Influence of the concentration of metipranolol eye drops on the drug concentration in human aqueous humour

The concentration of the metabolite of the beta-blocker metipranolol was determined in the aqueous humour of 89 cataract patients. At 1, 2 or 5 h before surgery, they received one drop (30 l) of a 0.1 % or 0.3% solution of the drug. At 1, 2 or 5 h after the application of 0.1 % metipranolol eye drops, desacetylmetipranolol concentrations of 624.55, 235.29 and 88.02 ng/ ml, respectively, were measured. At the same intervals after the instillation of 0.3% metipranolol eye drops, the respective values of 1289.20, 1120.88 and 327.36 ng/ ml were found. The metabolite concentration in the eye drops and the values measured show no consistent correlation.Offprint requests to: H. Bleckmann     

Transformation of rodent fibroblasts by herpes simplex virus: presence of morphological transforming region 1 (MTR 1) is not required for the maintenance of the transformed state.

Studies on the mechanisms of transformation of mammalian cells by herpes simplex virus (HSV) in vitro have been prevented so far by the extremely low transformation frequencies obtained in monolayer culture. Here we present a transformation system that relies on the direct seeding in soft agar of infected single cells, thus avoiding negative interactions between normal and transformed cells. We took advantage of HSV-I temperature-sensitive mutants at the UL9 locus, which codes for a DNA-binding protein necessary for viral DNA replication. At the non-permissive temperature, viral DNA synthesis and late gene expression are prevented. Viral gene expression is restricted to immediate early and early genes. Induction of transformation was highly efficient in our one-step transformation system. It depended on intact viral particles and viral DNA. Immediate early and/or early viral gene expression was sufficient to induce transformation. Colonies were stably transformed and did not show any rescue of viable virus after temperature downshift and co-cultivation with susceptible cells. Transformed cells maintained the transformed state in the absence of viral DNA. Our data therefore support the "hit-and-run" hypothesis for the transforming effect of HSV.     

Calcitonin Gene-related Peptide Stimulates the Induction of c-fos Gene Expression in Rat Astrocyte Cultures

The action of calcitonin gene-related pepide (CGRP) was studied on c-fos gene expression in rat astrocyte cultures. A strong and transient increase in c-fos mRNA was observed in cultured astrocytes after treatment with CGRP. Quantitative Northern blot analysis revealed an increase of c-fos mRNA within 15 min, a peak after 30 min with a 10 - 15 fold increase over unstimulated cells and a subsequent decline. Induction of the c-fos gene by CGRP was concentration-dependent, half maximal stimulation of c-fos mRNA being obtained with 100 nM CGRP. The CGRP effect appeared to be mediated by a CGRP receptor and calcitonin was found to mimic only weakly the action of CGRP on cultured astrocytes. Calcitonin transiently induced c-fos gene expression with a similar time course to CGRP, but its effect was much less pronounced. Agents affecting the intracellular cyclic AMP level, forskolin and Ro 20-1724, stimulated c-fos mRNA in a strong and transient fashion with a temporal sequence similar to the response to CGRP. Further, the phosphodiesterase inhibitor Ro 20-1724 potentiated the action of CGRP on c-fos mRNA induction, suggesting a role for cyclic AMP in the action of CGRP. The present results indicate that CGRP may play a physiological role as a regulator of astrocyte gene expression.     

Enzymic control of irreversible binding of metabolically activated benzo(a)pyrene in perfused rat liver by monooxygenase activity

Addition of [³H]-benzo(a)pyrene to the perfusion medium of isolated rat livers results in irreversible binding of radioactivity to DNA, RNA and protein. Binding to DNA accounted for about 0.1% of the total radioactivity which was bound in livers from animals treated with oil or saline and was increased by a factor of 3–5 after pretreatment of the animals with -naphthoflavone or with phenobarbital. When the inhibitiors of monooygenase activity, -naphthoflavone or metyrapone, were present in the perfusion medium, irreversible binding was reduced in livers from both -naphthoflavone- and phenobarbital-pretreated animals, irrespective of the inhibitor used.In livers from animals treated with oil or saline protein and a RNA fraction containing tightly associated protein were able to bind [³H]-benzo(a)pyrene metabolites to about the same extent but after induction by pretreatment with -naphthoflavone binding to the RNA fraction was enhanced to a much higher extent than binding to the protein fraction. Pretreatment with phenobarbital did not result in an increased irreversible binding to RNA and protein.A considerable amount of 15–25% of the total radioactivity added to the perfusion medium was excreted into the bile after treatment of the animals with the tested inducers of monooxygenase activity compared to an excretion of 3% in animals treated with oil or saline.The results indicate that nucleic acid and protein adduct formation in the liver is controlled by the action of the cytochrome P-450-dependent monooxygenases.In part subject of the doctoral thesis of Erik Klaus, Fachbereich Biologie, University of Mainz     

An in vitro model for videoimaging of human bladder smooth muscle cell contractions

Knowledge regarding human bladder smooth muscle cell (SMC) physiology is very limited. Only a few specific medical therapies for bladder disorders have therefore been established. The objective of this study was to develop a model for videomicroscopy of bladder SMC contractions. Cells were isolated from human cystoprostatectomy specimens and cultured in a modified EMEM medium. These cells were identified as SMCs by means of immunohistochemistry. For videomicroscopy, the culture flasks were coated with a viscous agent to allow cell contraction. Contractions were visualized by means of a cell culture microscope with a time-lapse videosystem. For cholinergic stimulation of the cells, acetylcholine, in concentrations ranging from 100 μM to 10 mM, was applied. The percentage of contracting cells within the observation field was evaluated for quantitative analysis. In control experiments without contractile stimulant 6% of the cells were observed to contract. Stimulation with acetylcholine induced a significant dose-dependent increase to 47% in contracting cells. These results demonstrated that videomicroscopy is an appropriate tool to investigate the contraction mechanisms of bladder SMCs. This model offers the possibility of studying drug effects on the human detrusor in vitro. Received: 16 September 1999 / Accepted: 1 May 2000     

Methylated DNA collected by tampons--a new tool to detect endometrial cancer.

This proof of principle study aimed to define a new and simple strategy for detection of endometrial cancer using epigenetic markers. We investigated DNA isolated from vaginal secretion collected from tampon for aberrant methylation of five genes (CDH13, HSPA2, MLH1, RASSF1A, and SOCS2) using MethyLight in 15 patients with endometrial cancer and 109 patients without endometrial cancer. All endometrial cancer patients revealed three or more methylated genes, whereas 91% (99 of 109) of the patients without endometrial cancer had no or fewer than three genes methylated in their vaginal secretion. The methods developed in this study provide the basis for a prospective clinical trial to screen asymptomatic women who are at high risk for endometrial cancer.     

The use of the source-skin distance measuring bridge indeed reduces skin teleangiectasia after interstitial boost in breast conserving therapy.

BACKGROUND AND PURPOSE: In 1990 the skin source measuring bridge was proposed as a tool to measure (1) the distance between the interstitial implant and the overlying skin during brachytherapy boost treatment as well as (2) the distances between the lateral source end and the exit point of the guide needle. The present study reports on the clinical experience using the source skin measuring bridge with respect to incidence and grade of teleangiectasia, and their relation to source skin distances and doses. PATIENTS AND METHODS: Two hundred and twenty-two breast cancer patients (229 breasts) treated between 1983 and 1996 with breast conserving therapy including a brachytherapy boost were scored on the occurrence of teleangiectasia. The minimum distance between the sources (above implant and laterally) and the skin surface were measured. RESULTS: If no bridge was used the appearance of teleangiectasia in the epiderm above the implant is 77, 63 and 50% for boost doses of 25, 20 and 15 Gy, respectively. For brachytherapy boost doses of 25 and 20 Gy and distances smaller than 10mm between the implant and the overlying epiderm, as determined with the skin source measuring bridge, the appearance of teleangiectasia was 78 and 46%, respectively. When respecting provisional dosimetry to spare the skin for a boost dose of 15 Gy, resulting in distances between 10 and 15 mm for the implant overlying skin and distances between 5 and 10 mm for the lateral skin, teleangiectasia can be reduced to a minimum (6.3% above and 3.3% laterally). While in a univariate analysis several parameters (use of the bridge, boost dose, boost modality, external beam therapy modality) were predictive factors, the use of the bridge remained the only significant variable in a multivariate analysis. CONCLUSIONS: The skin source measuring bridge reduces teleangiectasia after interstitial brachytherapy boost treatment. A hypothesis made previously relating teleangiectasia and source skin distances was verified and extended. Even when 3D planning is used, the bridge allows for a provisional calculation of the security margins between source positions and the skin at the time of BT implantation to assure a correct needle positioning from the beginning, instead of correcting dwell times later on to avoid unnecessary high skin doses.