The chicken beta-globin 5'HS4 boundary element blocks enhancer-mediated suppression of silencing.

A constitutive DNase I-hypersensitive site 5' of the chicken beta-globin locus, termed 5'HS4 or cHS4, has been shown to insulate a promoter from the effect of an upstream enhancer and to reduce position effects on mini-white expression in Drosophila cells; on the basis of these findings, it has been designated a chromatin insulator. We have examined the effect of the cHS4 insulator in a system that assays both the level of gene expression and the rate of transcriptional silencing. Because transgenes flanked by insulator elements are shielded from position effects in Drosophila cells, we tested the ability of cHS4 to protect transgenes from position effects in mammalian cells. Flanking of an expression vector with the cHS4 insulator in a colony assay did not increase the number of G418-resistant colonies. Using lox/cre-based recombinase-mediated cassette exchange to control integration position, we studied the effect of cHS4 on the silencing of an integrated beta-geo reporter at three genomic sites in K562 erythroleukemia cells. In this assay, enhancers act to suppress silencing but do not increase expression levels. While cHS4 blocked enhancement at each integration site, the strength of the effect varied from site to site. Furthermore, at some sites, cHS4 inhibited the enhancer effect either when placed between the enhancer and the promoter or when placed upstream of the enhancer. These results suggest that the activity of cHS4 is not dominant in all contexts and is unlikely to prevent silencing at all genomic integration sites.

Morphological changes after percutaneous transluminal angioplasty.

BACKGROUND: Percutaneous transluminal angioplasty (PTA) dilates constricted arteries at the circle of Willis to reverse cerebral ischemia caused by cerebral vasospasm. Although 90% of the patients show angiographic improvement after PTA, only 70% show clinical improvement. Why some patients do not improve after PTA is unknown. We report on a 48-year-old woman who failed to improve after PTA and died from aneurysm rerupture. Pathologic studies were performed to determine why PTA failed to reverse the symptoms of cerebral ischemia. METHODS: The arteries of the brain were studied by light microscopy using Gomori's trichrome stain. The arteries were also studied by scanning and transmission electron microscopy. RESULTS: The arteries that were dilated with PTA showed compression of the connective tissue, stretching of the internal elastic lamina, and a combination of compression and stretching of the smooth muscle. The small arteries and arterioles that had been treated with an infusion of intraarterial papaverine were constricted with a thickened intimal layer. CONCLUSION: The persistence of cerebral vasospasm in small and perforating arteries may contribute to the failure of cerebral ischemia to reverse after PTA.

Transluminal angioplasty and intra-arterial papaverine for the treatment of cerebral vasospasm after ruptured arteriovenous malformations.

BACKGROUND: This is the first report on the use of intra-arterial papaverine and percutaneous transluminal angioplasty in two patients with severe, symptomatic cerebral vasospasm who suffered ruptured arteriovenous malformations (AVMs). CASE DESCRIPTIONS: The source of hemorrhage was a venous aneurysm in the first case and a pedicular aneurysm of the distal posterior inferior cerebellar artery in the second case. In both cases, the AVMs were located in the superior vermis and there was minimal subarachnoid hemorrhage. The first patient underwent removal of the AVM before the period of cerebral vasospasm and the second patient underwent removal of the AVM after the cerebral vasospasm had resolved. The outcome was excellent in the first patient and poor in the second patient. CONCLUSION: Arteriovenous malformation with ruptured aneurysms may be at high risk for cerebral vasospasm even when there is minimal subarachnoid hemorrhage. We recommend early treatment of AVMs with ruptured pedicular, intranidal, or venous aneurysms to avoid rebleeding and to allow for aggressive treatment of cerebral vasospasm. The management of cerebral vasospasm after AVM rupture is discussed.

Cell proliferation rate by MIB-1 immunohistochemistry predicts postradiation recurrence in prostatic adenocarcinomas.

We examined whether the cell proliferation index by MIB-1, HER-2/neu gene amplification, Gleason grade, and pretreatment level of serum prostate specific antigen (PSA) correlated with postradiation recurrence (PRR) in patients with prostatic adenocarcinoma. Formalin-fixed, paraffin-embedded tissue sections from 42 pretreated cases of prostatic adenocarcinoma (38 needle biopsy and 4 transurethral resection specimens) were immunostained for MIB-1 (MMI, Ventana Medical Systems, Tucson, Ariz). HER-2/neu gene amplification was analyzed by fluorescence in situ hybridization using the Oncor unique sequence probe (Oncor, Gaithersburg, Md). The cell proliferation index by MIB-1 was determined by labeling index; levels of HER-2/neu were analyzed semiquantitatively. Twenty-three of 42 patients (55%) were considered to have PRR on the basis of consecutive elevations of serum levels of PSA to greater than 1.5 ng/mL after completion of treatment (mean follow-up time, 33.4 months). The cell proliferation index correlated with PRR on both univariate and multivariate analyses. Of the 23 tumors that showed PRR, 18 (78%) revealed a high cell proliferation index, compared with 6 of 19 cases (32%) that showed no PRR. Twelve of 23 cases of prostatic adenocarcinoma (52%) in the recurrent group showed HER-2/neu gene amplification, compared with 5 of 19 (26%) in the nonrecurrent group; these findings reached near significance on univariate analysis. Pretreatment levels of serum PSA also reached significance on multivariate analysis. In this preliminary study, the cell proliferation index by MIB-1 reached independent significance in predicting PRR in patients with prostatic adenocarcinoma, whereas HER-2/neu amplification by fluorescence in situ hybridization reached near significance.

Childhood melanoma: a clinicopathological study of 22 cases.

Childhood melanoma is a rare disease with an estimated incidence of one per million per year. Careful study of childhood melanoma patients is critical due to the limited data currently available pertaining to this disease. Twenty-two children 15 years of age or under with malignant melanoma were treated at the Pigmented Lesion Clinic of Massachusetts General Hospital over a 33-year period. The medical records of all patients were reviewed, as well as the histologic characteristics of the lesions. Patients who were initially diagnosed with malignant melanoma but on review found to have Spitz naevi were not included in our study. Ten patients were boys and 12 were girls. The median ages of the boys and girls in our study were 12.9 and 13.6 years, respectively. Among the classified primary melanomas, 10 were superficial spreading, three were borderline/minimal deviation, two were nodular and one was melanoma in situ. Four of 22 patients had a documented family history of melanoma, and two additional patients had a family history of dysplastic naevi. A majority of lesions (14/22) arose in association with a precursor lesion. Two children died of disease at 1 and at 7 years following initial diagnosis. Eight patients had documented metastases. Since the majority of melanomas arose in association with a precursor lesion, follow-up of children with congenital and/ or dysplastic naevi is recommended. An interesting finding was the sometimes paradoxical behaviour of relatively thin lesions with metastases. Thus, a high index of suspicion is needed by the clinician confronted with melanocytic lesions of childhood. We found children from age 12 to 15 to be more at risk for the development of melanoma than younger children. Melanoma presenting before the age of 10 is very unusual.

Overlapping thin-section fast spin-echo MR of the large vestibular aqueduct syndrome.

PURPOSE: To evaluate a high-resolution, thin-section fast spin-echo MR imaging technique of the inner ear to identify the large vestibular aqueduct syndrome seen on temporal bone CT scans. METHODS: We retrospectively reviewed the temporal bone CT scans of 21 patients with hearing loss and enlarged bony vestibular aqueducts by CT criteria. High-resolution fast spin-echo MR imaging was then performed on these patients using dual 3-inch phased-array receiver coils fixed in a temporomandibular joint holder and centered over the temporal bones. MR imaging included axial and oblique sagittal fast spin-echo sequences. The diameter of the midvestibular aqueduct on CT scans and the signal at the level of the midaqueduct on MR images were measured on axial sequences, then compared. High-resolution MR imaging with the same protocol was performed in 44 control subjects with normal ears, and similar measurements were taken. RESULTS: The average size of the enlarged bony vestibular aqueduct on CT scans was 3.7 mm, and the average width of the signal from within the enlarged aqueduct on MR images was 3.8 mm. Statistical analysis showed excellent correlation. MR images alone displayed the enlarged extraosseous endolymphatic sac, which accompanies the enlarged aqueduct in this syndrome. Five ears in three patients with enlarged bony vestibular aqueducts on CT scans showed no evidence of an enlarged endolymphatic duct or sac on MR images. An enlarged endolymphatic sac was seen on MR images in one patient with a bony vestibular aqueduct, which had normal measurements on CT scans. MR imaging alone identified a single case of mild cochlear dysplasia (Mondini malformation). In the 88 normal ears studied, the average size of the endolymphatic sac at its midpoint between the common crus and the external aperture measured on MR images was 0.8 mm (range, 0.5 to 1.4 mm). In 25% of the normal ears, no signal was seen from within the vestibular aqueduct. CONCLUSION: Thin-section, high-resolution fast spin-echo MR imaging of the inner ear is complementary to CT in studying patients with the large vestibular aqueduct syndrome, as MR imaging better displays the soft tissue and fluid of the membranous labyrinth.

Normal-pressure hydrocephalus: evaluation with cerebrospinal fluid flow measurements at MR imaging.

PURPOSE: To evaluate magnetic resonance (MR) imaging-based quantitative phase-contrast cerebrospinal fluid (CSF) velocity imaging for prediction of successful shunting in patients with normal-pressure hydrocephalus (NPH). MATERIALS AND METHODS: Eighteen patients (mean age, 73 years) with NPH underwent routine MR imaging and CSF velocity MR imaging before ventriculoperitoneal (VP) shunting. The calculated CSF stroke volume and the aqueductal CSF flow void score were compared with the surgical results. RESULTS: All 12 patients with CSF stroke volumes greater than 42 microL responded favorably to CSF shunting. Of the six patients with stroke volumes of 42 microL or less, three improved with shunting while three did not. The relationship between CSF stroke volume greater than 42 microL and favorable response to VP shunting was statistically significant (P < .05). There was no statistically significant relationship between aqueductal CSF flow void score and responsiveness to shunting. CONCLUSION: CSF velocity MR imaging is useful in the selection of patients with NPH to undergo shunt formation.

Transcriptional enhancers act in cis to suppress position-effect variegation.

We have examined the basis of enhancer effects on gene expression by altering the action of enhancers on expression of a stably integrated reporter gene. We used two distinct experimental approaches: recombinase-mediated deletion of an enhancer and modulation of the activity of another enhancer composed of downstream metal response elements (MREs). The flp recombinase was used to delete the 5'HS2 globin enhancer from a site downstream of beta-geo at nine separate integration sites in K562 erythroleukemia cells. In no case does deletion of 5'HS2 have a significant effect on the level of expression; however, the deletion does increase dramatically the rate at which expression of beta-geo is silenced. Zinc stimulation of a metallothionein enhancer has no effect on the level of reporter expression, but slows the rate of silencing. Silencing in both cases is highly site dependent, and resembles position-effect variegation (PEV). These results strongly support a binary mode of enhancer action, as in both cases the enhancer maintains reporter expression without a strong effect on the level of expression. Taken together, these findings suggest that transcriptional activators have a direct interaction with repressive chromatin structures, which is independent of an effect on the rate of transcription. We propose that cis-acting transcriptional control elements may act primarily through this mechanism.