A DNA methylation biomarker of alcohol consumption.

The lack of reliable measures of alcohol intake is a major obstacle to the diagnosis and treatment of alcohol-related diseases. Epigenetic modifications such as DNA methylation may provide novel biomarkers of alcohol use. To examine this possibility, we performed an epigenome-wide association study of methylation of cytosine-phosphate-guanine dinucleotide (CpG) sites in relation to alcohol intake in 13 population-based cohorts (ntotal=13 317; 54% women; mean age across cohorts 42-76 years) using whole blood (9643 European and 2423 African ancestries) or monocyte-derived DNA (588 European, 263 African and 400 Hispanic ancestry) samples. We performed meta-analysis and variable selection in whole-blood samples of people of European ancestry (n=6926) and identified 144 CpGs that provided substantial discrimination (area under the curve=0.90-0.99) for current heavy alcohol intake (⩾42 g per day in men and ⩾28 g per day in women) in four replication cohorts. The ancestry-stratified meta-analysis in whole blood identified 328 (9643 European ancestry samples) and 165 (2423 African ancestry samples) alcohol-related CpGs at Bonferroni-adjusted P<1 × 10-7. Analysis of the monocyte-derived DNA (n=1251) identified 62 alcohol-related CpGs at P<1 × 10-7. In whole-blood samples of people of European ancestry, we detected differential methylation in two neurotransmitter receptor genes, the γ-Aminobutyric acid-A receptor delta and γ-aminobutyric acid B receptor subunit 1; their differential methylation was associated with expression levels of a number of genes involved in immune function. In conclusion, we have identified a robust alcohol-related DNA methylation signature and shown the potential utility of DNA methylation as a clinically useful diagnostic test to detect current heavy alcohol consumption.

[Enophthalmos correction in complex orbital floor reconstruction : computer-assisted, intraoperative, non-contact, optical 3D support]. / Enophthalmuskorrektur bei komplexer Orbitarekonstruktion : Computerassistierte, intraoperative, berührungsfreie, optische 3D-Unterstützung.

In the case of displacement of the globe such as enophthalmos induced by trauma, the patient is affected on both counts: function and aesthetics. To prevent double vision or conspicuous asymmetry, exact correction of the globe position is required. The aim of this case report is to demonstrate an intraoperative computer-assisted, non-contact, optical 3D procedure for identification of the globe position to aid in placing the eyeball in the position required in complex reconstruction of the orbital floor. A 33-year-old man presented with a sunken eye on the right side in the horizontal and vertical plane 6 months after having undergone surgery elsewhere for a zygomatico-orbital fracture, also including the orbital floor. The patient was affected by double vision and a noticeable defective globe position. In planning the correction of the globe position, a three-dimensional image of the face with opened eyes was made with the optical sensor. Automatic comparison of symmetry revealed enophthalmos of 4 mm on relative en- and exophthalmometry. The decision was made to lift the orbital floor with a split calvarial bone graft. During surgery the position of the globe was also controlled by the three-dimensional optical technique. At the end of surgery there was exophthalmos of 1 mm. Six weeks after surgery the patient was not affected by any double vision. After 3 and 24 months enophthalmos was 1 mm. This case demonstrates how the non-ionizing, non-contact, optical 3D technique can help in planning, intraoperative transformation, and clinical monitoring to identify the correct position of the corneal vertex in complex orbital floor reconstruction.

[Management of conjunctival malignant melanoma associated with primary acquired melanosis (PAM) using 0.02% mitomycin C eyedrops]. / Therapie maligner Melanome der Konjunktiva bei primär erworbener Melanose (PEM) mit Mitomycin-C-0,02%-Augentropfen.

BACKGROUND: The therapy of malignant diseases of the conjunctiva with local chemotherapy is an extension of the therapeutic options in this field. We report on our experience in the therapy of malignant melanomas of the conjunctiva associated with primary acquired melanosis (PAM). METHODS AND PATIENTS: Between March 1998 and April 2001, 13 patients with malignant melanoma of the conjunctiva associated with PAM (6 female; 7 male; mean age 57+/-13 years) were treated with local chemotherapy. The tumor was classified as stage pT2 (pN0, pM0) in seven patients, stage pT3 (pN0, pM0) in three patients and in the remaining three patients the lid was involved in the malignant process (pT4, pN0, pM0). Local chemotherapy (mitomycin C 0.02% eyedrops 5 times a day) was applied after incisional biopsies in 2 cycles for 14 days with a 14-day break. In 4 patients a third cycle was included. RESULTS: Regression of the tumor was observed after completion of the therapy in all cases. Severe ocular or systemic secondary effects were not seen in our patients. Nine patients were without recurrence within the follow-up time. In three patients, a recurrence of the disease was observed. In these cases, the eyelid was involved in the process. CONCLUSION: Local chemotherapy with mitomycin C is a useful option in the treatment of malignant melanomas of the conjunctiva associated with PAM if the tumor stage is pT3 or less. From our point of view the combination with incisional biopsy is of great benefit. Prognosis of conjunctival malignant melanomas involving the lid margin (pT4) is poor.

Structural determinants of a glucocorticoid receptor recognition element.

Analysis of the relative inducibility of an extensive series of mutant glucocorticoid response elements (GREs) defines features critical to the constitution of an active GRE. Assuming that function as a GRE reflects binding of glucocorticoid receptor, our activity data are consistent with the recognition of the GRE as two hexamer half-sites, each half-site recognized by a single subunit of a receptor dimer, probably in a cooperative fashion. Integrity of both half-sites is necessary for an active element, and spacing of the half-sites is critical. The identity of 1 basepair within the hexamer half-site is unconstrained; the receptor probably makes no base-specific contacts at this position. In contrast, at other positions within the half-site, limited substitutions (if any) can be tolerated. These results along with data from certain insertion mutations suggest that the receptor recognizes each hexamer half-site as two separable subelements. A further implication is that the DNA-binding domain of the glucocorticoid receptor is composed of distinct subdomains, which interact with the subelements of the recognition sequence.

Mapping contacts between unpurified human progesterone receptor and the hormone response element of mouse mammary tumor virus.

Binding of steroid hormone receptors to specific recognition sites of hormone-inducible genes is one of the events required for hormonal regulation of gene transcription. We have employed an immunoprecipitation assay to map the interaction between unpurified human progesterone receptors from crude nuclear extracts of T47D cells and the hormone response element of the mouse mammary tumor virus (MMTV). DNase I footprints and methylation interference patterns are similar to those reported with highly purified rabbit progesterone receptors, suggesting that both human and rabbit receptors recognize similar features in the hormone response element. More importantly, these patterns suggest that if other factors are associated with unpurified nuclear receptor, they do not alter the contacts made by receptor nor do they make contacts themselves with MMTV DNA in a manner detected by DNase I or methylation interference assays. The sites of interaction of receptors bound with the clinically important progestin antagonist, RU 486, are comparable to those observed with an agonist-receptor complex. These results suggest that the antagonist prevents receptor action at a step after its recognition and binding to specific sites on a hormone-responsive enhancer element.

A quantitative comparison of dual control of a hormone response element by progestins and glucocorticoids in the same cell line.

Progesterone receptor-containing T47D human breast cancer cells are responsive to progestins but fail to respond to other steroid hormones, in particular dexamethasone, because they have no measurable levels of receptors for estrogens, androgens, or glucocorticoids. To quantitatively study dual responsiveness of the mouse mammary tumor virus (MMTV) promoter to progestins and glucocorticoids, we have stably transfected T47D cells with a glucocorticoid receptor (GR) expression vector. A cloned derivative (A1-2) was isolated that expresses a normal, full length GR, as assessed by steroid binding and Western immunoblot with a monoclonal anti-GR antibody. Moreover, GR is expressed at levels (80,000-100,000 molecules per cell) comparable to the high levels of endogenous progesterone receptor (200,000 molecules per cell). In A1-2 cells transiently transfected with an MMTV-chloramphenicol acetyl transferase reporter gene, induction by glucocorticoid was substantially greater (5-fold) than induction mediated by progestins. These results suggest that glucocorticoids may be the primary regulator of MMTV.

Dual function of a nuclear factor I binding site in MMTV transcription regulation.

Using linker-scanning mutagenesis we had previously identified four elements within the MMTV LTR which are necessary for transcriptional stimulation by glucocorticoid hormones. Two of them overlapped with regions to which the glucocorticoid receptor binds in vitro. The third element contained a NF-I binding site, and the fourth the TATA box. Here we show that mutations that abolish in vitro binding of NF-I had a negative effect also on the basal activity of the MMTV promoter of LTR-containing plasmids stably integrated in Ltk- fibroblasts. The analysis of double mutants altered in the NF-I plus either one of the receptor binding elements further demonstrated that the NF-I site functionally cooperated with the proximal (-120) element, which alone was extremely inefficient in stimulation. The stronger distal (-181/-172) element was independent of NF-I and showed functional cooperativity with the proximal hormone-binding element.

Human progesterone receptor binding to mouse mammary tumor virus deoxyribonucleic acid: dependence on hormone and nonreceptor nuclear factor(s).

Using crude progesterone receptor preparations from T47D human breast cancer cells, we show by immunoprecipitation assay that receptor specifically and with high affinity recognizes the hormone response element (HRE) of the mouse mammary tumor virus (MMTV). The use of crude preparations minimizes alterations of receptors or loss of associated factors that may occur during purification. Specific binding was obtained at 1:1 molar ratios of receptor to DNA, and HRE sequences are recognized with an affinity at least 3 orders of magnitude greater than nonspecific DNA. We have compared the DNA-binding activities of different forms of progesterone receptors. The unliganded 8S cytosol receptor had low but detectable binding activity for MMTV DNA. Addition of hormone to cytosol produced a small but consistent 2.5-fold increase. In vitro methods of transforming cytosol receptors from an 8S to a 4S species failed to increase DNA-binding further. By contrast, 4S receptors bound by R5020 in whole cells and extracted from nuclei by salt, displayed a substantially higher (average, 11-fold) binding activity than an equal number of unliganded cytosol receptors. The dissociation constants for cytosol and nuclear receptor binding to MMTV DNA were similar (approximately 2 x 10(-9) M). Thus, nuclear receptors possess a higher capacity for binding to specific recognition sequences. These results suggest that hormone or a hormone-dependent mechanism increases the intrinsic DNA-binding activity of receptors independent of receptor transformation from 8S to 4S. Further experiments indicate that a nonreceptor activity in nuclear extracts can increase the sequence-specific DNA-binding activity of cytosol receptors. This activity is present in both T47D cells and receptor-negative MDA-231 cells. We conclude that the higher DNA-binding activity of the nuclear receptor-hormone complex is due in part to receptor interaction with other nuclear proteins or factors. Such interactions may function to maintain receptors in a disaggregated active complex or to stabilize their binding to specific DNA sites.

Functional analysis of the glucocorticoid regulatory elements present in the mouse mammary tumor virus long terminal repeat. A synthetic distal binding site can replace the proximal binding domain.

Transcription of mouse mammary tumor virus DNA is stimulated by steroid hormones. The DNA sequences involved in this regulation are located in the viral long terminal repeat between positions -200 and -50 with respect to the transcription initiation site. In this region four, one distal and three proximal, in vitro binding sites for the glucocorticoid hormone-receptor complexes have been identified. We have prepared a series of 5' and 3' deletions of this region, using the exonuclease ExoIII. Combination of suitable 5' and 3' fragments enabled us to reconstitute the entire long terminal repeat with small internal deletions. The mutated long terminal repeats linked to the coding region of the Herpes simplex virus thymidine kinase gene were introduced into LTK- aprt- cells by transfection. Transcription from the mouse mammary tumor virus promoter in the presence or absence of hormone was assayed by nuclease S1 mapping. Deletion of the proximal in vitro binding sites resulted in a decrease in hormonal inducibility. When a synthetic oligonucleotide harboring the sequence of the distal in vitro binding site was inserted at the site of the proximal ones, hormone response was restored. This indicated that the distal binding site can replace the proximal ones in their hormone-regulatory function. However, insertion at the same site of an oligonucleotide containing the sequence 5' TGTTCT 3' found in all four binding sites, did not restore the hormone response, indicating that sequences flanking the TGTTCT motif are required for hormone response. Insertion of an unrelated DNA fragment at the site of the proximal binding element deletion completely abolished the hormone response. Analyses of different proximal binding-site deletion and insertion mutants suggested the presence of a transcriptional element located downstream from the most proximal hormone-receptor binding site.

Distinct sequence elements involved in the glucocorticoid regulation of the mouse mammary tumor virus promoter identified by linker scanning mutagenesis.

In the proviral DNA of mouse mammary tumor virus (MMTV), sequences up to approximately equal to 200 base-pairs from the RNA start site are required for stimulation of transcription by glucocorticoid hormones in cultured cells. A total of 26 mutant plasmids with clustered point mutations or small deletions in the hormone control region of the MMTV long terminal repeat were constructed, linked to the coding portion of the Herpes simplex virus thymidine kinase gene, and introduced by transfection into LTK- cells. Transcription from mutant DNA in the presence or absence of hormone was quantified by S1 nuclease protection assays. Our analysis revealed the presence of at least three control elements that affect the extent of transcription stimulation by glucocorticoid hormones: (1) a distal element, between -181 and -172 base-pairs from the RNA initiation site. Linker scanning mutants in this segment have a reduction of up to 20-fold in the hormone response with respect to wild type. (2) An element around position -120, defined by a mutation of 4 base-pairs between -121 and -117, which causes a fivefold reduction. (3) An element from approximately equal to -78 to -70, defined by a mutant with also a roughly fivefold lower stimulation. The first two are included in areas that have been shown by others to interact in vitro with hormone-receptor complexes; the last one overlaps the in vitro binding site of a nuclear protein factor. A mutant lacking all three elements (-193 to -70) is completely non-inducible by glucocorticoids. Together with earlier results obtained with 5' deletion mutants, the data show that the largest contribution to the stimulatory response is made by the distal element, which however does require the presence of both more-proximal ones for the response to be maximal. In the absence of the distal one, the two proximal elements together produce a residual stimulation in the order of 5 to 10% of wild type, while the -70 element alone is ineffective. In addition, we show that a functional TATA homology is required for maximum stimulation. It appears that transcriptional regulation of MMTV by glucocorticoid hormones is achieved by the concerted action of multiple sequence modules, not all of which correspond to receptor binding sites in vitro.

Naphthylphthalamic acid-binding sites in cultured cells from Nicotiana tabacum.

Naphthylphthalamic acid (NPA), an inhibitor of polar auxin transport, binds with high affinity to membrane preparations from callus and cell suspension cultures derived from Nicotiana tabacum (K d approx. 2·10(-9) M). The concentration of membrane-bound binding sites is higher in cell suspension than in callus cultures. The binding of NPA to these sites seems to be a simple process, in contrast to the binding of the synthetic auxin naphthylacetic acid (1-NAA) to membrane preparations from callus cultures, which is more complex (A.C. Maan et al., 1983, Planta 158, 10-15). Naphthylacetic acid, a number of structurally related compounds and the auxin-transport inhibitor triiodobenzoic acid were all able to compete with NPA for the same binding site with K d values ranging from 10(-6) to 10(-4) M. On the other hand, NPA was not able to displace detectable amounts of NAA from the NAA-binding site. A possible explantation is the existence of two different membrane-bound binding sites, one exclusively for auxins and one for NPA as well as auxins, that differ in concentration. The NPA-binding site is probably an auxin carrier.