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Results for “Enhancer mapping”

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PubMed2003

Characterization of green seed, an enhancer of abi3-1 in Arabidopsis that affects seed longevity.

Seeds are usually stored in physiological conditions in which they gradually lose their viability and vigor depending on storage conditions, storage time, and genotype. Very little is known about the underlying genetics of seed storability and seed deterioration. We analyzed a mutant in Arabidopsis disturbed in seed storability. This mutant was isolated as a grs (green-seeded) mutant in an abi3-1 (abscisic acid 3) mutant background. Genetic and physiological characterization showed that the monogenic grs mutant was not visibly green seeded and mapped on chromosome 4. This enhancer mutation did not affect the ABA sensitivity of seed germination or seed dormancy but was found to affect seed storability and seedling vigor. Seed storability was assessed in a controlled deterioration test, in which the germination capacity of the mutant decreased with the duration of the treatment. The decrease in viability and vigor was confirmed by storing the seeds in two relative humidities (RHs) for a prolonged period. At 60% RH, the mutant lost germinability, but storage at 32% RH showed no decrease of germination although seed vigor decreased. The decrease in viability and vigor could be related to an increase in conductivity, suggesting membrane deterioration. This was not affected by light conditions during imbibition, expected to influence the generation of active oxygen species. During seed maturation, ABI3 regulates several processes: acquiring dormancy and long-term storability and loss of chlorophyll. Our results indicate that GRS is a common regulator in the latter two but not of dormancy/germination.

Abscisic Acid↗

PubMed1988

Functional anatomy of the simian virus 40 late promoter.

We have examined the control sequences for the late promoter function of simian virus 40 (SV40) in COS-1 cells which produce SV40 T antigen constitutively. Plasmids were constructed by cloning mutant late promoter segments upstream from sequences coding for the bacterial chloramphenicol acetyltransferase (CAT) gene, and were converted to "double-origin" type by inserting functional replication origin segments downstream from the CAT gene for replicative competence when necessary. The late promoter activity was determined by transient expression assay of the CAT mRNA and enzyme activity levels following DNA-mediated gene transfer into COS-1 cells. We find that the minimal replication origin and the 21-bp repeat containing T antigen and transcription factor Sp1 binding sites, respectively, are dispensable for late promoter function provided that one copy of the 72-bp repeat enhancer is present. We have mapped within the 72-bp repeat the major late promoter component in a 68-bp fragment (located between nucleotides 205 and 272), and found an overlapping 55-bp fragment (located between nucleotides 179 and 234) to have about one-fifth of the late promoter activity. Both the 68- and 55-bp fragments lack some of the core sequence elements required of the 72-bp repeat for transcriptional enhancer activity, and lack the ability to enhance the activity of the SV40 early promoter. The results suggest that the organization of functional units of the 72-bp repeat required for transcriptional enhancement of the early promoter is different from that required for late promoter function. The 21-bp repeat was found to have some late promoter activity located within the origin-distal copy in the absence of the 72-bp repeat. In association with the 21-bp repeat, the otherwise dispensable origin-proximal 22-bp of the 72-bp repeat containing activator protein AP-1 binding site augmented late promoter activity by three- to fourfold.

Antigens, Viral, Tumor↗

PubMed2001

A strategy to identify positional candidate genes conferring Marek's disease resistance by integrating DNA microarrays and genetic mapping.

Marker-assisted selection (MAS) to enhance genetic resistance to Marek's disease (MD), a herpesvirus-induced T cell cancer in chicken, is an attractive alternative to augment control with vaccines. Our earlier studies indicate that there are many quantitative trait loci (QTL) containing one or more genes that confer genetic resistance to MD. Unfortunately, it is difficult to sufficiently resolve these QTL to identify the causative gene and generate tightly linked markers. One possible solution is to identify positional candidate genes by virtue of gene expression differences between MD resistant and susceptible chicken using deoxyribonucleic acid (DNA) microarrays followed by genetic mapping of the differentially-expressed genes. In this preliminary study, we show that DNA microarrays containing approximately 1200 genes or expressed sequence tags (ESTs) are able to reproducibly detect differences in gene expression between the inbred ADOL lines 63 (MD resistant) and 72 (MD susceptible) of uninfected and Marek's disease virus (MDV)-infected peripheral blood lymphocytes. Microarray data were validated by quantitative polymerase chain reaction (PCR) and found to be consistent with previous literature on gene induction or immune response. Integration of the microarrays with genetic mapping data was achieved with a sample of 15 genes. Twelve of these genes had mapped human orthologues. Seven genes were located on the chicken linkage map as predicted by the human-chicken comparative map, while two other genes defined a new conserved syntenic group. More importantly, one of the genes with differential expression is known to confer genetic resistance to MD while another gene is a prime positional candidate for a QTL.

Animals↗

PubMed2003

Feasibility of abdomino-pelvic T1-weighted real-time thermal mapping of laser ablation.

PURPOSE: To prove the hypothesis that T1-weighted (T1W) thermal mapping is reliable and achievable in magnetic resonance (MR)-guided laser tumor ablation. MATERIALS AND METHODS: Near real-time gray and color-scale T1W thermal maps in 111 MR-guided laser thermal ablations (LTA) of liver, kidney, and uterine tumors were studied. After laser fiber placement, near real-time gray and color-scale thermal maps were produced. Previous work showed T1 signal is inversely proportional to temperature below 55 degrees C (the point of irreversible necrosis). RESULTS: Thermal mapping was successful in 84% of uterine, 74% of hepatic, and 20% of renal ablations. For hepatic and uterine tumors, size and conspicuity of thermal lesions were significantly greater on subtracted colorized images rather than gray-scale raw image mapping (P = 0.001, paired Student's t-test). Patient movement (N = 24), fiber charring N = 2), magnetic field distortion, and reconstruction errors (N = 2) caused mapping failure. For both renal and hepatic tumors, comparison of near real-time T1W colorized thermal map and follow up gadolinium-enhanced MR imaging revealed moderate correlation (Pearson correlation coefficient of 0.55 and 0.5, respectively). CONCLUSION: In hepatic, renal, and uterine thermal maps, the color scale produced significantly greater sized lesions with significantly greater conspicuity than the gray scale. T1W thermal mapping was reliable and successfully achieved in 73.7% of procedures, and predicted the ablated area of the tumor moderately well.

Carcinoma, Hepatocellular↗

PubMed1995

Derivation and partial analysis of two highly active myeloma cell transfectants.

Vectors have been designed to optimise the expression of heterologous proteins in transfected mouse myeloma cells. The over-ridingly important DNA element contained in these constructs is the classical mouse immunoglobulin heavy chain enhancer. It is shown that even in the absence of a well-known promoter element, the enhancer can drive gene expression in stable cell transfectants and the main transcriptional start site utilized in such situations has been mapped to within the previously defined enhancer region. Using chicken lysozyme as a reporter function in these vectors, two transfected myeloma cell clones have been isolated which secrete this protein at levels 50-100-times as high as those usually obtained with the same vectors and it is shown that in molar terms this is at least as high as endogenous immunoglobulin produced by a related line. Analysis of these lines show that in one case only a single copy, and in the other two to three copies, of the apparently unrearranged vector have integrated at a single locus within the genome. Possible explanations for the high-level expression are discussed.

Animals↗

PubMed2025

Genetic-epigenetic interactions (meQTLs) in orofacial clefts etiology.

OBJECTIVES: Nonsyndromic orofacial clefts (OFCs) involve complex genetic and environmental factors, with over 60 risk loci accounting for only a minority of estimated heritability and residing in non-coding regions with unclear functional relevance. We hypothesize that some genetic variants alter orofacial cleft risk by modifying DNA methylation (DNAm) at regulatory sequences essential for craniofacial development, acting as methylation quantitative trait loci (meQTLs). METHODS: We analyzed 10 well-established OFC-associated SNPs against genome-wide DNAm profiles in 409 cases and 456 controls, identifying 23 potential meQTLs. We validated findings using 358 cleft-discordant sibling pairs analyzed with quantitative MethyLight assays. Cross-referencing with the mQTL Database assessed temporal patterns across human development. Functional annotation used GeneHancer and craniofacial enhancer databases. RESULTS: Nine meQTLs were successfully replicated, including the highly significant rs987525 (8q24) - cg16561172 (MYC) association (P = 9.610E-6). This association mapped to a mesendoderm-active enhancer upstream of MYC, providing mechanistic explanation for the longstanding 8q24 cleft locus. Additional validated associations involved MAFB-PLCG1, NOG-PPM1E, FOXE1-FRZB, and SPRY2-LGR4 interactions. Independent differential methylation analysis revealed significant differences between discordant siblings at three CpG sites. Cross-referencing confirmed concordance with population-level methylation effects, with childhood representing the critical developmental window for most associations. CONCLUSIONS: This systematic meQTL characterization in OFCs demonstrates that genetic variants influence disease risk through epigenetic mechanisms. The 8q24-MYC regulatory pathway evidence provides crucial mechanistic insight into a major OFC risk locus. These findings bridge genetic associations with functional consequences, address missing heritability challenges, and suggest potential biomarkers and therapeutic targets for OFC prevention and treatment.

Journal Article↗

PubMed1988

Capturing nuclear sequence-specific DNA-binding proteins by using simian virus 40-derived minichromosomes.

We have used recombinant simian virus 40 (SV40) minichromosomes to retrieve sequence-specific DNA-binding proteins derived from the cell nucleus of COS-7 cells. We showed that the transcription factors AP-1 and Sp1 are stably bound to the SV40 DNA late in viral infection. Under similar conditions, minichromosomes carrying the rat insulin (rINS1) enhancer, which is under negative regulation in COS-7 cells, bound two proteins which mapped to distinct regions of the rINS1 enhancer. The SV40 P element competed for one of these proteins which bound to the region from -198 to -230. This factor may be related to AP-1. The other factor selectively bound a regulatory element in the region from -92 to -124 of the insulin enhancer. These proteins may play a role in regulating the rINS1 enhancer function.

Animals↗

PubMed1993

Ocular effects of topical administration of the phosphodiesterase III inhibitor milrinone in rabbits and cats.

Increases in intracellular cAMP levels have previously been shown to decrease intraocular pressure (IOP) and increase ocular blood flow (OBF). However, the ocular effects of milrinone, which increases intracellular cAMP levels via selective cAMP PDE III inhibition, have not been investigated. The purpose of this study was to investigate the ocular effects of topically administered milrinone at different concentrations in rabbits and cats. When compared to vehicle in conscious rabbits, topical administration of milrinone at 0.03% decreased IOP (-14.1 +/- 2.6% vs. -7.4 +/- 3.7%, max. changes expressed as mean +/- SEM), at 0.1% increased IOP (10.4 +/- 8.5% vs. -1.7 +/- 4.1%), and at 0.01% and 1% did not significantly affect IOP. Neither pupil size nor central corneal thickness were affected by milrinone. Additionally, there were no signs of inflammation and no effects on corneal clarity. In anesthetized cats, topical administration of milrinone at 0.01-0.3% increased OBF (38.9 +/- 6.0% for milrinone vs. -7.4 +/- 4.4% for vehicle), and at 0.03%-0.3% decreased mean arterial pressure (-19.0 +/- 5.6 vs. 3.0 +/- 4.1 mmHg) in a dose-related manner. The durations of OBF enhancement (1.5-2.5 h) and MAP reduction (less than 30 min to 2 h) were also dose-dependent. In conclusion, milrinone induced biphasic IOP effects: IOP was decreased at 0.03% but increased at 0.1%. Milrinone at 0.01% increased OBF, possibly via a local vasodilator effect, and at 0.03-0.3% increased OBF, possibly via local and systemic effects. These data suggest that cAMP PDE III inhibitors such as milrinone may have efficacy as agents which enhance ocular blood flow following topical ocular application.

Administration, Topical↗

PubMed2004

Study of association between alpha-synuclein gene polymorphism and methamphetamine psychosis/dependence.

Methamphetamine (MAP) dissipates proton gradients across the membranes of synaptic vesicles, enhances cytoplasmic dopamine (DA) concentrations, and causes calcium-independent, nonvesicular DA release into synapses. MAP is taken into the cytosol by the dopamine transporter (DAT) on the synaptic terminals of DA neurons, and endogenous DA is concurrently released through the transporter by carrier exchange mechanisms, resulting in a robust increase in DA concentration in the synaptic clefts. The enhanced DA release through DAT by MAP is the main mechanism for the reinforcing effects of MAP. The complexes of alpha-synuclein and DAT facilitate membrane clustering of the DAT, thereby accelerating DA uptake in vitro. alpha-Synuclein has been shown to be overexpressed in the midbrain DA neurons of chronic cocaine abusers. The present study was performed to study the association between the alpha-synuclein gene polymorphisms and MAP psychosis/dependence in Japanese population. Since the T10A7 polymorphic site at the 5' end of the noncoding exon 1' in the alpha-synuclein gene is highly polymorphic, we analyzed the noncoding exon 1' and intron 1, including this polymorphic site by sequencing. We confirmed four single nucleotide polymorphisms (SNPs) within 1.38 kbp of the T10A7 polymorphic site. No significant difference was found in genotype or allele frequencies in the T10A7 polymorphic site between MAP psychotic/dependent and control subjects. We found significant association between three SNPs in the vicinity of this polymorphic site in intron 1 and MAP psychosis/dependence in female subjects, but not in males. These results suggest an association of the alpha-synuclein gene polymorphisms with MAP psychosis/dependence in our female subjects. Further analyses are necessary to clarify the gender difference, by using a larger sample size and/or different ethnic groups, as well as functional variations in the alpha-synuclein gene.

Adult↗

PubMed2025

H3K4me2 distinguishes a distinct class of enhancers during the maternal-to-zygotic transition.

After egg fertilization, an initially silent embryonic genome is transcriptionally activated during the maternal-to-zygotic transition. In zebrafish, maternal vertebrate pluripotency factors Nanog, Pou5f3 (OCT4 homolog), and Sox19b (SOX2 homolog) (NPS) play essential roles in orchestrating embryonic genome activation, acting as "pioneers" that open condensed chromatin and mediate acquisition of activating histone modifications. However, some embryonic gene transcription still occurs in the absence of these factors, suggesting the existence of other mechanisms regulating genome activation. To identify chromatin signatures of these unknown pathways, we profiled the histone modification landscape of zebrafish embryos using CUT&RUN. Our regulatory map revealed two subclasses of enhancers distinguished by presence or absence of H3K4me2. Enhancers lacking H3K4me2 tend to require NPS factors for de novo activation, while enhancers bearing H3K4me2 are epigenetically bookmarked by DNA hypomethylation to recapitulate gamete activity in the embryo, independent of NPS pioneering. Thus, parallel enhancer activation pathways combine to induce transcriptional reprogramming to pluripotency in the early embryo.

Animals↗

PubMed2000

Determining the role of the Epstein-Barr virus Cp EBNA2-dependent enhancer during the establishment of latency by using mutant and wild-type viruses recovered from cottontop marmoset lymphoblastoid cell lines.

Epstein-Barr virus (EBV) nuclear antigen (EBNA) 2 (EBNA2) is involved in upregulating the expression of both EBNAs and latency-associated membrane proteins. Transcription of the six EBNA genes, which are expressed in EBV-immortalized primary B cells, arises from one of two promoters, Cp and Wp, located near the left end of the viral genome. Wp is exclusively used to drive EBNA gene transcription during the initial stages of infection in primary B cells; induction of transcription from Cp follows. We previously have mapped an EBNA2-dependent enhancer upstream of Cp (M. Woisetschlaeger et al., Proc. Natl. Acad. Sci. USA 88:3942-3946, 1991) and, more recently, have demonstrated that deletion of this enhancer results in EBV-immortalized lymphoblastoid cell lines (LCLs) that are heavily biased toward the use of Wp to drive transcription of the EBNA genes (L. Yoo et al., J. Virol. 71:9134-9142, 1997). To assess the immortalizing capacity of this mutant EBV and to monitor the early events after infection of primary B cells, B cells isolated from cottontop marmosets were used to generate LCLs immortalized with the Cp EBNA2 enhancer deletion mutant virus. As previously reported, all EBV-infected marmoset LCLs examined could be triggered to produce significant levels of virus. Infection of human B cells with wild-type or Cp EBNA2 enhancer mutant viruses recovered from marmoset B-cell lines demonstrated that (i) the Cp EBNA2 enhancer mutant virus immortalizes primary human B cells nearly as efficiently as wild-type virus and (ii) the Cp EBNA2-dependent enhancer plays an important role in the induction of Cp activity during the early stages of infection. The latter is consistent with the phenotype of LCLs immortalized with the Cp EBNA2 enhancer mutant EBV. Finally, using an established LCL in which EBNA2 function is regulated by beta-estradiol, we showed that the loss of EBNA2 function results in an approximately 4-fold decrease in the steady-state levels of Cp-initiated transcripts and a concomitant increase in the steady-state levels of Wp-initiated transcripts. Taken together, these results provide strong evidence that EBNA2 plays an important role in regulating Cp activity. These results also demonstrate that diminished induction of Cp activity does not appear to affect the ability of EBV to immortalize primary B cells in cultures. Finally, as shown here, infection of marmoset B cells with immortalization-competent mutants of EBV provides a convenient reservoir for the production of mutant viruses.

Animals↗

PubMed1981

Possible mechanisms of enkephalin action on hippocampal CA1 pyramidal neurons.

(1) Intracellular and extracellular recordings were made from CA1 pyramidal neurons in an in vitro rat hippocampal slice preparation, while [D-Ala2, D-Leu5]enkephalin (DADL) was applied by perfusion at a known concentration (1 to 5 X 10-7 M), in a small droplet, or by iontophoresis into the cellular and dendritic layers of the slice. The effects of DADL on synaptic potentials and membrane properties were studied in an effort to determine the mechanisms underlying its epileptogenic action in the hippocampus. (2) DADL increased the size and often the duration of excitatory postsynaptic potentials (EPSPs) generated on either the apical or basal dendrites; this resulted in an increased discharge probability for a constant orthodromic stimulus. Extracellular field potential recordings showed a larger population spike for a given size field EPSP. These effects of DADL could be reversed substantially by perfusion with naloxone (1 to 5 X 10-7 M) and appeared qualitatively different from the epileptiform actions of penicillin. (3) DADL did not appear to increase the intrinsic excitability of the soma membrane, since membrane potential, input resistance, spike threshold, and antidromic field potentials all were unchanged. In addition, the shape of the membrane charging curve during hyperpolarizing current injection was not changed noticeably by DADL. (4) At the concentrations tested, DADL did not attenuate recurrent inhibition in the CA1 region, as evaluated by comparing in the absence and presence of DADL: (a) antidromically evoked recurrent inhibitory postsynaptic potentials (IPSPs) and their dependence of membrane potential, (b) the reduction of a synaptically driven population spike by a prior antidromic volley, (c) iontophoretic GABA (gamma-aminobutyric acid) responses. Similarly, IPSPs evoked by orthodromic stimulation appeared either unaffected or occasionally enhanced by DADL. (5) By iontophoretic mapping, it was shown that the DADL-sensitive sites are limited to stratum oriens and stratum pyramidale. Local application of DADL into stratum radiatum was relatively ineffective in enhancing the efficacy of synapses located in this region. (6) The dendritic input-output relationship between the presynaptic fiber volley and the field EPSP was not changed by DADL. This finding and the results of the iontophoretic mapping experiments suggest that increased excitatory transmitter release was not involved. (7) The data are consistent with the proposal that DADL selectively attenuates a dendritic IPSP which is virtually invisible to the soma, although the possibility cannot be ruled out that DADL may, in addition, act to enhance the responsiveness of pyramidal dendritic membrane to excitatory synaptic activation.

Animals↗

PubMedDate not supplied

Breast cancer in limited-resource countries: treatment and allocation of resources.

Treating breast cancer under the constraints of significantly limited health care resources poses unique challenges that are not well addressed by existing guidelines. We present evidence-based guidelines for systematically prioritizing cancer therapies across the entire spectrum of resource levels. After consideration of factors affecting the value of a given breast cancer therapy (contribution to overall survival, disease-free survival, quality of life, and cost), we assigned each therapy to one of four incremental levels--basic, limited, enhanced, or maximal--that together map out a sequential and flexible approach for planning, establishing, and expanding breast cancer treatment services. For stage I disease, basic-level therapies are modified radical mastectomy and endocrine therapy with ovarian ablation or tamoxifen; therapies added at the limited level are breast-conserving therapy, radiation therapy, and standard-efficacy chemotherapy (cyclophosphamide, methotrexate, and 5-fluorouracil [CMF], or doxorubicin and cyclophosphamide [AC], epirubicin and cyclophosphamide [EC], or 5-fluorouracil, doxorubicin, and cyclophosphamide [FAC]); at the enhanced level, taxane chemotherapy and endocrine therapy with aromatase inhibitors or luteinizing hormone-releasing hormone (LH-RH) agonists; and at the maximal level, reconstructive surgery, dose-dense chemotherapy, and growth factors. For stage II disease, the therapy allocation is the same, with the exception that standard-efficacy chemotherapy is a basic-level therapy. For locally advanced breast cancer, basic-level therapies are modified radical mastectomy, neoadjuvant chemotherapy (CMF, AC, or FAC), and endocrine therapy with ovarian ablation or tamoxifen; the therapy added at the limited level is postmastectomy radiation therapy; at the enhanced level, breast-conserving therapy, breast-conserving whole-breast radiation therapy, taxane chemotherapy, and endocrine therapy with aromatase inhibitors or LH-RH agonists; and at the maximal level, reconstructive surgery and dose-dense chemotherapy and growth factors. For metastatic or recurrent disease, basic-level therapies are total mastectomy for ipsilateral in-breast recurrence, endocrine therapy with ovarian ablation or tamoxifen, and analgesics; therapies added at the limited level are radiation therapy and CMF or anthracycline chemotherapy; at the enhanced level, chemotherapy with taxanes, capecitabine, or trastuzumab, endocrine therapy with aromatase inhibitors, and bisphosphonates; and at the maximal level, chemotherapy with vinorelbine, gemcitabine, or carboplatin, growth factors, and endocrine therapy with fulvestrant. Compared with the treatment of early breast cancer, the treatment of advanced breast cancer is more resource intensive and generally has poorer outcomes, highlighting the potential benefit of earlier detection and diagnosis, both in terms of conserving scarce resources and in terms of reducing morbidity and mortality. Use of the scheme outlined here should help ministers of health, policymakers, administrators, and institutions in limited-resource settings plan, establish, and gradually expand breast cancer treatment services for their populations.

Aromatase Inhibitors↗

PubMed2001

Discretizing large traceable vessels and using DE-MRI perfusion maps yields numerical temperature contours that match the MR noninvasive measurements.

The success of hyperthermia treatments is dependent on thermal dose distribution. However, the three-dimensional temperature distribution remains largely unknown. Without this knowledge, the relationship between thermal dose and outcome is noisy, and therapy cannot be optimized. Accurate computations of thermal distribution can contribute to an optimized therapy. The hyperthermia modeling group in the Department of Radiotherapy, University Medical Center Utrecht devised a Discrete Vasculature [Kotte et al., Phys. Med. Biol. 41, 865-884 (1996)] model that accounts for the presence of vessel trees in the computational domain. The vessel tree geometry is tracked using magnetic resonance (MR) angiograms to a minimum diameter between 0.6 and 1 mm. However, smaller vessels (0.2-0.6 mm) are known to account for significant heat transfer. The hyperthermia group at Duke University Medical Center has proposed using perfusion maps derived from dynamic-enhanced magnetic resonance imaging to account for the tissue perfusion heterogeneity [Craciunescu et al., Int. J. Hyperthermia 17, 221-239 (2001)]. In addition, techniques for noninvasive temperature measurements have been devised to measure temperatures in vivo [Samulski et al., Int. J. Hypertherminal, 819-829 (1992)]. In this work, a patient with high-grade sarcoma has been retrospectively modeled to determine the temperature distribution achieved during a hyperthermia treatment. Available for this model were MR depicted geometry, angiograms, perfusion maps, as necessary for accurate thermal modeling, as well as MR thermometry data for validation purposes. The vasculature assembly through modifiable potential program [Van Leeuwen et al., IEEE Trans. Biomed. Eng. 45, 596-604 (1998)] was used in order to incorporate the traceable large vessels. Temperature simulations were made using different approaches to describe perfusion. The simulated cases were the bioheat equation with constant perfusion rates per tissue type, perfusion maps alone, tracked vessel tree and perfusion maps, and generated vessel tree. The results were compared with MR thermometry data for a single patient data set, concluding that a combination between large traceable vessels and perfusion map yields the best results for this particular patient. The technique has to be repeated on several patients, first with the same type of malignancy, and after that, on patients having malignancies at other different sites.

Algorithms↗

PubMed2005

Enhancing BOLD response in the auditory system by neurophysiologically tuned fMRI sequence.

Auditory neuroscience has not tapped fMRI's full potential because of acoustic scanner noise emitted by the gradient switches of conventional echoplanar fMRI sequences. The scanner noise is pulsed, and auditory cortex is particularly sensitive to pulsed sounds. Current fMRI approaches to avoid stimulus-noise interactions are temporally inefficient. Since the sustained BOLD response to pulsed sounds decreases with repetition rate and becomes minimal with unpulsed sounds, we developed an fMRI sequence emitting continuous rather than pulsed gradient sound by implementing a novel quasi-continuous gradient switch pattern. Compared to conventional fMRI, continuous-sound fMRI reduced auditory cortex BOLD baseline and increased BOLD amplitude with graded sound stimuli, short sound events, and sounds as complex as orchestra music with preserved temporal resolution. Response in subcortical auditory nuclei was enhanced, but not the response to light in visual cortex. Finally, tonotopic mapping using continuous-sound fMRI demonstrates that enhanced functional signal-to-noise in BOLD response translates into improved spatial separability of specific sound representations.

Acoustic Stimulation↗

PubMedDate not supplied

Colour Doppler flow mapping: providing an insight into cardiac hydrodynamics.

Colour Doppler flow mapping can now provide spatial velocity information in relation to surrounding structural detail as imaged by conventional echocardiography and, as such, represents a major advance in non-invasive cardiac imaging. This article describes the basic principles of colour Doppler flow mapping, details how the Doppler information is processed and displayed to arrive at the real-time two-dimensional flow-enhanced image. Since colour Doppler flow mapping is really the first available technique which allows detailed real-time velocity information to be displayed in vivo and in relation to intracardiac structures, it can allow important insight into the haemodynamics of intracardiac flow. With the application of digital computer analysis techniques it may also be possible to extract valuable quantitative information from colour Doppler flow mapping.

Blood Flow Velocity↗

PubMed1993

Characterization of the role of individual protein binding motifs within the hepatitis B virus enhancer I on X promoter activity using linker scanning mutagenesis.

A combination of linker scanning mutagenesis and deletional analyses has been used to determine the role of individual DNA-protein binding sites on expression from the hepatitis B virus (HBV) enhancer I-X promoter (map position (mp) 1042-1354, HBV adw2). Linker scanning mutation of the EF-C site caused a 67.5% drop in X promoter activity in HuH7 cells, but had no effect in HepG2 or HepSK cells. Mutation of the E element resulted in an approximately 50% reduction in X promoter activity in HuH7, HepG2, and HepSK cells. Deletional analysis showed that sequences upstream of the EF-C site (mp 1163) were required for full X promoter activity and implicated the NF-1a site as being sufficient for basal X promoter activity. However, PCR-directed linker scanning mutation of the NF-1a site did not cause a reduction in X promoter activity, indicating that this site was not an essential component of the X promoter. Taken together, these results indicated that multiple, partially redundant protein:DNA interactions in the enhancer I are essential for full X promoter activity. The lack of an essential basal promoter element supports the suggestion that the two separate HBV enhancer elements (enhI and enhII) were created by integration of the X gene into a primordial enhancer element.

Base Sequence↗

PubMed1993

Down-regulation of calcitonin gene transcription by vitamin D requires two widely separated enhancer sequences.

Transcription of the calcitonin (CT) gene is down-regulated by vitamin D in normal and transformed thyroid C cells. DNA transfer techniques have been previously used to map and characterize a cAMP-induced enhancer at nucleotides -255 to -129 and an enhancer of basal transcription at -1060 to -905 in the CT 5' flanking DNA. The same methods were used to identify a negative response element for vitamin D. Deletion mutants of a genomic fragment of CT extending from nucleotides -1460 to +90 were attached to a promoterless GH gene and transfected individually into the medullary thyroid carcinoma cell line TT. CT nucleotides -1460 to -129 induced significant basal transcription of the GH reporter gene in TT cells. Basal transcription was elevated 3-fold to 4-fold by treatment with cAMP analog. The biologically active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3, had a minor (20%) inhibitory effect on basal transcription but inhibited more than 60% of the cAMP-induced transcription. We further investigated the cAMP-induced response and found that transcriptional activity of the downstream cAMP-induced enhancer was greatly synergized in the presence of the upstream enhancer of basal transcription. The latter enhancer contained three functional CANNTG sequences designated E1 (nucleotides -1060 to -1030), E2 (nucleotides -940 to -920), and E3 (nucleotides -920 to -900). E2 and E3 were essential for maximal cAMP-induced transcription. Detailed mapping of the vitamin D response showed that a minimum requirement for inhibition of the cAMP-induced enhancer by vitamin D was a sequence overlapping E3 (nucleotides -920 to -829). We conclude that a negative response element to vitamin D is located between nucleotides -920 and -829 in the CT 5' flanking DNA. It is possible that vitamin D inhibits transcription by interfering with the synergistic interaction between the cAMP-induced enhancer and the enhancer of basal transcription.

24,25-Dihydroxyvitamin D 3↗