiesis in the granulocytic lineage. Compared with the erythroid (beta-globin) and B-cell (immunoglobulin) lineages, little is known of the regulatory sequences and transcription factors involved in the regulation of genes specific for granulopoiesis. We have approached this issue by identifying a strong enhancer for the murine MPO gene. A candidate enhancer region was mapped by the detection of a strong DNase I hypersensitive site, -3.4 to -3.2 kb upstream of the MPO gene. A 301 bp fragment encompassing the DNase I site was shown to have strong enhancer function in a transient assay following transfection of a reporter gene into a MPO-expressing cell (WEHI 3BD+), but was inactive in lymphoid cells. Analysis of sub-fragments revealed that the whole 301 bp fragment is required for maximal enhancer function.

Animals↗