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At least 721 records · Page 40Linked to original sources

PubMed2006

Hepatic metastases: detection with multi-detector row CT, SPIO-enhanced MR imaging, and both techniques combined.

PURPOSE: To retrospectively compare the accuracy in detection of hepatic metastases among contrast material-enhanced multi-detector row computed tomography (CT) alone, superparamagnetic iron oxide (SPIO)-enhanced magnetic resonance (MR) imaging alone, and a combination of contrast-enhanced CT and SPIO-enhanced MR imaging. MATERIALS AND METHODS: The ethics committee did not require its approval or informed consent for this retrospective study, which was compliant with Declaration of Helsinki principles. Data in 38 patients (22 men, 16 women; mean age, 64.5 years; range, 35-78 years) suspected of having hepatic metastases who underwent both contrast-enhanced CT and SPIO-enhanced MR imaging were retrospectively analyzed. Twenty-one of the 38 patients had 61 metastases. Seventeen of the 61 metastases were confirmed histologically; the remaining 44 metastases were defined with imaging follow-up. At MR imaging, SPIO-enhanced heavily T1-weighted images, T2*-weighted gradient echo images, and T2-weighted fast spin-echo images were evaluated. Contrast-enhanced multi-detector row CT images obtained in the portal phase were evaluated. Four blinded observers independently reviewed CT images, MR images, and the combination of CT and MR images. Diagnostic accuracy was evaluated by using the alternative free-response receiver operating characteristic (AFROC) method. Sensitivities and positive predictive values were also analyzed with the Fisher protected least significant difference test and generalized estimating equations. RESULTS: The mean area under the AFROC curve for the combined approach (0.70) was significantly higher than that for SPIO-enhanced MR imaging alone (0.58, P < .05, Fisher protected least significant difference test), and there was no significant difference between each of them and that for contrast-enhanced CT alone (0.66). For all lesions, the mean sensitivity of combined imaging (0.59) was significantly higher than that of CT (0.48) or MR imaging (0.43) alone (P < .05, Fisher protected least significant difference test and generalized estimating equations). For all lesions, the mean positive predictive values were 0.82, 0.89, and 0.81, for combined MR and CT, CT alone, and MR alone, respectively. CONCLUSION: The addition of SPIO-enhanced MR imaging to contrast-enhanced multi-detector row CT (ie, combined analysis of SPIO-enhanced MR images and contrast-enhanced CT images) can improve sensitivity in the detection of hepatic metastases, although this improvement in sensitivity was not significant at AFROC analysis.

Adult↗

PubMed1999

Brain: gadolinium-enhanced fast fluid-attenuated inversion-recovery MR imaging.

PURPOSE: To determine the clinical utility of gadolinium-enhanced fluid-attenuated inversion-recovery (FLAIR) magnetic resonance (MR) imaging of the brain by comparing results with those at gadolinium-enhanced T1-weighted MR imaging with magnetization transfer (MT) saturation. MATERIALS AND METHODS: In 105 consecutive patients referred for gadolinium-enhanced brain imaging, FLAIR and T1-weighted MR imaging with MT saturation were performed before and after administration of gadopentetate dimeglumine (0.1 mmol per kilogram of body weight). Pre- and postcontrast images were evaluated to determine the presence of abnormal contrast enhancement and whether enhancement was more conspicuous with the FLAIR or T1-weighted sequences. RESULTS: Thirty-nine studies showed intracranial contrast enhancement. Postcontrast T1-weighted images with MT saturation showed superior enhancement in 14 studies, whereas postcontrast fast FLAIR images showed superior enhancement in 15 studies. Four cases demonstrated approximately equal contrast enhancement with both sequences. Six cases showed some areas of enhancement better with T1-weighted imaging with MT saturation and other areas better with postcontrast fast FLAIR imaging. Superficial enhancement was typically better seen with postcontrast fast FLAIR imaging. CONCLUSION: Fast FLAIR images have noticeable T1 contrast making gadolinium-induced enhancement visible. Gadolinium enhancement in lesions that are hyperintense on precontrast FLAIR images, such as intraparenchymal tumors, may be better seen on T1-weighted images than on postcontrast fast FLAIR images. However, postcontrast fast FLAIR images may be useful for detecting superficial abnormalities, such as meningeal disease, because they do not demonstrate contrast enhancement of vessels with slow flow as do T1-weighted images.

Brain↗

PubMed2006

The role of contrast-enhanced sonography of focal liver lesions before percutaneous biopsy.

OBJECTIVE: The objective of our study was to evaluate the clinical utility of performing contrast-enhanced sonography before percutaneous biopsy of focal liver lesions. SUBJECTS AND METHODS: One hundred eighty-six patients with focal liver lesions detected on either sonography or contrast-enhanced CT were randomly divided into two groups: a group who underwent contrast-enhanced sonography and another who underwent unenhanced sonography. The contrast-enhanced sonography group (79 patients, 129 lesions) underwent SonoVue-enhanced sonography before biopsy, and the unenhanced sonography group (107 patients, 143 lesions) did not undergo contrast-enhanced sonography before biopsy. Conventional sonography was used in all patients to guide the biopsy procedures. The pathologic diagnosis was considered definitive and final if the biopsy result was malignant. If the initial biopsy result was benign or negative for malignancy, then the result was either confirmed or denied on the basis of contrast-enhanced CT, MRI, angiography, serum alpha-fetoprotein level, or clinical follow-up over a period of 6 months. In some patients with suspected malignancy, biopsy was repeated when considered necessary during the follow-up. The diagnostic accuracy of the initial biopsy was defined as the percentage of the total number of lesions that were correctly diagnosed at the initial biopsy. The difference in diagnostic accuracy between the two groups was analyzed to evaluate the value of performing contrast-enhanced sonography before biopsy. RESULTS: Of the 129 lesions in the contrast-enhanced sonography group, 28 (21.7%) were benign and 101 (78.3%) were malignant. Of the 143 lesions in the unenhanced sonography group, 36 (25.2%) were benign and 107 (74.8%) were malignant. There was no significant difference in the distribution of malignant and benign lesions in these two groups (p > 0.05). There was no statistically significant difference in the distribution of lesions by size between the contrast-enhanced and unenhanced sonography groups (chi(2) = 0.619, p > 0.05). The diagnostic accuracy of the initial biopsy was significantly higher in the contrast-enhanced sonography group than in the unenhanced sonography group (95.3% vs 87.4%, respectively; p < 0.05). The diagnostic accuracy of the initial biopsy for malignant lesions < or = 2.0 cm was also significantly higher in the contrast-enhanced sonography group than in the unenhanced sonography group (97.1% vs 78.8%, respectively; p < 0.05). No major complications occurred in our study except one case of pneumothorax in the unenhanced sonography group. CONCLUSION: Contrast-enhanced sonography before percutaneous focal liver lesion biopsy improved the diagnostic accuracy of the procedure by providing important intralesional information for differentiating viable, denaturalized, or necrotic tissue; consequently, by providing more accurate information about the site of biopsy even in lesions < or = 2.0 cm, contrast-enhanced sonography before biopsy reduced the number of puncture attempts.

Adolescent↗

PubMed2000

Selective intraarterial contrast-enhanced CT of pancreaticoduodenal tumors: early clinical experience in evaluating blood supply and detectability.

OBJECTIVE: The purpose of this study was to compare CT with selective intraarterial contrast enhancement with IV contrast-enhanced CT for diagnostic usefulness in the detection of tumors in the pancreaticoduodenal region. SUBJECTS AND METHODS: intraarterial contrast enhanced CT was performed in 36 patients with tumors of the pancreaticoduodenal region. Feeding arteries of the tumors and distribution of hyperattenuating areas on intraarterial contrast-enhanced CT were analyzed with various routes of contrast material injections. The intraarterial contrast-enhanced CT scans were compared with the IV contrast-enhanced CT scans. RESULTS: In all 29 patients with standard vascular anatomy, the right cephalic portion of the pancreatic head was enhanced on CT during common hepatic or gastroduodenal arteriography and the left caudal portion was enhanced on CT during superior mesenteric arteriography. The enhanced areas were complementary to each other in the whole pancreatic head, including the tumor. Tumor conspicuity from the surrounding pancreatic tissue on intraarterial contrast-enhanced CT was not superior to that on IV contrast-enhanced CT in all but four patients with cystic tumors. After intraarterial contrast-enhanced CT, three patients with tumors less invasive than pancreatic ductal carcinoma underwent local resection of their lesions. CONCLUSION: Intraarterial contrast-enhanced CT for pancreaticoduodenal tumors has potential technical problems and is not valuable in improving the detectability of tumors other than cystic lesions because the enhancement of the wall and septa of the tumor is emphasized. However, the feeding artery of the tumor and its surrounding tissue were clearly depicted.

Adult↗

PubMed2004

Multiarterial phase dynamic MRI of small early enhancing hepatic lesions in cirrhosis or chronic hepatitis: differentiating between hypervascular hepatocellular carcinomas and pseudolesions.

OBJECTIVE: The purpose of this study was to assess the value of multiarterial phase contrast-enhanced dynamic MRI of the whole liver obtained during a single breath-hold for small early enhancing hepatic lesions in patients with cirrhosis or chronic hepatitis, emphasizing the distinction between hypervascular hepatocellular carcinomas and pseudolesions. MATERIALS AND METHODS: The study population included 40 patients with cirrhosis or chronic hepatitis who had small early enhancing hepatic lesions (a total of 70 lesions: 40 hepatocellular carcinomas, 30 pseudolesions). All patients underwent multiarterial phase contrast-enhanced dynamic MRI (six phases) of the whole liver during a single breath-hold. RESULTS: Twenty-one (53%) of 40 hypervascular hepatocellular carcinomas showed rapid central washout after early enhancement of the lesion as well as peritumoral coronal enhancement, but these findings were not observed in any hypervascular pseudolesions (p < 0.001). In 19 hepatocellular carcinomas without rapid central washout, early enhancement of the lesion appeared at the second, third, or fourth phase (mean, 2.5 phases). In eight of these 19 hepatocellular carcinomas, lesion enhancement disappeared by the sixth phase. Conversely, in 30 hypervascular pseudolesions, early enhancement of the lesion appeared at the second, third, fourth, or fifth phase (mean, 3.0 phases). In 28 of these 30 pseudolesions, lesion enhancement continued until the sixth phase. CONCLUSION: Rapid central washout after the early enhancement of the lesion and coronal enhancement surrounding the lesion are highly specific and diagnostic findings of small hypervascular hepatocellular carcinomas if present at multiarterial phase contrast-enhanced dynamic MRI. Hypervascular pseudolesions tend to show prolonged enhancement during the arterial phase compared with hypervascular hepatocellular carcinomas.

Aged↗

PubMed1999

Surgically induced intracranial contrast enhancement: potential source of diagnostic error in intraoperative MR imaging.

BACKGROUND AND PURPOSE: Intraoperative MR imaging is being used increasingly during neurosurgical interventions. The aim of this study was to describe and classify different forms of surgically induced intracranial contrast enhancement observed during intraoperative MR examinations. METHODS: A total of 51 intraoperative MR examinations were performed to assess the extent of brain tumor removal. The intraoperative MR results (T1-weighted images, unenhanced and obtained serially after the IV administration of paramagnetic contrast material) were compared with preoperative and early postoperative MR findings. Animal experiments were conducted to obtain further evidence of the mechanism of surgically induced contrast enhancement. RESULTS: Four different types of surgically induced contrast enhancement were found: meningeal enhancement, increased enhancement of the choroid plexus, delayed enhancement at the resection margins, and immediate intraparenchymal contrast enhancement. The types of surgically induced contrast enhancement differ regarding their location, configuration, and time course. Their potential to be confused with contrast-enhancing, residual tumor also varies. Three of the four types of surgically induced contrast enhancement were reproducible in an animal model. CONCLUSION: Surgically induced contrast enhancement is a potential source of error in intraoperative MR imaging. Careful analysis of the location, configuration, and time course of intraoperatively observed intracranial enhancement is critical to avoid confusing surgically induced contrast enhancement with contrast-enhancing, residual tumor.

Animals↗

PubMed2001

A selective cyclooxygenase-2 inhibitor, NS-398, enhances the effect of radiation in vitro and in vivo preferentially on the cells that express cyclooxygenase-2.

It has been proposed that Cyclooxygenase (COX)-2 inhibitors may be able to enhance the effects of chemotherapeutic or radiation treatment; however, currently few studies have been reported that define the radiation-enhancing effect of COX-2 inhibitors. We conducted in vitro radiation survival experiments using rat intestinal epithelial cells which were stably transfected with COX-2 cDNA in the sense (RIE-S) and antisense (RIE-AS) orientations to investigate the potential radiosensitizing effect of the selective COX-2 inhibitor, NS-398. Apoptosis was measured using 7-aminoactinomycin-D with flow cytometry to investigate underlying mechanisms for the effect of NS-398 on radiosensitivity. The same experiments were repeated with NCI-H460 human lung cancer cells, which express COX-2 constitutively, and HCT-116 human colon cancer cells, which lack COX-2 expression. In vivo tumor growth delay assays were also performed with tumors formed by H460 and HCT-116 cells. No difference was observed in the intrinsic radiation sensitivity of RIE-S and RIE-AS cells exposed to radiation alone. However, 150-400 microM of NS-398 enhanced radiosensitivity in a concentration-dependent manner in RIE-S cells with dose enhancement ratios of 1.2-1.9 at a surviving fraction of 0.25. However, this effect was not shown in RIE-AS cells. NS-398 enhanced radiosensitivity in H460 cells with a dose enhancement ratio of 1.8 but protected HCT-116 cells from the effects of radiation. Radiation-induced apoptosis was enhanced by NS-398 in RIE-S and H460 cells but not in RIE-AS and HCT-116 cells. Additionally, this radiation-enhancing effect in RIE-S cells seemed to be attributable to some mechanisms other than the reversal of radioresistance induced by COX-2. NS-398 (36 mg/kg) enhanced the effect of radiation on H460 tumors in vivo by an enhancement factor of 2.5; however, it did not enhance the radiosensitivity of HCT-116 tumors (enhancement factor = 1.04). These in vitro and in vivo results suggest that selective COX-2 inhibitors enhance the effect of radiation on tumors that express COX-2 but not on COX-2-lacking tumors. This effect may be attributable to enhancement of radiation-induced apoptosis. Thus, selective COX-2 inhibitors may have potential as radiosensitizers for treatment of human cancers.

Animals↗

PubMed1994

Enhanced lumbar nerve roots in the spine without prior surgery: radiculitis or radicular veins?

PURPOSE: To evaluate the clinical significance of continuous intradural lumbosacral nerve root enhancement in symptomatic patients without prior lumbar surgery. METHODS: Fifty-three patients without prior back surgery, referred to our institution for evaluation of low-back pain and radiculopathy, were studied with gadolinium-enhanced MR (0.1 mmol/kg) of the lumbar spine. Scans were reviewed for the presence of lumbosacral nerve root enhancement and any associated nerve root compression. Results were correlated with clinical history and physical examinations. RESULTS: Seventeen continuously enhancing nerve roots and two enhancing fila terminale were observed in 13 patients. Eight of 17 (47%) had no referable symptoms. Nine of these nerve roots (53%) were not associated with any degree of nerve root compression. Seven cases (41%) were noted to have flow-related enhancement on the entry section of the T1-weighted axial sequence. CONCLUSIONS: Lumbosacral nerve root enhancement correlates poorly with clinical radiculopathy. The use of contrast enhancement to detect lumbosacral nerve root enhancement in cases in which the unenhanced scan is less than diagnostic is not warranted. The high association between lumbosacral nerve root enhancement and entry-section flow-related enhancement suggests that these enhancing structures within the cauda equina are vessels. It is likely that lumbosacral nerve root enhancement represents intravascular enhancement of radicular veins and not a breakdown in the blood-nerve barrier.

Adult↗

PubMed1997

MR contrast enhancement of the normal neonatal brain.

PURPOSE: To determine the pattern of enhancement on contrast-enhanced MR studies of the brain in neonates. METHODS: Contrast-enhanced brain MR studies of 16 neonates were reviewed retrospectively. All infants had normal neonatal courses, normal noncontrast MR findings, and normal neurologic examinations at age 12 months. All enhancing regions within the brain, dura, calvaria, and orbits were recorded. An enhancement factor, F = (Ic-Ip)/Ip, was calculated from region-of-interest intensity measurements in five regions of each hemisphere (basal ganglia, thalami, and three hemispheric locations), where Ic was signal intensity after contrast administration and Ip was the noncontrast signal intensity for each region. RESULTS: Enhancement was detected in the choroid plexus, pituitary infundibula, pineal glands, dura, veins and venous sinuses, cranial sutures, and irises of the orbital globes. No enhancement of the brain parenchyma was detected by visual inspection, although some change in signal intensity of the cerebral parenchyma was detected by the region-of-interest intensity measurements, with enhancement factors ranging from 0 to 0.08 (mean, 0.04). No consistent regional variation in enhancement was detected. Because the degree of enhancement was identical to that in the normal adult brain, the slight enhancement detected was attributed to contrast material in capillaries and small venules. CONCLUSION: In addition to the expected findings of enhancement of the pituitary stalk, the pineal gland, the choroid plexus, the dura, and the cerebral veins, we detected enhancement of the calvarial sutures and ocular irises. No evidence of enhancement of the cerebral parenchyma was detected, suggesting that the blood-brain barrier to gadolinium chelates is intact in the neonatal brain.

Adult↗

PubMed1989

Transactivation of the Xenopus rRNA gene promoter by its enhancer.

A key question concerning the mechanism of transcriptional activation by enhancers is about the role of the DNA that connects the enhancer to the promoter. The linking DNA will be important if a regulatory protein(s) binds to the enhancer and then tracks or slides along the DNA to the promoter, or if, on binding, the protein(s) alters the topological state of the DNA. By contrast, if the linking DNA loops out to allow the formation of a promoter-enhancer complex, or if the enhancer increases the local concentration of a transcription factor, co-linearity of the promoter and the enhancer will not be strictly required. In Xenopus laevis, the transcription of the ribosomal RNA genes is stimulated by an enhancer composed of repetitive sequences in the intergenic spacer regions. These repetitive elements contain 60 or 81 base pairs, and their activity is relatively independent of their position and orientation. When the enhancer and promoter sequences are each located on separate DNA molecules, however, the enhancer is no longer able to augment transcription. We have now tested whether or not this apparent requirement for having the enhancer and promoter in cis can be overcome by keeping them in close proximity while locating them separately on different molecules. This was achieved by generating multiply intertwined, dimeric-catenanes in which the enhancer and promoter were located in trans on different rings. By injecting these catenanes into frog oocytes and measuring the activity of the enhancers in a series of competition assays, we were able to demonstrate that such enhancers can augment transcription in vivo.

Animals↗

PubMed1992

Transcription of the hypersensitive site HS2 enhancer in erythroid cells.

In the human genome, the erythroid-specific hypersensitive site HS2 enhancer regulates the transcription of the downstream beta-like globin genes 10-50 kilobases away. The mechanism of HS2 enhancer function is not known. The present study employs RNA protection assays to analyze the transcriptional status of the HS2 enhancer in transfected recombinant chloramphenicol acetyltransferase (CAT) plasmids. In erythroid K562 cells in which the HS2 enhancer is active, the HS2 sequence directs the synthesis of long enhancer transcripts that are initiated apparently from within the enhancer and elongated through the intervening DNA into the cis-linked CAT gene. In nonerythroid HL-60 cells in which the HS2 enhancer is inactive, long enhancer transcripts are not detectable. Splitting the HS2 enhancer between two tandem Ap1 sites abolishes the synthesis of a group of long enhancer transcripts and results in loss of enhancer function and transcriptional silencing of the cis-linked CAT gene. In directing the synthesis of RNA through the intervening DNA and the gene by a tracking and transcription mechanism, the HS2 enhancer may (i) open up the chromatin structure of a gene domain and (ii) deliver enhancer binding proteins to the promoter sequence where they may stimulate the transcription of the gene at the cap site.

Base Sequence↗

PubMed1997

Evaluation of the post-operative lumbar spine with MR imaging. The role of contrast enhancement and thickening in nerve roots.

PURPOSE: Two new signs of lumbar nerve-root affection have been reported in recent years on the basis of MR examinations, namely: thickening in nerve roots; and contrast enhancement in nerve roots. The aim of this study was to assess contrast enhancement in nerve roots in a standardised way, and to evaluate the clinical significance of contrast enhancement and of nerve-root thickening in the symptomatic post-operative lumbar spine. MATERIAL AND METHODS: A total of 121 patients (who had previously been operated on for lumbar disc herniation) underwent 152 MR examinations, mainly on a 1.5 T system. Focal nerve-root enhancement was identified by visual assessment. Intradural enhancement was also quantified by pixel measurements that compared the affected nerve roots before and after contrast administration. Non-affected nerve roots were used as reference. RESULTS: Enhanced nerve roots in the dural sac increased at least 40-50% in signal intensity after contrast administration compared to pre-contrast images and also compared to non-affected nerve roots. Intradural nerve-root enhancement was seen in 10% of the patients and focal enhancement in the root sleeve was seen in a further 26%. Nerve-root thickening was seen in 30%. Good correlation with clinical symptoms was found in 59% of the patients with intradural enhancement, in 84% with focal enhancement, and in 86% with nerve-root thickening. The combination of thickening and enhancement in the nerve root correlated with symptoms in 86% of the patients. CONCLUSION: Nerve-root enhancement (whether focal or intradural) and thickening in the nerve root are significant MR findings in the post-operative lumbar spine. In combination with disc herniation or nerve-root displacement, these two signs may strengthen the indication for repeat surgery. However, root enhancement within 6 months of previous surgery may be a normal post-operative finding.

Adult↗

PubMed1997

Developmental acquisition of enhancer function requires a unique coactivator activity.

Enhancers are believed to stimulate promoters by relieving chromatin-mediated repression. However, injection of plasmid-encoded genes into mouse oocytes and embryos revealed that enhancers failed to stimulate promoters prior to formation of a two-cell embryo, even though the promoter was repressed in the maternal nucleus of both oocytes and one-cell embryos. The absence of enhancer function was not due to the absence of a required sequence-specific enhancer activation protein, because enhancer function was not elicited even when these proteins either were provided by an expression vector (GAL4:VP16) or were present as an endogenous transcription factor (TEF-1) and shown to be active in stimulating promoters. Instead, enhancer function in vivo required a unique coactivator activity in addition to enhancer-specific DNA binding proteins and promoter repression. This coactivator activity first appeared during mouse development in two- to four-cell embryos, concurrent with the major onset of zygotic gene expression. Competition between various enhancers was observed in these embryos, but not competition between enhancers and promoters, and competition between enhancers was absent in one-cell embryos. Moreover, enhancer function in oocytes could be partially restored by pre-injecting mRNA from cells in which enhancers were active, the same mRNA did not affect enhancer function in two- to four-cell embryos.

Animals↗

PubMed1998

Inactivation of a common epitope responsible for the induction of antibody-dependent enhancement of HIV.

BACKGROUND: The primary antigenic domain responsible for complement-mediated antibody-dependent enhancement (C'-ADE) of HIV and simian immunodeficiency virus resides in the principal immunodominant sequence of the transmembrane protein. OBJECTIVE: To identify whether there are amino-acid residues common to the epitopes of the known enhancing human monoclonal antibodies (MAb), and to provide a structural model for this functional region present on the HIV envelope. Since our model predicts that this region is involved in the association of gp120 with gp41, this association was monitored for each mutant. DESIGN: The binding of enhancing human MAb to point and deletion mutations within the enhancing domain was analyzed by two methods. The first analyzed binding to mutants expressed in COS cells: the second quantified the binding of four enhancing human MAb to each mutant gp160 versus wild-type control by enzyme-linked immunosorbent assay (ELISA). METHODS: Site-directed mutagenesis was used to produce specific deletions and point mutants, which were expressed in COS cells. Binding of MAb 50-69 and V3-loop MAb 5F7 were visualized in the wild-type and each of the mutant constructs by immunohistochemistry. Quantitative evaluation of enhancing human MAb binding to each mutant versus wild-type was performed by ELISA. A model for the enhancing domain and its relationship to gp120 association with gp41 was provided by molecular dynamics and ligand docking methods. RESULTS: All available enhancing human MAb known to bind to the principal immunodominant region of gp41 were unable to bind to deletions involving the disulfide loop, which in our molecular model provided the primary association site between gp120 and gp41. Point mutations in the loop blocked this association, but had a quantitatively smaller effect on the binding of the enhancing human MAb. A conservative W596Y mutation completely blocked the binding of all human MAb, but had no effect on gp120-gp41 association. CONCLUSIONS: A variety of mutations within the primary C'-ADE domain inhibit binding of enhancing human MAb as well as blocking the association of gp120 and gp41. A conservative W596Y mutation blocks binding of all enhancing human MAb with retention of gp120-gp41 association. These data are important to the design of vaccines in which the primary enhancing epitope is disarmed to prevent the subsequent induction of an amnestic response that could lead to viral enhancement of infection. The retention of the gp120-gp41 association is postulated to yield an immunogen similar to natural infection for both subunit and genetic vaccines.

Amino Acid Sequence↗

PubMed1993

Functional and defective components of avian endogenous virus long terminal repeat enhancer sequences.

Oncogenic avian retroviruses, such as Rous sarcoma virus (RSV) and the avian leukosis viruses, contain a strong enhancer in the U3 portion of the proviral long terminal repeat (LTR). The LTRs of a second class of avian retroviruses, the endogenous viruses (ev) lack detectable enhancer activity. By creating ev-RSV hybrid LTRs, we previously demonstrated that, despite the lack of independent enhancer activity in the ev U3 region, ev LTRs contain sequences that are able to functionally replace essential enhancer domains from the RSV enhancer. A hypothesis proposed to explain these data was that ev LTRs contain a partial enhancer that includes sequences necessary but not sufficient for enhancer activity and that these sequences were complemented by RSV enhancer domains present in the original hybrid constructs to generate a functional enhancer. Studies described in this report were designed to define sequences from both the ev and RSV LTRs required to generate this composite enhancer. This was approached by generating additional ev-RSV hybrid LTRs that exchanged defined regions between ev and RSV and by directly testing the requirement for specific motifs by site-directed mutagenesis. Results obtained demonstrate that ev enhancer sequences are present in the same relative location as upstream enhancer sequences from RSV, with which they share limited sequence similarity. In addition, a 67-bp region from the internal portion of the RSV LTR that is required to complement ev enhancer sequences was identified. Finally, data showing that CArG motifs are essential for high-level activity, a finding that has not been previously demonstrated for retroviral LTRs, are presented.

Avian Leukosis Virus↗

PubMed1990

Immunoglobulin heavy-chain enhancer is required to maintain transfected gamma 2A gene expression in a pre-B-cell line.

The immunoglobulin heavy-chain (IgH) enhancer serves to activate efficient and accurate transcription of cloned IgH genes when introduced into B lymphomas or myelomas. The role of this enhancer after gene activation, however, is unclear. The endogenous IgH genes in several cell lines, for example, have lost the IgH enhancer by deletion and yet continue to be expressed. This might be explained if the role of the enhancer were to establish high-level gene transcription but not to maintain it. Alternatively, other enhancers might lie adjacent to endogenous IgH genes, substituting their activity for that of the lost IgH enhancer. To address both of these alternatives, we searched for enhancer activity within the flanking regions of one of these IgH enhancer-independent genes and designed an experiment that allowed us to consider separately the establishment and maintenance of expression of a transfected gene. For the latter experiment we generated numerous pre-B cell lines stably transformed with a gamma 2a gene. In this gene, the IgH enhancer lay at a site outside the heavy-chain transcription unit, between DH and JH gene segments. After expression of the transfected gene was established, selective conditions were chosen for the outgrowth of subclones that had undergone D-J joining and thus IgH enhancer deletion. Measurements of gamma 2a expression before and after enhancer deletion revealed that the enhancer was required for maintenance of expression of the transfected gene. The implication of this finding for models of enhancer function in endogenous genes is discussed.

Animals↗

PubMed1996

Intronic and flanking sequences are required to silence enhancement of an embryonic beta-type globin gene.

In the course of studying regulatory elements that affect avian embryonic rho-globin gene expression, the multipotential hematopoietic cell line K562 was transiently transfected with various rho-globin gene constructs containing or lacking an avian erythroid enhancer element. Enhanced levels of rho gene expression were seen from those constructs containing an enhancer element and minimal 5' or 3' flanking rho sequences but were not seen from enhancer-containing constructs that included extensive 5' and 3' flanking sequences. Deletion analysis localized 5' and 3' "enhancer-silencing elements" to -2140 to -2000 and +1865 to +2180 relative to the mRNA cap site. A third element required for enhancer silencing was identified within the second intron of the rho gene. The treatment of K562 cells with hemin, which induces erythroid differentiation, partially alleviated the enhancer-silencing effect. The silencer elements were able to block enhancement from a murine erythroid enhancer, but not from a nonerythroid enhancer. Electrophoretic mobility shift assays demonstrated that the transcription factor YY1 is able to bind both the 5' and 3' enhancer silencer elements; a point mutation of the single overlapping YY1/NF-Y binding site in the 3' element completely abolished the enhancer-silencing effect. These results demonstrate a complex enhancer silencer that requires 5' flanking, intronic, and 3' flanking sequences for a single regulatory effect on a eukaryotic gene.

Animals↗

PubMed1997

Identification of a new class of exonic splicing enhancers by in vivo selection.

In vitro selection strategies have typically been used to identify a preferred ligand, usually an RNA, for an identified protein. Ideally, one would like to know RNA consensus sequences preferred in vivo for as-yet-unidentified factors. The ability to select RNA-processing signals would be particularly beneficial in the analysis of exon enhancer sequences that function in exon recognition during pre-mRNA splicing. Exon enhancers represent a class of potentially ubiquitous RNA-processing signals whose actual prevalence is unknown. To establish an approach for in vivo selection, we developed an iterative scheme to select for exon sequences that enhance exon inclusion. This approach is modeled on the in vitro SELEX procedure and uses transient transfection in an iterative procedure to enrich RNA-processing signals in cultured vertebrate cells. Two predominant sequence motifs were enriched after three rounds of selection: a purine-rich motif that resembles previously identified splicing enhancers and a class of A/C-rich splicing enhancers (ACEs). Individual selected ACEs enhanced splicing in vivo and in vitro. ACE splicing activity was competed by RNAs containing the purine-rich splicing enhancer from cardiac troponin T exon 5. Thus, ACE activity is likely to require a subset of the SR splicing factors previously shown to mediate activity of this purine-rich enhancer. ACE motifs are found in two vertebrate exons previously demonstrated to contain splicing enhancer activity as well as in the well-characterized Drosophila doublesex (dsx) splicing enhancer. We demonstrate that one copy of the dsx repeat enhances splicing of a vertebrate exon in vertebrate cells and that this enhancer activity requires the ACE motif. We suggest the possibility that the dsx enhancer is a member of a previously unrecognized family of ACEs.

Animals↗