Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA.

We show that RNA polymerase (Pol) II prevents erroneous transcription in vitro with different strategies that depend on the type of DNARNA base mismatch. Certain mismatches are efficiently formed but impair RNA extension. Other mismatches allow for RNA extension but are inefficiently formed and efficiently proofread by RNA cleavage. X-ray analysis reveals that a TU mismatch impairs RNA extension by forming a wobble base pair at the Pol II active center that dissociates the catalytic metal ion and misaligns the RNA 3' end. The mismatch can also stabilize a paused state of Pol II with a frayed RNA 3' nucleotide. The frayed nucleotide binds in the Pol II pore either parallel or perpendicular to the DNA-RNA hybrid axis (fraying sites I and II, respectively) and overlaps the nucleoside triphosphate (NTP) site, explaining how it halts transcription during proofreading, before backtracking and RNA cleavage.

Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif.

Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA-RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5--the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5.

Molecular basis of transcriptional mutagenesis at 8-oxoguanine.

Structure-function analysis has revealed the mechanism of yeast RNA polymerase II transcription at 8-oxoguanine (8-oxoG), the major DNA lesion resulting from oxidative stress. When polymerase II encounters 8-oxoG in the DNA template strand, it can misincorporate adenine, which forms a Hoogsteen bp with 8-oxoG at the active center. This requires rotation of the 8-oxoG base from the standard anti- to an uncommon syn-conformation, which likely occurs during 8-oxoG loading into the active site. The misincorporated adenine escapes intrinsic proofreading, resulting in transcriptional mutagenesis that is observed directly by mass spectrometric RNA analysis.

Structure-function studies of the RNA polymerase II elongation complex.

RNA polymerase II (Pol II) is the eukaryotic enzyme that is responsible for transcribing all protein-coding genes into messenger RNA (mRNA). The mRNA-transcription cycle can be divided into three stages: initiation, elongation and termination. During elongation, Pol II moves along a DNA template and synthesizes a complementary RNA chain in a processive manner. X-ray structural analysis has proved to be a potent tool for elucidating the mechanism of Pol II elongation. Crystallographic snapshots of different functional states of the Pol II elongation complex (EC) have elucidated mechanistic details of nucleotide addition and Pol II translocation. Further structural studies in combination with in vitro transcription experiments led to a mechanistic understanding of various additional features of the EC, including its inhibition by the fungal toxin alpha-amanitin, the tunability of the active site by the elongation factor TFIIS, the recognition of DNA lesions and the use of RNA as a template.

The role of double covalent flavin binding in chito-oligosaccharide oxidase from Fusarium graminearum.

ChitO (chito-oligosaccharide oxidase) from Fusarium graminearum catalyses the regioselective oxidation of N-acetylated oligosaccharides. The enzyme harbours an FAD cofactor that is covalently attached to His94 and Cys154. The functional role of this unusual bi-covalent flavin-protein linkage was studied by site-directed mutagenesis. The double mutant (H94A/C154A) was not expressed, which suggests that a covalent flavin-protein bond is needed for protein stability. The single mutants H94A and C154A were expressed as FAD-containing enzymes in which one of the covalent FAD-protein bonds was disrupted relative to the wild-type enzyme. Both mutants were poorly active, as the k(cat) decreased (8.3- and 3-fold respectively) and the K(m) increased drastically (34- and 75-fold respectively) when using GlcNac as the substrate. Pre-steady-state analysis revealed that the rate of reduction in the mutant enzymes is decreased by 3 orders of magnitude when compared with wild-type ChitO (k(red)=750 s(-1)) and thereby limits the turnover rate. Spectroelectrochemical titrations revealed that wild-type ChitO exhibits a relatively high redox potential (+131 mV) and the C154A mutant displays a lower potential (+70 mV), while the H94A mutant displays a relatively high potential of approximately +164 mV. The results show that a high redox potential is not the only prerequisite to ensure efficient catalysis and that removal of either of the covalent bonds may perturb the geometry of the Michaelis complex. Besides tuning the redox properties, the bi-covalent binding of the FAD cofactor in ChitO is essential for a catalytically competent conformation of the active site.

Hereditary posterior microphthalmos with papillomacular fold and high hyperopia.

Five patients had a bilateral hereditary ocular syndrome composed of posterior microphthalmos with a papillomacular fold and high hyperopia. Anterior segment dimensions were near normal; the vitreous compartment was markedly fore-shortened. A papillomacular retinal fold extending from the center of the fovea toward the optic nerve head was present. Visual acuity ranged from 0.05 (20/400) to 0.6 (20/33); refractive errors ranged from + 11.25 to + 17.50 diopters. An autosomal recessive pattern of inheritance is postulated.

Biometric studies of the eyeball and retinal detachment.

The relationship between axial length and equatorial diameter and eye volume has been studied in 4 groups of eyes: intact emmetropic eyes, emmetropic eyes with retinal detachment, eyes with moderate myopia and retinal detachment, and eyes with high myopia and retinal detachment. It was found that eyes with detached retinas, regardless of whether they are myopic or emmetropic, are of significantly different dimensions from emmetropic control eyes. There is usually a significant increase in equatorial diameter and therefore in equatorial circumference. The results indicate that eyes with detached retinas differ from normal eyes in their abnormally large size.

[Stability of postoperative visual acuity after rt-PA -SF6-treatment of submacular hemorrhage in age-related macular degeneration]. / Stabilität des postoperativen Visus nach rt-PA-SF6-Behandlung der submakulären Hämorrhagie bei altersabhängiger Makuladegeneration.

PURPOSE: Treatment of submacular hemorrhage with recombinant tissue plasminogen activator(rt-PA) and SF6 induces blood displacement out of the fovea by maintaining prone positioning. The underlying disease, e.g. the choroidal neovascularisation remains untreated. Nevertheless, it has been noted that the visual results of this method are more favorable than those expected in the natural course. Contributing long-term results the present study intends to give more information concerning this phenomenon. METHOD: A total of 45 consecutive eyes were treated using a standardized procedure (duration of hemorrhage < or =1 week, area of hemorrhage 1-10 disc areas, 50 gammag rt-PA, 0.5 ml SF6 24 h later, prone positioning for 3 days). Those with a follow-up of > or =6 months were included in the study. RESULTS: From 25 patients a total of 25 eyes fulfilled the criteria. Postoperative visual acuity(3-7 days) was improved or unchanged in 19 eyes and worse in 6 eyes. Comparison of postoperative visual acuity after follow-up of an average of 14.2 months (range 6-28 months) revealed an improvement in 10 eyes, no change in 7 eyes and a deterioration in 8 eyes. A second treatment was performed in 2 eyes and photocoagulation in 1 eye. CONCLUSION: After more than 1 year the postoperative visual acuity was stable or even better in about two-thirds of the eyes therefore treatment with rt-PA and SF6 may also influence the choroidal neovascularisation.

[First unilateral, later bilateral optic neuropathy. Amiodarone as the cause?]. / Zunächst einseitige, später beidseitige Optikopathie. Amiodaron als Ursache?

CASE REPORT: Amiodarone has many severe side-effects and can cause keratopathy and neuropathy. We report the case of a 79-year-old male patient with progressive visual loss and papilloedema of the right eye during therapy with amiodarone. To reduce papilloedema he was treated with corticosteroids. Amiodarone therapy was then discontinued, but there was no considerable improvement and after 6 weeks visual loss occurred in the left eye as well. CONCLUSION: With regard to the findings we consider this case to be an amiodarone-induced optic neuropathy. According to our knowledge only 19 cases have been reported in the literature but the results of one study indicate that the incidence of optic neuropathy is as high as 1.79%. The pathophysiology of the optic nerve damage remains unclear and an effective treatment is not available. It might be possible that the accelerated elimination of amiodarone with colestyramine could be suitable to prevent optic neuropathy of the second eye.

[Sclera fixation of posterior chamber lenses in vitrectomized eyes. Perfluorocarbon as an operative aid]. / Sklerafixation von Hinterkammerlinsen in vitrektomierten Augen. Perfluorkarbon als operatives Hilfsmittel.

INTRODUCTION: Accurate positioning of transsclerally fixated intraocular lenses in vitrectomized eyes is difficult to achieve because of the lack of stability of the globe. External stabilization with a Flieringa device is widely used. Internal stabilization, however, with liquid perfluorocarbon seems to be more advantageous. METHODS: Endostabilization with perfluorocarbon after pars plana vitrectomy prior to transscleral fixation of a posterior chamber lens was performed in 18 eyes. The intraocular lens floats on the surface of the perfluorocarbon. The level of the liquid can be adjusted to position the lens accurately. Transscleral fixation of the intraocular lens was followed by silicone oil injection in 4 eyes and by BSS injection in 14 eyes. RESULTS AND CONCLUSIONS: Endostabilization of the globe with liquid perfluorocarbon serves as a diaphragm preventing hemorrhage into the vitreous cavity and sliding of the intraocular lens, thus permitting proper positioning. There were no complications either intraoperatively, or postoperatively. Perfluorocarbon is a considerable aid in facilitating the positioning of a transsclerally fixated intraocular lens.

[Late dislocation of the capsular bag after phacoemulsification with endocapsular IOL in pseudoexfoliation syndrome]. / Späte Luxation des Kapselsackes nach Phakoemulsifikation mit endokapsulärer IOL beim Pseudoexfoliationssyndrom.

UNLABELLED: PES has been associated with weakness of zonular attachments to the ciliary body and with late dislocation of the IOL following cataract extraction with in-the-bag IOL implantation. We evaluated the frequency of PES in patients with IOL dislocation following phacoemulsification cataract surgery. PATIENTS AND METHODS: We retrospectively reviewed the records of five patients who had undergone phacoemulsification, followed by implantation of a posterior chamber IOL in the capsular bag during the years 1992 through 1996 and who showed IOL dislocation during follow-up. We looked for clinical signs of PES that had been noted prior to or after IOL dislocation. RESULTS: Detailed review of the records of these patients identified PES as a coexistent condition in all five. The most impressive clinical feature was shrinkage of the capsular bag to conform to the size and contour of the implanted IOL. CONCLUSIONS: In accordance with many histopathological and clinical studies supporting our findings, we suggest that shrinkage of the capsular bag can be reduced by not using foldable IOL, by early anterior YAG capsulotomy or by the use of an IOL with a larger optic.

[Induced astigmatism in cataract surgery. Scleral tunneling incisions of 5.5 mm and 6.5 mm after 1-year follow-up]. / Induzierter Astigmatismus in der Kataraktchirurgie. Skerale Tunnelinzisionen von 5,5 mm und 6,5 mm nach 1jähriger Verlaufskontrolle.

To evaluate the effect of scleral pocket incisions closed with a single horizontal suture on postoperative astigmatism, 97 patients were enrolled in a prospective study. After 1 year of follow-up the data of 80 patients could be analysed. Routine phacoemulsification was performed in all patients consecutively by one surgeon. 40 patients received a posterior chamber lens with a 5 mm by 6 mm oval polymethylmethacrylate (PMMA) optic (group I), and 40 patients received an intraocular lens with a 6 mm diameter round PMMA optic (group II). The incisions were 5.5 mm and 6.5 mm, respectively. Follow-up visits including keratometry were scheduled 1 day, 5 days, 3 months, 6 months and 1 year postoperatively. The induced astigmatism was calculated using vector analysis. One day after operation the mean induced cylinder was 1.22 D in group I and 1.06 D in group II. After 5 days it amounted to 1.09 D (group I) and 1.03 D (group II), and at 3 months it was 1.07 D and 1.00 D, respectively. Six months after operation the induced cylinder was 1.04 D (group I) and 0.96 D (group II), and at 1 year it was 1.02 D and 0.81 D. There was no statistically significant difference between the groups at any time (Wilcoxon test, P > 0.05). We conclude that scleral pocket incisions closed with a single horizontal suture induce about 1 D of corneal astigmatism, with stability over time. There is no clinical advantage in reducing the incision width by using oval optics.

[Disruption of the blood-aqueous barrier after implantation of sclera-fixed posterior chamber lenses. Early postoperative phase and long-term outcome]. / Beeinträchtigung der Blut-Kammerwasser-Schranke nach Implantation von sklerafixierten Hinterkammerlinsen. Frühe postoperative Phase und Langzeitergebnisse.

The implantation of transsclerally sutured posterior chamber lenses (PCL) leads to greater trauma to the eye than endocapsular PCL implantation. Persistent breakdown of the blood-aqueous barrier might impair the postoperative long-term prognosis. Using laser-tyndallometry, we quantified the disorder of the barrier function during the early postoperative phase and in the long-term postoperative course for both surgical procedures. During the first 3 postoperative days, flare values were three times higher in the group with transsclerally sutured PCL than in the conventional PCL group. Cell counts after transscleral suture fixation (27.7 +/- 18.3/0.075 mm3) decreased slightly during the first 5 postoperative days. In contrast, the conventional PCL group regained the preoperative level (2.5 +/- 5.1) after 3 days. After 3 months, no significant differences in flare values and cell counts were seen between the two groups. After implantation of transsclerally sutured posterior chamber lenses, breakdown of the blood-aqueous barrier was initially more pronounced than in conventional PCL implantation. However, the barrier function was re-established equally in both groups in the long term. No signs of a persistent disorder of the barrier were found.

[Lacrimal duct recanalization with balloon catheter]. / Tränenwegrekanalisation mit Ballonkatheter.

In dilation of nasolacrimal duct stenosis materials now being used originally were developed for percutaneous transluminal coronary angioplasty (PTCA). Suitable modifications of the angioplasty equipment could improve therapeutic results and thus influence the area of indication of this new technique. In addition to the standard PTCA equipment, we used a cannula designed to fit the guide wire, leading to less traumatic intubation of the lacrimal duct and allowing radiographic control of the recanalization procedure. Five patients showing relative stenosis and ten patients showing complete obstruction of the nasolacrimal duct were treated by dacryocystoplasty. Clinical and radiographic follow-up was done over a period of 6 months. In a total of seven patients, dilation proved successful. Out of these, four showed stenosis (80% recanalization rate) and three complete obstruction (30% recanalization rate) before dilation. Recanalization by dilation using balloon catheters seems to be possible even in cases of complete nasolacrimal duct obstruction. However, the success rate is considerably lower than in relative stenosis.

Mechanism of transcriptional stalling at cisplatin-damaged DNA.

The anticancer drug cisplatin forms 1,2-d(GpG) DNA intrastrand cross-links (cisplatin lesions) that stall RNA polymerase II (Pol II) and trigger transcription-coupled DNA repair. Here we present a structure-function analysis of Pol II stalling at a cisplatin lesion in the DNA template. Pol II stalling results from a translocation barrier that prevents delivery of the lesion to the active site. AMP misincorporation occurs at the barrier and also at an abasic site, suggesting that it arises from nontemplated synthesis according to an 'A-rule' known for DNA polymerases. Pol II can bypass a cisplatin lesion that is artificially placed beyond the translocation barrier, even in the presence of a G.A mismatch. Thus, the barrier prevents transcriptional mutagenesis. The stalling mechanism differs from that of Pol II stalling at a photolesion, which involves delivery of the lesion to the active site and lesion-templated misincorporation that blocks transcription.

[Laser radiation protection in medicine]. / Laserstrahlenschutz in der Medizin.

In medicine, lasers with different wave lengths are applied for therapeutic purposes. The human eye is especially endangered by laser rays with a wave length between 400 and 1400 nm. This is due to the fact that these laser rays are focused in the retina by the refracting media of the eye, so that the energy dose is considerably increased. If these lesions are situated in the most sensitive point of the retina, this may cause a markedly deteriorated vision. The necessary protective measures in the use of laser rays are based on a classification of lasers in four degrees according to the risk involved. The essential measures of protection are a shielding by filters, protective glasses and the determination of a so-called laser zone.

[Vitrectomy and subsequent photocoagulation in proliferative diabetic retinopathy with vitreous hemorrhage]. / Vitrektomie und anschliessende Photokoagulation bei proliferativer diabetischer Retinopathie mit Glaskörpereinblutung.

Forty eyes in 39 patients with proliferative diabetic retinopathy, vitreal hemorrhage and progressive neovascularization in at least 3 fundus quadrants were treated with panretinal xenon photocoagulation immediately after pars plana vitrectomy. Lens extraction was performed on 7 eyes and 2 eyes were aphakic. Six months after treatment, 68% of the eyes showed an improvement in vision, 25% had vitreal rebleeding and 11% developed rubeosis iridis. While treatment had no effect on peripheral rubeosis iridis in 5 eyes, all 7 eyes with pupillary rubeosis iridis showed complete regression. The results indicate the clinical value of combining, pars plana vitrectomy with panretinal xenon photocoagulation in selected patients.

[Photocoagulation in diabetic rubeosis iridis and neovascular glaucoma]. / Photokoagulation bei diabetischer Rubeosis iridis und neovaskulärem Glaukom.

This retrospective study was performed to examine the effect of retinal coagulation on the course of iris neovascularization and neovascular glaucoma in patients with diabetic retinopathy. Sixty-four eyes in 42 patients were examined. Following retinal coagulation iris neovascularization regressed in 47 eyes (73%), while 17 eyes (27%) showed no improvement. In eyes with neovascular glaucoma, a drop in intraocular pressure was noted. Visual acuity decreased in 60% of the treated eyes. Regression of iris neovascularization was related to the number of burns applied.

[Daunomycin in vitrectomy of complicated diabetic retinal detachment]. / Daunomycin bei Vitrektomie komplizierter diabetischer Netzhautablösungen.

Fourteen eyes with combined traction and rhegmatogenous retinal detachments from proliferative diabetic retinopathy were treated with pars plana vitrectomy (PPV) and daunomycin applied to the vitreous cavity using the technique described by Wiedemann et al. for the treatment of proliferative retinopathy. The operations were completed by filling the vitreous with silicone. The aim of our study was to investigate the effect of daunomycin and the reproliferation rate. After a follow-up period of at least 3 months, the post-operative results were compared with a control group of similar eyes treated with PPV and silicone tamponade alone. No evidence was found to the effect that daunomycin affects the rate of reproliferation.

Retinal enzyme activities under hypoxic conditions prior to neovascularization.

In an effort to demonstrate biochemical changes occuring in malperfused retinas, oxygen-induced retrolental fibroplasia was produced in kittens. Immediately before the onset of vasoproliferations, that is in the stage of maximal hypoxia, the activities of lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PDH), malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) were determined. The activity of MDH was found to be significantly decreased, whereas the activities of the other enzymes showed no major changes.