Matter-wave interference of a native polypeptide.

The de Broglie wave nature of matter is a paradigmatic example of quantum physics and it has been exploited in precision measurements of forces and fundamental constants. However, matter-wave interferometry has remained an outstanding challenge for natural polypeptides, building blocks of life, which are fragile and difficult to handle. Here, we demonstrate the wave nature of gramicidin, a natural antibiotic composed of 15 amino acids. Its center of mass is delocalized over more than 20 times the molecular size in our time-domain Talbot-Lau interferometer. We compare the observed interference fringes with a model that includes both a rigorous treatment of the peptide's quantum wave nature as well as a quantum chemical assessment of its optical properties to distinguish our result from classical predictions. The realization of quantum optics with this prototypical biomolecule paves the way for quantum-assisted measurements on a large class of biologically relevant molecules.

Tailored photocleavable peptides: fragmentation and neutralization pathways in high vacuum.

Photocleavable tags (PCTs) have the potential for excellent spatio-temporal control over the release of subunits of complex molecules. Here, we show that electrosprayed oligopeptides, functionalized by a tailored ortho-nitroarylether can undergo site-specific photo-activated cleavage under UV irradiation (266 nm) in high vacuum. The comparison of UV photodissociation (UVPD) and collision-induced dissociation (CID) points to the thermal nature of the cleavage mechanism, a picture corroborated by the temperature dependence of the process. Two competing photodissociation pathways can be identified. In one case a phenolate anion is separated from a neutral zwitterion. In the other case a neutral phenol derivative leaves a negatively charged peptide behind. To understand the factors favoring one channel over the other, we investigate the influence of the peptide length, the nature of the phenolic group and the position of the nitro-group (ortho vs. para). The observed gas phase cleavage of a para-nitro benzylic ether markedly differs from the established behavior in solution.

Sustained elevation of NF-kappaB DNA binding activity in radiation-induced lung damage in rats.

PURPOSE: To characterize the cellular distribution and DNA binding activity of the nuclear factor kappaB (NF-KappaB) in a model of radiation-induced lung damage in the rat. MATERIAL AND METHODS: The right lung of Fischer rats was irradiated with a single dose of 20 Gy. The cellular distributions of NF-KappaB proteins and mRNA were detected with immunohistochemistry and in-situ hybridization respectively. The DNA binding activity of NF-KappaB, nuclear and cytoplasmic levels of NF-KappaB proteins, and kinase activity towards IkappaBalpha (IKappaBAlpha) were determined using electrophoretic mobility shift assays (EMSA), Western blots and kinase assays, respectively. The mRNA level of interleukin 6 (IL-6) was determined using quantitative room temperature polymerase chain reaction. RESULTS: There was a continuous elevation of NF-KappaB DNA binding activity in the rat lung after ionizing irradiation over 6 months. The irradiated lung tissue exhibited an increased kinase activity towards IKappaBAlpha and a selective loss of nuclear IKappaBAlpha. The NF-KappaB-DNA binding complex switched from p50-p65 heterodimers in normal lung tissue to p50 homodimers in irradiated lung tissue. The increased level of IL-6 mRNA suggests transcriptional activation of NF-KappaB-dependent genes in the irradiated rat lung. CONCLUSIONS: The DNA binding activity of NF-KappaB is continuously activated after irradiation of the rat lung by loss of nuclear IKappaBAlpha. This might play a role in sustaining chronic inflammation and hyperproliferation of mesenchymal cells after irradiation.

Protecting against promiscuity: the regulatory role of insulators.

Eukaryotic genomes contain transcriptional regulatory elements that alter promoter activity through long-range interactions. Many control elements show a broad range of promoter interactions, suggesting that these elements are capable of inappropriate transcription. The identification of a novel class of directing regulatory elements, called insulators, has provided clues into mechanisms used in eukaryotic genomes to maintain transcription fidelity. Insulators contribute to the organization of independent domains of gene function by restricting enhancer and silencer function. This review describes the properties of insulators and related elements that have been isolated from several eukaryotic genomes. Two classes of models of insulator function are considered. These models provide insights into possible mechanisms used by these diverse elements to provide regulatory autonomy.

Impact of the tumour bed effect on microenvironment, radiobiological hypoxia and the outcome of fractionated radiotherapy of human FaDu squamous-cell carcinoma growing in the nude mouse.

PURPOSE: To investigate the impact of the tumour bed effect (TBE) on histological parameters of the micromilieu, radiobiological hypoxic fraction and local control after fractionated irradiation in FaDu squamous-cell carcinoma in the nude mouse. This tumour has previously shown a clear-cut TBE caused by increased necrotic cell loss at a constant cell production rate in the viable tumour compartment. MATERIALS AND METHODS: Human FaDu tumours were studied in the NMRI nude mouse. Tumours were transplanted either into unirradiated subcutaneous (s.c.) tissues (controls) or s.c. tissues pre-irradiated with 12.5 Gy (TBE group). In both groups we measured the volume doubling time (VDT), potential doubling time (T(pot)), relative necrotic area, and in the viable tumour compartment the relative vascular area (9F1 mAb), relative hypoxic area (NITP or pimonidazole), relative perfused area (Hoechst 33342), and the perfused fraction of vasculature. The tumour control dose 50% (TCD 50), radiobiological hypoxic fraction (rHF) and dose-modifying factors (DMF) for the comparison of tumours in the TBE and control groups were determined from local tumour control data after treatment with single doses under ambient conditions or under clamp hypoxia, and after irradiation with 30 fractions under ambient conditions within 6 weeks using maximum-likelihood analysis. RESULTS: A clear-cut TBE (VDT = 4.0 days (95%CI 2.9;4.4) for the control group versus 7.2 days (6.4;8.9) for the TBE group; p <0.0001) caused by increased necrosis (mean relative necrotic area of 12% (5;20)) versus 33% (10;41); p = 0.07) at a constant cell production rate (T(pot) = 2.2 days (1.4;2.3) versus 2.2 days (1.7;2.6); p = 0.30) was confirmed. Histological analysis of the micromilieu within the vital subarea revealed no systematic differences between the TBE and control groups. The rHF of 2% (0.1;27) for control tumours was lower than the 15% (95% CI 2;91) for the TBE group, but this difference was nonsignificant (p = 0.12). Compared with control tumours, the TCD50 for irradiation under clamped hypoxia was in a statistical trend lower for tumours in the TBE group (DMF 1.11 (0.98;1.28), p = 0.09). After fractionated irradiation, tumours of the TBE group were significantly more radiosensitive (TCD50 56.6 Gy (46;70) versus 78.7 Gy (63;100); p = 0.003). CONCLUSIONS: The results on FaDu tumours growing in pre-irradiated tissues indicate that increased necrosis caused by impairment of the vascular supply may increase the radiosensitivity of tumours treated by fractioned irradiation.

Patient position reproducibility in fractionated stereotactically guided conformal radiotherapy using the BrainLab mask system.

PURPOSE: Dedicated mask systems nowadays allow the use of stereotactic radiotherapy in fractionated regimes, therefore combining the advantages of high precision radiotherapy with the biological benefit of fractionation. Therefore the knowledge of institution specific isocenter accuracy is essential for decision-making about margins to be allowed to form the planning target volume. PATIENTS AND METHOD: Measurements of isocenter deviations during fractionated treatments were performed in 33 patients using the simulator Simulix-xy (Oldelft) in connection with the BrainLab angiographic localizer-box as well as port-films. In both cases repeated images were overlaid by use of anatomical landmarks with a methodical accuracy in the order of 0.5 mm. RESULTS: Both methods yield random isocenter deviations of less then 2 mm (standard deviation) in all three directions and no significant systematic deviations. These values are in the order of the accuracy of the method, obtained by comparison of two independent investigators, as well as they are comparable with the literature. CONCLUSIONS: The accuracy of less than 2 mm indicates safety margins of 3-4 mm as sufficient for clinical routine to cover the target in 95.5% of all set-ups (2 SD).

Long-range repression by multiple polycomb group (PcG) proteins targeted by fusion to a defined DNA-binding domain in Drosophila.

A tethering assay was developed to study the effects of Polycomb group (PcG) proteins on gene expression in vivo. This system employed the Su(Hw) DNA-binding domain (ZnF) to direct PcG proteins to transposons that carried the white and yellow reporter genes. These reporters constituted naive sensors of PcG effects, as bona fide PcG response elements (PREs) were absent from the constructs. To assess the effects of different genomic environments, reporter transposons integrated at nearly 40 chromosomal sites were analyzed. Three PcG fusion proteins, ZnF-PC, ZnF-SCM, and ZnF-ESC, were studied, since biochemical analyses place these PcG proteins in distinct complexes. Tethered ZnF-PcG proteins repressed white and yellow expression at the majority of sites tested, with each fusion protein displaying a characteristic degree of silencing. Repression by ZnF-PC was stronger than ZnF-SCM, which was stronger than ZnF-ESC, as judged by the percentage of insertion lines affected and the magnitude of the conferred repression. ZnF-PcG repression was more effective at centric and telomeric reporter insertion sites, as compared to euchromatic sites. ZnF-PcG proteins tethered as far as 3.0 kb away from the target promoter produced silencing, indicating that these effects were long range. Repression by ZnF-SCM required a protein interaction domain, the SPM domain, which suggests that this domain is not primarily used to direct SCM to chromosomal loci. This targeting system is useful for studying protein domains and mechanisms involved in PcG repression in vivo.

Differences in insulator properties revealed by enhancer blocking assays on episomes.

Insulators are genomic elements that define domains of transcriptional autonomy. Although a large number of insulators have been isolated, it is unclear whether these elements function by shared molecular mechanisms. Novel applications of FLP recombinase technology were used to dissect and compare the function of the Drosophila: gypsy and scs insulators. Inter actions between FLP monomers bound to chromosomally integrated FRT sites were unimpeded by either insulator, demonstrating that these insulators do not establish a chromosomal environment capable of disrupting all types of protein-protein interactions. The gypsy insulator blocked enhancer-activated transcription on FLP-generated extra-chromosomal episomes, whereas the scs insulator displayed silencing effects. These data indicate that these insulators differ in the mechanisms used to prevent enhancer function. That the gypsy insulator blocked enhancer-promoter communication within small episomes suggests that these effects may be accomplished without a global reorganization of chromatin structure. Instead, the gypsy insulator may disrupt enhancer-activated transcription by direct interference with transmission of the enhancer signal to the promoter.

Long-term career attainments of deaf and hard of hearing college graduates: results from a 15-year follow-up survey.

This article reports on the results of a national longitudinal survey of 240 college graduates with hearing loss. Results confirm that economic benefits resulted from these alumni's postsecondary training. Most respondents were relatively successfully employed and satisfied with life. Over time, increasing numbers had completed higher degrees and secured white-collar positions. Between 1988 and 1998, men in the study sample made more consistent earnings gains than their female counterparts. Larger proportions of deaf alumni had earned advanced degrees and secured white-collar jobs than hard of hearing alumni. Deaf alumni also earned more. Results also showed that recipients of associate's degrees earned more than recipients of bachelor's degrees. Implications of the findings for secondary educators, vocational rehabilitation counselors, and postsecondary service providers are discussed. Recommendations are made on how to improve career decision making by deaf and hard of hearing adolescents, enrich the career potential of deaf and hard of hearing women, and increase the productivity of workers with hearing loss.

Lack of effect of small high-dose volumes on the dose-response relationship for the development of fibrosis in distant parts of the ipsilateral lung in mini-pigs.

PURPOSE: Multi-field radiation therapy for intrathoracic tumours results in a heterogeneous dose distribution in lung tissue. This study investigated whether irradiation of small lung volumes with high fibrogenic doses affects the dose-response relationship for development of fibrosis in distant parts of the ipsilateral lung of mini-pigs. MATERIALS AND METHODS: The whole right lung of 26 'Mini-Lewe' pigs was irradiated with homogeneous doses of between 25 Gy and 40 Gy given in five equal fractions using opposing anterior-posterior portals and a linear accelerator. Another 32 animals were irradiated with a constant dose of 35 Gy to a small house-shaped high-dose field (base 3.0 cm, height 4 cm) located 3 cm caudolateral to the right hilus, while the surrounding right lung received either no irradiation or homogeneous doses of between 20 Gy and 30 Gy. The radiation fields were simulated and port films were obtained for each of the 10 fields in all pigs. Fibrosis was quantified 9 months after irradiation by determination of the hydroxyproline (HP) content of the 32 high-dose volumes and in the lung apex and the basolateral lung of all 58 pigs. Based on the reference value for the HP-ratio, i.e. the HP-concentration of the right lung over the left lung, obtained in 12 unirradiated control animals, the experimental results were converted into quantal data for probit analysis, a responder being an animal with an HP-ratio > 1.33. RESULTS: A dose-response relationship for the HP-ratio was obtained in the different lung sites and irradiation groups. For a given dose level the mean HP-ratios and response rates did not differ systematically between the lung apex and the basolateral lung. Probit analysis of the pooled data produced ED50 values of 21.8 Gy (95% CI 12-37) for irradiation without a high-dose volume and 25.9 Gy (24-28) for irradiation with a high-dose volume. These values are not significantly different. The results from both irradiation groups could be well fitted by a common dose-response curve with an ED50 value of 26.1 Gy. Unexpectedly, the response rates in the high-dose volume increased with increasing dose to the surrounding right lung. Analysis of the port films provided an explanation for this finding: inaccuracies in daily field positioning. When this error was corrected for by use of the mean dose to the high-dose volume, a dose-response curve with an ED50 of 25.2 Gy (22-29) was determined for the high-dose volume. CONCLUSIONS: The results of the study indicate that the irradiation of a small lung volume with high fibrogenic doses does not affect the dose-response relationship for development of fibrosis in distant parts of the ipsilateral lung.

Down-regulation of SP1 DNA binding activity in the process of radiation-induced pulmonary fibrosis.

PURPOSE: To determine changes in the expression and function of the transcription factor SP1 in radiation-induced pulmonary fibrosis. MATERIALS AND METHODS: The right lungs of female Fischer rats were irradiated with a fibrogenic single dose of 20 Gy gamma-irradiation. SP1 mRNA and protein expression was determined by Northern and Western blotting, respectively, between 30 min and 12 weeks after irradiation. Cellular localization of SP1 protein was characterized by immunohistochemistry (peroxidase labelling). SP1 DNA binding activity was studied with electrophoretic mobility shift assays (EMSA). RESULTS: Eight weeks after irradiation, pulmonary fibrosis was first observed. SP1 DNA binding activity showed a short-term increase from 30 min to 12 h after irradiation. Thereafter it remained quite stable until 1 month after irradiation. However, 2 months after irradiation, SP1 DNA binding activity was no longer detectable. The SP1 mRNA level was not reduced at this time, nor was there a reduction in its size. However, Western blotting revealed the occurrence of at least two slightly smaller additional bands 2 months after irradiation whereas the original SP1 band vanished. This suggests a degradation event of SP1 taking place near one or both ends of the protein. Most of the SP1 protein was found in type II pneumocytes and alveolar macrophages of the normal and fibrotic lung. Bronchial epithelial cells were also positive. In the fibrotic lung, proliferating fibroblasts also become positive. CONCLUSIONS: The functional knockout of the transcription factor SP1, in the process of irradiation-induced pulmonary fibrosis, is demonstrated. This should help elucidate the severe disturbances in transcriptional regulation, cellular proliferation and differentiation occurring in the lung at long intervals after irradiation.

Enhancer blocking by the Drosophila gypsy insulator depends upon insulator anatomy and enhancer strength.

Insulators are specialized DNA sequences that prevent enhancer-activated transcription only when interposed between an enhancer and its target promoter. The Drosophila gypsy retrotransposon contains an insulator composed of 12 degenerate binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein that are separated by AT-rich DNA possessing sequence motifs common to matrix/scaffold attachment regions (MARs/SARs). To further understand mechanisms of insulator function, the parameters required for the gypsy insulator to prevent enhancer-activated transcription were examined. Synthetic binding regions were created by reiteration of a single Su(Hw) binding site that lacked the MAR/SAR motifs. These synthetic binding regions reconstituted insulator activity, suggesting that the property of enhancer blocking may be distinct from matrix association. We found that the number and spacing of Su(Hw) binding sites within the gypsy insulator, as well as the strength of the enhancer to be blocked, were important determinants of insulator function. These results provide a link between transcription and insulation, suggesting that these processes may be mechanistically interconnected.

[Benign proximal esophageal stenosis--mostly a complication of gastroesophageal reflux disease]. / Die benigne proximale Osophagusstenose--zumeist Komplikation der gastroösophagealen Refluxkrankheit.

BACKGROUND AND OBJECTIVE: Benign stenoses can occur anywhere in the oesophagus, but are most common in its distal part as a result of gastro-oesophageal reflux (GOR). It was the aim of this study to evaluate retrospectively the causes and incidence of benign stenosis of the proximal oesophagus (SPR) as well as its endoscopic and drug treatment. PATIENTS AND METHODS: Between December 1989 and December 1997 a total of 17,413 patients were referred to the authors' hospital for oesophago-gastroduodenoscopy, 1024 of them (6%) for clarification of heartburn, regurgitation and/or dysphagia. 53 of these patients (5%) were found to have benign stenosis of the oesophagus requiring bougie dilatation, located in the lower third in 29 (55%), in the middle third in six (11%) and in the upper third in 18 (34%) patients. Causes of stenosis in the upper third were peptic stricture in nine (50%), heterotopic gastric mucosa in three (17%), caustic corrosion in three (17%), post-radiation in two (11%), and the result of web formation in one (6%). Endoscopic bougie dilatation was performed in all these patients, those with GOR additionally receiving 40 mg omeprazole daily. RESULTS: In those patients with nonpeptic benign stenosis/stricture lasting improvement of symptoms was achieved with one to three dilatation. But those with GOR needed a mean of 13 dilatations during a follow-up period averaging 61 months. Barrett's oesophagus (replacement of squamous by columnar epithelium) was found in five patients. No case of dysplasia was discovered. Laparoscopic fundoplication was performed in one woman in whom bougie dilatation had failed. Remission was maintained, as needed, by bougie and omeprazole in eight patients. CONCLUSION: In benign stenosis of the upper oesophagus endoscopic dilatation is the treatment of choice. In cases of peptic aetiology the administration of proton pump inhibitors is the optimal adjuvant method.

Core promoter elements can regulate transcription on a separate chromosome in trans.

Transvection can cause the expression of a gene to be sensitive to the proximity of a homolog. It can account for many cases of intragenic complementation at the Drosophila yellow gene, where one mode of transvection involves the action of enhancers in trans on a promoter present on a separate chromosome. Our goal was to identify cis-acting elements that regulate the trans action of enhancers. Using gene replacement, we altered two core promoter elements at yellow and tested the resulting alleles for their ability to support transvection. We found that the TATA box and initiator element can regulate transvection.

An analysis of transvection at the yellow locus of Drosophila melanogaster.

Studies of a wide variety of organisms have shown that homologous sequences can exert a significant impact on each other, resulting in changes in gene sequence, gene expression, chromatin structure, and global chromosome architecture. Our work has focused on transvection, a process that can cause genes to be sensitive to the proximity of a homologue. Transvection is seen at the yellow gene of Drosophila, where it mediates numerous cases of intragenic complementation. In this article, we describe two approaches that have characterized the process of transvection at yellow. The first entailed a screen for mutations that support intragenic complementation at yellow. The second involved the analysis of 53 yellow alleles, obtained from a variety of sources, with respect to complementation, molecular structure, and transcriptional competence. Our data suggest two ways in which transvection may be regulated at yellow: (1) a transcriptional mechanism, whereby the ability of an allele to support transvection is influenced by its transcriptional competency, and (2) a structural mechanism, whereby the pairing of structurally dissimilar homologues results in conformational changes that affect gene expression.

Two modes of transvection: enhancer action in trans and bypass of a chromatin insulator in cis.

Ed Lewis introduced the term "transvection" in 1954 to describe mechanisms that can cause the expression of a gene to be sensitive to the proximity of its homologue. Transvection since has been reported at an increasing number of loci in Drosophila, where homologous chromosomes are paired in somatic tissues, as well as at loci in other organisms. At the Drosophila yellow gene, transvection can explain intragenic complementation involving the yellow2 allele (y2). Here, transvection was proposed to occur by enhancers of one allele acting in trans on the promoter of a paired homologue. In this report, we describe two yellow alleles that strengthen this model and reveal an unexpected, second mechanism for transvection. Data suggest that, in addition to enhancer action in trans, transvection can occur by enhancer bypass of a chromatin insulator in cis. We propose that bypass results from the topology of paired genes. Finally, transvection at yellow can occur in genotypes not involving y2, implying that it is a feature of yellow itself and not an attribute of one particular allele.

A comparison of omeprazole, lansoprazole and pantoprazole in the maintenance treatment of severe reflux oesophagitis.

BACKGROUND: Proton pump inhibitors are effective for the healing of oesophagitis. Standard doses of omeprazole, lansoprazole or pantoprazole are sufficient for healing in mild to moderate cases of oesophagitis. AIM: To compare the efficacy of double the standard doses of omeprazole, lansoprazole or pantoprazole for maintenance treatment of severe oesophagitis complicated by a stricture. METHODS: Thirty-six patients with reflux oesophagitis and stricture confirmed by endoscopy were included in a prospective study comparing three maintenance therapies. In all cases weekly dilatation of the stenosis was performed and patients were treated with omeprazole 20 mg b.d. until healing of oesophagitis and dysphagia relief were achieved. Thirty participants responded to therapy and were then randomly assigned to 4 weeks of maintenance treatment with omeprazole (20 mg b.d.; n = 10), lansoprazole (30 mg b.d.; n = 10) or pantoprazole (40 mg b.d.; n = 10). Subsequently, endoscopies were performed-the endoscopists were blinded to the therapy assignment. The endpoints were defined as the absence of oesophagitis, oesophageal stricture and complaints. RESULTS: After 4 weeks of treatment, the number of patients remaining in remission (no oesophagitis or stricture and no symptoms) was nine out of 10 (90%) in the omeprazole group, two out of 10 (20%) in the lansoprazole group (P < 0.01) and three out of 10 (30%) in the pantoprazole group (P < 0.01). CONCLUSIONS: In our study omeprazole was superior to either lansoprazole or pantoprazole in the maintenance treatment of complicated gastro-oesophageal reflux disease.

Fractionation effect on radiation-induced growth retardation of tibia in rabbits and rats.

A study of the sensitivity to fractionation of the growing tibia of rabbits and rats was conducted by comparing the growth of the treated right bone to that of the untreated left side in each individual animal using radiographic measurements. The experimental endpoint was the percentage of normal growth 24 weeks after irradiation in rabbits and 14 weeks after treatment in rats. The results show clear dose-response relationships in all experimental arms. A clear-cut fractionation effect was demonstrated in both species. The alpha/beta-ratios determined by maximum likelihood analysis according to the LQ-model with graded responses were 3.2 Gy (95% C.I. 1.1; 5.6) in rabbits and 6.9 Gy (5.3; 8.7) in rats, when all data were included in the calculations. When single-dose data were excluded the alpha/beta-values were -0.6 Gy (-3.1; 2.3) in rabbits and 5.0 Gy (3.5; 7.0) in rats. Our data provide further evidence that low doses per fraction should be used when irradiation of the epiphysis cannot be avoided in pediatric patients.

Polycomb group repression is blocked by the Drosophila suppressor of Hairy-wing [su(Hw)] insulator.

The suppressor of Hairy-wing [SU(HW)] binding region disrupts communication between a large number of enhancers and promoters and protects transgenes from chromosomal position effects. These properties classify the SU(HW) binding region as an insulator. While enhancers are blocked in a general manner, protection from repressors appears to be more variable. In these studies, we address whether repression resulting from the Polycomb group genes can be blocked by the SU(HW) binding region. The effects of this binding region on repression established by an Ultrabithorax Polycomb group Response Element were examined. A transposon carrying two reporter genes, the yellow and white genes, was used so that repression and insulation could be assayed simultaneously. We demonstrate that the SU(HW) binding region is effective at preventing Polycomb group repression. These studies suggest that one role of the su(Hw) protein may be to restrict the range of action of repressors, such as the Polycomb group proteins, throughout the euchromatic regions of the genome.