Ultra-high resolution array painting facilitates breakpoint sequencing.

OBJECTIVE: To describe a considerably advanced method of array painting, which allows the rapid, ultra-high resolution mapping of translocation breakpoints such that rearrangement junction fragments can be amplified directly and sequenced. METHOD: Ultra-high resolution array painting involves the hybridisation of probes generated by the amplification of small numbers of flow-sorted derivative chromosomes to oligonucleotide arrays designed to tile breakpoint regions at extremely high resolution. RESULTS AND DISCUSSION: How ultra-high resolution array painting of four balanced translocation cases rapidly and efficiently maps breakpoints to a point where junction fragments can be amplified easily and sequenced is demonstrated. With this new development, breakpoints can be mapped using just two array experiments: the first using whole-genome array painting to tiling resolution large insert clone arrays, the second using ultra-high-resolution oligonucleotide arrays targeted to the breakpoint regions. In this way, breakpoints can be mapped and then sequenced in a few weeks.

Pseudoainhum in chronic psoriasis.

Pseudoainhum is a term used to describe the presence of constricting bands of the extremities due to a variety of underlying causes. Progression of the lesions can cause irreversible damage and autoamputation of the affected digit. This report documents a rare association of pseudoainhum and psoriasis and emphasizes the importance of recognizing this condition.

Virtual coupling of pyran protons in the 1H NMR spectra of C- and N-glucuronides [correction]: dependence on substitution and solvent.

We have observed that certain C- and N-glucuronides prepared as intermediates for breast cancer preventives demonstrate non-first order 1H NMR spectra that are not the result of impurities or degradation but are instead due to virtual coupling in the pyran proton network. This virtual coupling shows the expected dependence on solvent and field strength and, more importantly, on the nature of the C-1 substitution. Although the hybridization of the atom bonded to C-1 may play a role, it appears that steric and/or electronic factors, which have the effect of increasing Delta(v)/J for H-3 and H-4, are critical for eliminating the spectral complexity. These observations, which appear to be fairly general, suggest that this phenomenon should be considered when addressing the purity of pharmaceutical agents containing these types of structural units.

Development and deployment of an internet-based data management system for use by the Asthma Clinical Research Network.

Data management system development for the first Asthma Clinical Research Network (ACRN) study began at the data coordinating center (DCC) in May 1995 with the requirement for delivery of a production system by November 1995. Special methods had to be used to establish an internet local area network (LAN), place clinical client systems, and achieve an accelerated software development cycle. The development of a fully integrated data management system prior to the start of the study was not possible. Therefore an early analysis focused on identifying discrete groupings of data management functions that would allow development of distinct database modules to provide specific functionality such as subject randomization, subject registration, and data entry. The modules were categorized as either being associated with clinical centers or the DCC so that the clinical center modules could be developed and delivered to meet the start date of the study. In the second phase of development during the relatively slow patient-enrollment period, the DCC functional modules were delivered discretely over time. While at the time this development model was a necessity due to limited DCC resources, it continues to be used today as it permits the DCC to implement studies more rapidly and efficiently for the ACRN. This paper describes the methodologies used to develop an internet-based LAN, establish clinical center client systems, establish DCC client and server operations, and develop a data management system. It describes the circumstances that contributed to the development of these systems and the special methodologies developed. The technical aspects of the data management system and LAN are presented as well as a description of the requirements and constraints analysis used to develop the hardware and software systems.

Modem remote support of pulmonary-function testing and quality control systems.

The data coordinating center (DCC) of the Asthma Clinical Research Network (ACRN) is responsible for the support of 11 pulmonary-function testing systems and two quality control systems. Pulmonary-function data from these systems are used as outcome indicators in studies conducted by the ACRN. Each of these systems is composed of a spirometer, a personal computer for data acquisition from the spirometer, a modem, and a printer. These systems are located at six clinical centers nationwide. An analysis conducted at the beginning of the first ACRN protocol identified the following requirements: (1) standard pulmonary-function testing, (2) standard methacholine-challenge testing, (3) the ability to handle simultaneous multiple protocols as well as have data from non-ACRN subjects, (4) the ability to separate data from different protocols as well as separate ACRN and non-ACRN data, (5) the ability to transmit data from the remote clinical centers to the DCC, (6) the ability to ensure quality data and to report on those results, and (7) the ability to provide remote support.

Papular acantholytic dyskeratosis of the vulva.

We describe two patients with unusual asymptomatic, papular lesions on the vulva, clinically resembling lichen planus, the histology of which revealed unexpected findings of suprabasilar clefting, acantholysis and dyskeratotic cells giving rise to corps ronds and grains together with hyperkeratosis and parakeratosis, features originally associated with a diagnosis of Darier's disease. Focal acantholytic dyskeratosis has been described in a wide variety of inflammatory and neoplastic processes including those involving mucous membranes and has been attributed various diagnostic labels. We feel that the findings in our patients are consistent with a diagnosis of papular acantholytic dyskeratosis of the vulva, a rare condition, which was first described in 1984.

An unhydrolyzable analogue of N-(4-hydroxyphenyl)retinamide. synthesis and preliminary biological studies.

The synthesis of a nonhydrolyzable, carbon-linked analogue (4-HBR) of the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) using Umpolung methods is described. Preliminary studies of biological activity show 4-HBR is similar to 4-HPR in its actions although a potentially relevant and desirable difference is its reduced suppression of plasma vitamin A levels. These results show that 4-HPR does not have to be hydrolyzed to retinoic acid to produce its chemotherapeutic effects.

Chemotherapeutic evaluation of 4-hydroxybenzylretinone (4-HBR), a nonhydrolyzable C-linked analog of N-(4-hydroxyphenyl) retinamide (4-HPR) against mammary carcinogenesis.

The antitumor effects of N-(4-hydroxyphenyl)retinamide (4-HPR), and its stable C-linked analog, 4-hydroxybenzylretinone (4-HBR) on the regression of established 7,12-dimethylbenz(a)anthracene(DMBA)-induced rat mammary tumors were compared. 4-HBR is a stable and nonhydroyzable derivative which cannot be converted in vivo to retinoic acid (RA). The results indicate that 4-HBR decreased mammary tumor volumes to the same extent as equimolar concentration (2 mmol/kg diet) of 4-HPR (-45% for 4-HBR vs. -42% for 4-HPR, p<0.01). Both 4-HPR and 4-HBR bind very poorly to nuclear retinoid receptors RARs and RXRs. The similarity of physicochemical properties of 4-HPR and 4-HBR as well as their equal antitumor potency suggests that 4-HPR like 4-HBR, is acting directly rather than through hydrolysis to free RA. Treatment with 4-HPR caused an almost 65% decrease in serum retinol levels. These results suggest that 4-HBR may have a significant chemotherapeutic advantage over 4-HPR, as the nonhydrolyzable analog may not cause night blindness which occurs as a significant side effect of 4-HPR usage.

NMR studies of retinoid-protein interactions: the conformation of [13C]-beta-ionones bound to beta-lactoglobulin B.

PURPOSE: Vitamin A (retinol) and its metabolites comprise the natural retinoids. While the biological action of these molecules are thought to be primarily mediated by ca. 55 kDa nuclear retinoic acid receptors, a number of structurally similar 15-20 kDa proteins are involved in the transport, and possibly metabolism, of these compounds. The milk protein beta-lactoglobulin B (beta-LG) is an 18 kDa protein which binds retinol and may be involved in oral delivery of retinol to neonates. beta-LG also binds drugs and other natural products and is of potential interest as a protective delivery vehicle. METHODS: To examine the conformation of the model retinoid beta-ionone both in solution and when bound to beta-LG, NMR and computational methods have been employed. RESULTS: Taken together, NMR studies of beta-ionone in solution measuring scalar and dipolar coupling, as well as CHARMm calculations, suggest beta-ionone prefers a slightly twisted 6-s-cis conformation. Isotope-edited NMR studies of 13C-labeled beta-ionones bound to beta-LG, primarily employing the HMQC-NOE experiment, suggest beta-ionone also binds to beta-LG in its 6-s-cis conformation. CONCLUSIONS: The methods employed here allow estimates of protein-bound ligand conformation. However, additional sites of ligand labeling will be necessary to aid in binding site localization.

Chemopreventive activity of a C-glucuronide analog of N-(4-hydroxyphenyl) retinamide-O-glucuronide against mammary tumor development and growth.

The long term chemopreventive effects of the N-(4-hydroxyphenyl) retinamide-O-glucuronide (4-HPROG), and its stable C-linked benzyl glucuronide analog, retinamidobenzyl glucuronide (4-HPRCG) on the growth and development of 7,12-dimethylbenz[a]anthracene-induced mammary tumors were compared. The retinamidobenzyl glucuronide is stable toward acid hydrolysis and resists the actions of beta-glucuronidase. The results indicate that the C-linked glucuronide analog, 4-HPRCG has a greater chemopreventive potency than an equimolar concentration of 4-HPROG. Tumor latency was 15% longer in rats fed 2 mmol/kg diet of 4-HPRCG as compared to 4-HPROG. At 80 days post DMBA-intubation, tumor incidence was 57% and 27% in the 4-HPROG and 4-HPRCG treated rats, respectively. Tumor multiplicity was also markedly decreased in the 4-HPRCG treated rats. At 80 days post DMBA intubation the control rats had an average of 1.43 tumors/rat compared to 0.71 and 0.36 tumors/rat in the 4-HPROG and 4-HPRCG respectively. The higher potency and low toxicity of 4-HPRCG suggest that this stable analog may have an in vivo chemopreventive advantage over its analog, 4-HPROG. The results also demonstrated that these glucuronide analogs do not bind effectively in vitro either to the nuclear retinoid receptors or to the cellular retinoid binding proteins. Regardless of the mode of action of these retinoids, they are clearly effective chemopreventive agents.

Screening for malignant melanoma using instant photography.

OBJECTIVE: To assess the use of instant photography, in addition to clinical grading, as a method of screening for malignant melanoma during routine health examinations. SETTING: A health screening clinic with an average throughput of about 12,000 patients a year. METHODS: Suspicious pigmented skin lesions were judged clinically using the revised seven point checklist scoring system. They were then photographed with a Polaroid camera and the prints were graded independently by two consultant dermatologists with a special interest in malignant melanoma. A copy of the print was also given to the patient to keep for observation of any change in the lesion. RESULTS: Over a 45 month period 39,922 patients of both sexes were screened and 1052 skin lesions were clinically assessed and photographed. Fourteen malignant melanomas were diagnosed--all, except one, were thin lesions with a good prognosis. CONCLUSIONS: The clinical opinions of non-dermatologists using the revised seven point checklist proved disappointing in screening because of the large number of benign lesions that were given high scores. Photography, on the other hand, detected 11 melanomas and succeeded in separating the majority of banal lesions from potentially malignant ones, thus greatly reducing the need for specialist referral. Nevertheless, three melanomas were missed on purely photographic grading, which emphasises the danger of placing too much reliance solely on a two dimensional image. Finally, the possession of a personal copy of the photograph by the patient proved popular and led to a diagnosis of melanoma in two instances. This procedure merits further study.

Melanoma yield, number of biopsies and missed melanomas in a British teaching hospital pigmented lesion clinic: a 9-year retrospective study.

The demand for pigmented lesion clinics (PLCs) is increasing in view of improved skin cancer awareness following public health education campaigns. These clinics offer an effective way of screening a large number of patients. However, there is no evidence, as yet, that they have an impact on mortality due to malignant melanoma. With the lack of follow-up inherent to these busy screening clinics, there is some concern that melanomas may be missed. This study reports on 7874 patient visits to a PLC in a teaching hospital between 1985 and 1994. In total, 1705 biopsies were performed over the 9-year period. Lesions were more likely to be biopsied in men compared with women. The yield for picking melanomas was one in 36 patient visits. The mean age of patients attending the PLC was 10 years less than the mean age for population-based melanomas. Melanoma thickness did not significantly change over the 9-year period. Only 0.2% of patients (14 cases) re-presented to the PLC for a second or third visit with a final diagnosis of melanoma, but for five of these patients, the interval between the two visits was over 2 years. Most of these 're-attending' melanomas were early lesions. PLCs offer a fast, safe and efficient service for the screening of pigmented lesions but their role in reducing mortality due to malignant melanoma remains to be established. It is likely that these clinics have an important role in terms of public health education regarding sun avoidance and early recognition of skin cancer.

N-linked glycoside and glucuronide conjugates of the retinoid, acitretin, are biologically active in cornea and conjunctiva.

The purpose of this study was to test two water-soluble, synthetic retinoids, glucoseamido acitretin and glucuronamido acitretin, for biological activity in cells of the cornea and conjunctiva. Vitamin A-deficient, xerophthalmic rats were treated topically with these retinoids, and corneas were examined histologically for effects on epithelial keratinization. The effect of these retinoids on the proliferation of rabbit conjunctival fibroblasts in culture was also investigated. Glucoseamido acitretin treatment restored a normal cornea after eight to nine days of treatment, while no improvement was observed in the vehicle-treated corneas. Likewise, glucuronamido acitretin application restored a normal corneal surface and reversed keratinization after eight to ten days of treatment. These retinoids caused no irritation of the eye or ocular adnexa. In culture, exposure of conjunctival fibroblasts to glucoseamide acitretin inhibited cell proliferation. Cultures exposed to glucoseamido acitretin at 10(-8) M or 10(-6) M had cell densities 77.3% and 51.9% of control, respectively, after seven days. Glucuronamido acitretin also inhibited cell proliferation. Cultures exposed to glucuronamido acitretin at 10(-8) M had a cell density of 69.2% of control at day seven, while at 10(-6) M this retinoid completely inhibited cell proliferation. These results show that glucoseamide acitretin and glucuronamido acitretin are biologically active in the cornea and conjunctiva, and may be considered for ophthalmic use in diseases involving abnormalities of ocular surface cell differentiation or hyperproliferation of fibroblasts.

Chemotherapeutic evaluation of N-(4-hydroxyphenyl) retinamide-O-glucuronide in the rat mammary tumor model.

The growth inhibitory effects of N-(4- Hydroxyphenyl) retinamide (4-HPR) and its glucuronide derivative, N-(4-Hydroxyphenyl) retinamide-O-glucuronide (4-HPROG) on established DMBA induced rat mammary tumors were compared. The results indicate that the glucuronide analog had a greater antitumor potency than equimolar concentration of the free retinoid. Tumor regression occurred in 75% of the rats fed 2 mmol/Kg diet of 4-HPROG. In a 6-week study, the maximum tolerated dietary dose (MTD) was found to be 3.5 mmol/Kg diet for 4-HPR and 5 mmol/Kg diet in the case of 4-HPROG. The higher potency and lower toxicity of the glucuronide suggests that this conjugate may have an in vivo chemotherapeutic advantage over the parent free retinoid.