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Results for “Enhancer mapping”

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At least 1,135 records · Page 63Linked to original sources

PubMed2003

Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-kappa B signaling pathways in murine RAW 264.7 macrophages.

Extracellular nucleotides such as ATP are present in abundance at sites of inflammation and tissue damage, and these agents exert a potent modulatory effect on macrophage/monocyte function via the nucleotide receptor P2X(7). In this regard, after exposure to bacterial LPS, P2X(7) activation augments expression of the inducible nitric oxide (NO) synthase and production of NO in macrophages. Because P2X(7) has been reported to stimulate certain members of the MAP kinase family (ERK1/2) and can enhance the DNA-binding activity of NF-kappa B, we tested the hypothesis that LPS and nucleotides regulate NF-kappa B-dependent inflammatory events via cross talk with MAPK-associated pathways. In this regard, the present studies revealed that cotreatment of macrophages with LPS and the P2X(7)-selective ligand 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) results in the cooperative activation of NF-kappa B DNA-binding activity and a sustained attenuation of levels of the NF-kappa B inhibitory protein I kappa B alpha. Interestingly, a persistent reduction in I kappa B alpha levels is also observed when the MEK1/2 inhibitor U0126 is coadministered with LPS, suggesting that components of the MEK/ERK pathway are involved in regulating I kappa B alpha protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in I kappa B alpha levels is supported by the finding that Ras activation, which is upstream of MEK/ERK activation, is reduced upon macrophage cotreatment with BzATP and LPS compared with the effects of BzATP treatment alone. These data are consistent with the concept that the Ras/MEK/ERK pathways are involved in regulating NF-kappa B/I kappa B-dependent inflammatory mediator production and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF-kappa B and Ras/MEK/MAPK-associated pathways.

Adenosine Triphosphate↗

PubMed2003

The effect of relaxation on the epitope mapping by saturation transfer difference NMR.

The effect of longitudinal relaxation of ligand protons on saturation transfer difference (STD) was investigated by using a known binding system, dihydrofolate reductase and trimethoprim. The results indicate that T1 relaxation of ligand protons has a severe interference on the epitope map derived from a STD measurement. When the T1s of individual ligand protons are distinctly different, STD experiments may not give an accurate epitope map for the ligand-target interactions. Measuring the relaxation times prior to mapping is strongly advised. A saturation time shorter than T1s is suggested for improving the potential epitope map. Reduction in temperature was seen to enhance the saturation efficiency in small to medium size targets.

Animals↗

PubMed1988

Identification of protein-binding sites in the hepatitis B virus enhancer and core promoter domains.

We have investigated the role of liver-specific trans-acting factor(s) in the regulation of hepatitis B virus (HBV) gene expression. A recorder plasmid (pEcoAluCAT; HBV nucleotides 1 through 1878) was constructed containing the HBV enhancer and the promoter region of the pregenomic RNA, which was ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene. Upon transfecting this plasmid into various cell lines, the CAT gene was expressed only in cells of liver origin. Moreover, competition cotransfections with pEcoAluCAT and plasmids containing HBV enhancer sequences in human hepatoblastoma-derived HepG2 cells indicated the presence of titratable trans-acting factor(s) in these cells. Gel mobility shift assays using HBV enhancer and core promoter domains confirmed the existence of sequence-specific DNA-binding proteins in liver cell nuclear extract which bound to these regions. These binding sites encompass 17- and 12-nucleotide palindromes in the HBV enhancer and core promoter domains, respectively, when mapped by the methylation interference assay.

Base Sequence↗

PubMed1995

Structural Organization of the Spliced Immediate-Early Gene Complex that Encodes the Major Acidic Nuclear (IE1) and Transactivator (IE2) Proteins of African Green Monkey Cytomegalovirus.

Total immediate-early (IE) RNA synthesized after infection with African green monkey cytomegalovirus (SCMV) in the presence of cycloheximide contained a major 2.3-kb mRNA species that acted as template for in vitro synthesis of a single 94-kD nuclear protein. The same IE RNA hybridized predominantly to a 1.8-kb subregion of the 220-kb genome which mapped 1.5 kb to the left of the in vitro transcription start site and TATATAA motif previously associated with the powerful MIE (IE94) enhancer region. However, DNA sequence and S1-mapping analysis of a 5-kb region downstream from the promoter revealed the existence of a far upstream noncoding first exon and four additional spliced exons capable of encoding two alternative protein products with shared N-terminal domains. This region is similar in structure to that of the MIE gene complex of human cytomegalovirus (HCMV), including being highly CpG suppressed. Exons 2, 3, and 4 encode an acidic protein equivalent to the 68-kD IE1 protein (UL123) of HCMV and exons 2, 3, and 5 encode a protein equivalent to the 80-kD IE2 (UL122) DNA-binding protein of HCMV. Transcripts from across the IE2 region were detected within the cycloheximide RNA, but they were present at 10- to 20-fold lower abundance than IE1 transcripts. The proposed 547-codon IE1 (IE94) acidic phosphoprotein of SCMV displays minimal residual homology with the IE1 protein of HCMV, but both associate with metaphase chromosomes and have large C-terminal glutamic-acid-rich domains. In contrast, the proposed 583-codon IE2 protein of SCMV displays extensive amino acid similarity to the HCMV IE2 transcriptional regulatory protein especially within C-terminal domains that are known to play a major role in promoter targeting for both transactivation and negative autoregulation functions. Copyright 1995 S. Karger AG, Basel

Journal Article↗

PubMed1998

Pheromone-dependent G1 cell cycle arrest requires Far1 phosphorylation, but may not involve inhibition of Cdc28-Cln2 kinase, in vivo.

In yeast, the pheromone alpha-factor acts as an antiproliferative factor that induces G1 arrest and cellular differentiation. Previous data have indicated that Far1, a factor dedicated to pheromone-induced cell cycle arrest, is under positive and negative posttranslational regulation. Phosphorylation by the pheromone-stimulated mitogen-activated protein (MAP) kinase Fus3 has been thought to enhance the binding of Far1 to G1-specific cyclin-dependent kinase (Cdk) complexes, thereby inhibiting their catalytic activity. Cdk-dependent phosphorylation events were invoked to account for the high instability of Far1 outside early G1 phase. To confirm any functional role of Far1 phosphorylation, we undertook a systematic mutational analysis of potential MAP kinase and Cdk recognition motifs. Two putative phosphorylation sites that strongly affect Far1 behavior were identified. A change of serine 87 to alanine prevents the cell cycle-dependent degradation of Far1, causing enhanced sensitivity to pheromone. In contrast, threonine 306 seems to be an important recipient of an activating modification, as substitutions at this position abolish the G1 arrest function of Far1. Only the phosphorylated wild-type Far1 protein, not the T306-to-A substitution product, can be found in stable association with the Cdc28-Cln2 complex. Surprisingly, Far1-associated Cdc28-Cln2 complexes are at best moderately inhibited in immunoprecipitation kinase assays, suggesting unconventional inhibitory mechanisms of Far1.

Animals↗

PubMedDate not supplied

Evidence for conformationally different states of interleukin-10: binding of a neutralizing antibody enhances accessibility of a hidden epitope.

We present the mapping of two anti-human interleukin-10 (hIL-10) antibodies (CB/RS/2 and CB/RS/11) which have been described as binding their antigen cooperatively. The epitopes were identified using hIL-10-derived overlapping peptide scans prepared by spot synthesis. To identify residues essential for binding within the two epitopes, each position was replaced by all other L-amino acids. The epitope-derived peptides were further characterized with respect to antibody affinity and their inhibition of the antibody-hIL-10 interaction. One antibody (CB/RS/11) binds to residues which are completely buried in the X-ray structure of IL-10. Accessibility of this hidden epitope is enhanced upon binding of the antibody CB/RS/2, which recognizes a discontinuous epitope located nearby. The recognition of the hidden CB/RS/11 epitope, as well as the cooperative binding behaviour of the two antibodies, provides evidence that IL-10 can adopt a conformational state other than that observed in the crystal structure.

Amino Acid Sequence↗

PubMed1993

A cell-type-specific transcription enhancer of type 16 human papillomavirus (HPV 16)-P97 promoter is defined with HPV-associated cellular events in human epithelial cell lines.

Expression of the early genes of human papillomavirus type 16 (HPV16) is known to be highly restricted to epithelial cells. This report describes the molecular basis of HPV16 epitheliotropism at the transcriptional level. A series of 5'-end deletion mutants of the long control region (LCR) located upstream of the HPV16 early genes were tested in a transient expression assay using various human epithelial and fibroblastic cell lines. The specific region termed the cell-type-dependent regulatory element (CTRE) was found to function as an enhancer in some of HPV-associated human epithelial cell lines, but not at all in HPV-free human epithelial cell lines. No LCR-associated enhancer activity was detectable in fibroblastic cell lines. The CTRE was mapped at a distinct region upstream from the previously proposed proximal keratinocyte dependent enhancer (KD). The CTRE augmented transcription initiated from the autologous P97 promoter as well as from a heterologous promoter in a orientation-independent manner. Within the CTRE, there are three copies of the consensus nuclear factor-1 (NF1) binding site. Using CTRE containing mutations in each of the NF1-sites, it was demonstrated that all of these NF1-sites were necessary for a full enhancer activity in the HPV-associated human epithelial cell line. This suggests that the CTRE plays a critical role in the cell-type specific expression of HPV16-early gene at the level of transcriptional regulation.

Animals↗

PubMed1992

Three-dimensional in vivo mapping of brain lesions in humans.

We describe a multistep technique for three-dimensional reconstruction and analysis of brain lesions in vivo, based on the manipulation of magnetic resonance raw data obtained with a special protocol. The technique permits the direct visual identification of neuroanatomical landmarks in each brain specimen and eliminates the need to rely on averaged templates of human brain sections, which can be a source of lesion localization error. The technique also allows for the bidirectional cross-reference between data points in two-dimensional slices and in volume reconstruction and for the projection of subcortical structures onto the three-dimensional cortical surface. The three-dimensional lesion mapping technique can be applied to research with the lesion method in both human and nonhuman primates, to the planning of neurosurgical lesion approach, and to the teaching of neuroanatomy.

Brain Diseases↗

PubMed2000

In vivo intermolecular double-quantum imaging on a clinical 1.5 T MR scanner.

A novel MRI method based on the intermolecular double-quantum coherence (DQC) for soft tissues is described. DQC images of human brain were obtained for the first time on a whole-body 1.5 T scanner. The combination of quantum and classical formalisms was used to characterize multiple-quantum coherences, and to aid in the design of a DQC imaging sequence. The theoretical analysis suggests that signals from the intermolecular DQCs have higher sensitivity than those from the zero-quantum coherence (ZQC) for human brain, and the sensitivity increases with increased field strength. The DQC signal may provide a new form of contrast for MRI.

Brain↗

PubMed2000

Correction for B(1) and B(0) variations in quantitative T(2) measurements using MRI.

A new method is described for compensating for the bias introduced by variations in radiofrequency (RF) field strength and main magnetic field strength when making quantitative T(2) measurements using MRI. Field measurements made during the MRI study are used in combination with a signal model for off-resonance and imperfect RF pulses to correct the estimated T(2) value at every voxel. Applicable to both multicomponent and conventional single-component T(2) studies, the method has been validated experimentally using paramagnetic salt solutions in a multicompartment phantom. Studies of the human head are used to demonstrate the method in practice.

Artifacts↗

PubMed2000

New image contrast mechanisms in intermolecular double-quantum coherence human MR imaging.

We have developed a novel magnetic resonance imaging (MRI) method based on the intermolecular double-quantum coherence (DQC) in humans. Combined quantum mechanical and classical formalisms were used to characterize the signal and to aid in the design of a DQC imaging sequence with conventional or echoplanar acquisitions. Imaging contrast was evaluated in volunteers using a 1.5-T clinical scanner. The results demonstrated that the DQC images have contrasts fundamentally different from the conventional images based on single-quantum coherence (SQC). Both our theoretical analysis and experiments suggest that signals from DQCs have a higher signal-to-noise ratio than those from zero-quantum coherence (ZQC) for human brain imaging. The new contrast in DQC imaging may be useful for the detection of varying microstructures, potentially improving the detection of tumors without the need for contrast agents and providing a higher sensitivity and selectivity to magnetic susceptibility distributions in functional MRI brain studies.

Brain↗

PubMed2003

The OBF-1 gene locus confers B cell-specific transcription by restricting the ubiquitous activity of its promoter.

Transcription of the gene encoding the transcriptional coactivator Oct-binding factor 1 (OBF-1)/OCA-B/Bob.1 is largely restricted to B cells. During B cell development OBF-1 expression shows two peaks, one in immature B cells in the bone marrow and the other in germinal center B cells. Promoter analysis has identified a cAMP response element (CRE)-binding site present in the OBF-1 proximal promoter that is crucial for activity in B cells and for the induction of OBF-1 expression upon stimulation with CD40 ligand/IL-4. Here we address the question of how transcription of the OBF-1 gene is restricted to B cells. Surprisingly, in transient transfection assays the OBF-1 proximal promoter exhibited an equally strong activity in B and non-B cells. In contrast, upstream promoter regions displayed B cell-specific properties, partly overlapping with DNaseI hypersensitive sites identified in this study. In mice, expression of a neomycin resistance gene under the control of a Polyoma enhancer/TK promoter cassette was restricted to B cells when integrated into the OBF-1 locus, but was ubiquitous when integrated into two other loci, Oct-1 or the large subunit of RNA polymerase II.Therefore, lineage commitment of the OBF-1 gene is promoter independent and is achieved by regulating the entire locus in a B cell-specific manner.

Animals↗

PubMed2003

Determining and optimizing the precision of quantitative measurements of perfusion from dynamic contrast enhanced MRI.

PURPOSE: To examine the sensitivity of quantitative dynamic contrast enhanced MRI (DCE-MRI) perfusion maps to errors in the various source images and to determine optimal imaging parameters for reducing this sensitivity. MATERIALS AND METHODS: A detailed analysis of the precision of a DCE-MRI protocol was performed using the "propagation of errors" technique to investigate the effect of errors in the source images on errors in K(trans). Optimal parameter values and interactions between parameters were examined. The propagation of errors analysis was validated by Monte-Carlo simulations. RESULTS: The precision of K(trans) was found to be most sensitive to artifacts in the tissue portion of the baseline images and least sensitive to noise in the arterial portion of the dynamic images. The tip-angle strongly affected the precision, with the optimum being a function of tissue T1(0). CONCLUSION: Protocol optimization requires matching the tip-angle to the anticipated T1(0) of the tissue of interest; however such optimization yields a relatively small improvement. Future developmental efforts would be most productively focused on minimizing the artifact level.

Artifacts↗

PubMed2006

Report on a multicenter fMRI quality assurance protocol.

Temporal stability during an fMRI acquisition is very important because the blood oxygen level-dependent (BOLD) effects of interest are only a few percent in magnitude. Also, studies involving the collection of groups of subjects over time require stable scanner performance over days, weeks, months, and even years. We describe a protocol designed by one of the authors that has been tested for several years within the context of a large, multicenter collaborative fMRI research project (FIRST-BIRN). A full description of the phantom, the quality assurance (QA) protocol, and the several calculations used to measure performance is provided. The results obtained with this protocol at multiple sites over time are presented. These data can be used as benchmarks for other centers involved in fMRI research. Some issues with the various protocol measures are highlighted and discussed, and possible protocol improvements are also suggested. Overall, we expect that other fMRI centers will find this approach to QA useful and this report may facilitate developing a similar QA protocol locally. Based on the findings reported herein, the authors are convinced that monitoring QA in this way will improve the quality of fMRI data.

Brain Mapping↗

PubMed2006

The fast automatic algorithm for correction of MR bias field.

PURPOSE: To develop a method for efficient automatic correction of slow-varying nonuniformity in MR images. MATERIALS AND METHODS: The original MR image is represented by a piecewise constant function, and the bias (nonuniformity) field of an MR image is modeled as multiplicative and slow varying, which permits to approximate it with a low-order polynomial basis in a "log-domain." The basis coefficients are determined by comparing partial derivatives of the modeled bias field with the original image. RESULTS: We tested the resulting algorithm named derivative surface fitting (dsf) on simulated images and phantom and real data. A single iteration was sufficient in most cases to produce a significant improvement to the MR image's visual quality. dsf does not require prior knowledge of intensity distribution and was successfully used on brain and chest images. Due to its design, dsf can be applied to images of any modality that can be approximated as piecewise constant with a multiplicative bias field. CONCLUSION: The resulting algorithm appears to be an efficient method for fast correction of slow varying nonuniformity in MR images.

Algorithms↗

PubMed2002

Imaging cortical anatomy by high-resolution MR at 3.0T: detection of the stripe of Gennari in visual area 17.

The brain can be parcellated into numerous anatomical and functional subunits. The classic work by Brodmann (Vergleichende Lokalisationslehre der Grosshirnrinde in ihren Prinzipien dargestellt auf Grund des Zellenbaues. Leipzig: Barth; 1909) identified areas of the cerebral cortex based on histological differences. An alternative to his cytoarchitectonic approach is the myeloarchitectonic approach. MRI has excellent white/gray matter contrast in the brain due to the presence of myelin, and thus seems uniquely suited for in vivo studies of cortical myeloarchitecture. Here it is demonstrated that the stripe or stria of Gennari can be consistently detected in human occipital cortex. T(1)-weighted images obtained at 3T from six of 10 normal volunteers, with resolutions of 350 x 350 x 600 mu clearly demonstrate this myelin-rich intracortical layer. It is concluded that the striate cortex (area 17 of Brodmann) of the human brain can be delineated in vivo on T(1)-weighted images, potentially enabling detection of specific cortical boundaries within individual brains.

Brain Mapping↗

PubMed2001

Spiral-in/out BOLD fMRI for increased SNR and reduced susceptibility artifacts.

BOLD fMRI is hampered by dropout of signal in the orbitofrontal and parietal brain regions due to magnetic field gradients near air-tissue interfaces. This work reports the use of spiral-in trajectories that begin at the edge of k-space and end at the origin, and spiral in/out trajectories in which a spiral-in readout is followed by a conventional spiral-out trajectory. The spiral-in trajectory reduces the dropout and increases the BOLD contrast. The spiral-in and spiral-out images can be combined in several ways to simultaneously achieve increased signal-to-noise ratio (SNR) and reduced dropout artifacts. Activation experiments employing an olfaction task demonstrate significantly increased activation volumes due to reduced dropout, and overall increased SNR in all regions.

Amygdala↗

PubMed2005

High-resolution T1 and T2 mapping of the brain in a clinically acceptable time with DESPOT1 and DESPOT2.

Variations in the intrinsic T(1) and T(2) relaxation times have been implicated in numerous neurologic conditions. Unfortunately, the low resolution and long imaging time associated with conventional methods have prevented T(1) and T(2) mapping from becoming part of routine clinical evaluation. In this study, the clinical applicability of the DESPOT1 and DESPOT2 imaging methods for high-resolution, whole-brain, T(1) and T(2) mapping was investigated. In vivo, 1-mm(3) isotropic whole-brain T(1) and T(2) maps of six healthy volunteers were acquired at 1.5 T with an imaging time of <17 min each. Isotropic maps (0.34 mm(3)) of one volunteer were also acquired (time <21 min). Average signal-to-noise within the 1-mm(3) T(1) and T(2) maps was approximately 20 and approximately 14, respectively, with average repeatability standard deviations of 46.7 ms and 6.7 ms. These results demonstrate the clinical feasibility of the methods in the study of neurologic disease.

Brain↗