Involvement of ubiquitination and sumoylation in bladder lesions induced by persistent long-term low dose ionizing radiation in humans

PURPOSE: We determined whether ubiquitination and sumoylation processes are up-regulated in bladder urothelium by chronic, long-term, persistent low doses of ionizing radiation in male patients with benign prostate hyperplasia and females with chronic cystitis living more than 19 years in 137Cs contaminated areas after the Chernobyl accident in Ukraine. MATERIALS AND METHODS: Bladder urothelial biopsies from 45 patients were subjected to histopathological and immunohistochemical study of Ub, SUMO1, SUMO E2 conjugating enzyme Ubc9, and the cell cycle inhibitors p53 and p27(Kip1). RESULTS: Of 25 group 1 patients from radio contaminated areas chronic proliferative atypical cystitis (Chernobyl cystitis), featuring multiple foci of dysplasia, and carcinoma in situ were observed in 23 (92%) and 19 (76%), respectively, in addition to 1 small pTa grade 1 urothelial carcinoma. Chronic cystitis with areas of dysplasia and urothelial hyperplasia were detected in 2 (10%) and 3 (15%), respectively of the 20 patients in control group 2 from clean (without radio contamination) areas of Ukraine. Greatly increased levels of Ub, SUMO1, Ubc9 and p53 as well as decreased levels of p27(Kip1) were evident in patients in group 1 compared to those in group 2 (all p <0.001). CONCLUSIONS: These findings support the hypothesis that up-regulated ubiquitination and sumoylation processes might be an adaptive response to unscheduled proteolysis of aberrant p53 and p27(Kip1) cell cycle regulators occurring with long-term low dose rate ionizing radiation exposure with a possible contribution to urothelial carcinogenesis.     

Upregulation of fibroblast growth factor receptor 3 and epidermal growth factor receptors, in association with Raf-1, in urothelial dysplasia and carcinoma in situ after the Chernobyl accident

The present study was carried out in order to examine the molecular status of selected growth factor receptors (GFR) in urinary bladder lesions, recently described by our group as representing 'Chernobyl cystitis'. Fibroblast growth factor receptor 3 (FGFR3), epidermal growth factor receptor 1 (EGFR1), EGFR2neu (a member of the same family), p53 and Raf-1 serine/threonine kinase expression were evaluated immunohistochemically in urinary bladder biopsies from 22 men with benign prostate hyperplasia (group 1). For comparison, 16 men with benign prostate hyperplasia and five women with chronic cystitis living in non-radio-contaminated areas of the country were also investigated as controls (group 2). Additionally, 14 patients with dysplasia, carcinoma in situ (CIS) and primary urothelial carcinoma (UC) operated before the Chernobyl accident as well as 23 patients with UC living in the radio-contaminated areas were included as pre- and post-Chernobyl UC groups 1 and 2, respectively. Chronic proliferative atypical cystitis ('Chernobyl cystitis') was observed in group 1 patients. Foci of dysplasia and CIS were found in 22 (100%) and 19 (86%) of the 22 cases, respectively; moreover, two small UC were also detected. Elevated levels of FGFR3, EGFR2/neu, p53 and to a lesser extent EGFR1 and Raf-1 expression in the urothelial dysplasia and CIS were evident for patients of group 1. Statistically significant differences in immunohistochemical scores for FGFR3, EGFR1, p53 and Raf-1 were observed between groups 1 and 2 and between group 1 and the post-Chernobyl UC group 2, where a change in expression of EGFR2/neu was also noted. A significant decrease in FGFR3 expression in additional pre-Chernobyl UC group 1 with dysplasia, CIS and UC compared with group 1 Chernobyl cystitis cases was detected. Our findings suggest that FGFR and EGFR signaling pathways, associated with p53 and Raf-1 activation, may contribute to multistage urothelial carcinogenesis caused by irradiation, through autocrine or paracrine growth stimulation.     

Microcystin-LR induced cellular effects in mammalian and fish primary hepatocyte cultures and cell lines: a comparative study

The impact of microcystin-LR, one of the most common cyanobacterial toxins, on liver and gut cells originating from mammals and fish was compared. Upon exposure of human and rainbow trout (Oncorhynchus mykiss) cell lines up to 2.5 microM microcystin-LR, no alteration in cell viability was observed as assessed with three fluorescent indicators dyes, CFDA-AM, Alamar Blue and neutral red. The lack of sensitivity of the trout cell lines coincided with an absence of detectable mRNA levels of organic anion transporter polypeptide (OATP), which is implicated in the uptake of microcystin-LR. In contrast to the cell lines, primary rainbow trout and mouse hepatocytes showed damage to subcellular structures, particularly the lysosomes, as indicated by neutral red. This led us to propose a thus far undetected role of lysosomes as targets and mediators of microcystin-LR elicited cellular damage. An inhibitor of OATP, rifampicin, partly protected hepatocytes from this damage. Yet, the sensitivity of rainbow trout hepatocytes rapidly declined in culture, accompanied by decreasing levels of OATP mRNA. The sensitivity of mouse hepatocytes toward microcystin-LR also declined in culture but overall was about 25-fold greater than that of the trout cells. These differences mirror those observed in vivo and suggest the use hepatocytes for deciphering the species differences.     

Sedation during colonoscopy

In order to perform a proper screening for colonic cancer, repeated colonoscopies are required. Comfort during colonoscopy is very important, so that the patient will accept repeated procedures. Currently, there are 3 types of sedation used during colonoscopy: general anesthesia performed by an anesthesiologist; sedo-analgesia performed by an anesthesiologist or by a gastroenterologist; sedo-analgesia performed by a trained nurse. Sedo-analgesia is the most frequently used type of sedation during colonoscopy worldwide. It is realized by combining midazolam with propofol and/or fentanyl (alfentanyl) or pethidine. According to the data obtained from 34 centers performing colonoscopy in Romania, in 2003, 22,162 colonoscopies were performed: 54.5% without anesthesia, 39.5% with sedation with midazolam and 6% with sedo-analgesia. In a study performed in our department we noticed a significant improvement in the outcome of the colonoscopy when sedo-analgesia was used on a regular basis. The percentage of total colonoscopies (excluding those that could not be continued due to stenosis) was 84.2% when sedation was seldomly performed and 92.3% when sedo-analgesia was regularly used (p=0.042). We believe that the strategy of sedation during colonoscopy in Romania should be changed so that all the patients should benefit from sedo-analgesia, proved to be safe and cost/efficient.     

McKittrick-Wheelock syndrome - a rare cause of acute renal failure

AIM: Fluid and electrolyte hypersecretion in the villous adenoma of the rectum is presented in the case of a 74 year old man presenting with a severe fluid imbalance. CASE REPORT: The patient had a 2-year history of mucous diarrhea and, on admission, presented prerenal uremia, hyponatremia and severe hypokalemia. At sigmoidoscopy, a 6/4 cm villous adenoma of the rectum was found. The increased loss of volume, followed by exhaustion of the physiological compensation mechanisms, led to a life-threatening hypokalemia, as well as to acute renal failure. Conservative treatment was followed by a temporary improvement of the renal function. Alternative treatment was: endocavitary irradiation, endoscopic resection and radical tumor surgery. The surgical removal of the adenoma led to complete recovery of the symptoms. CONCLUSION: The McKittrick-Wheelock syndrome can be a problem of difficult diagnosis, both for the gastroenterologist and also for the nephrologist. The patient may develop severe complications, which require a sustained treatment.     

CO<Subscript>2</Subscript> laser increases the regenerative capacity of human adipose-derived stem cells by a mechanism involving the redox state and enhanced secretion of pro-angiogenic molecules

CO₂ laser has a beneficial effect on stem cells by mechanisms that are not clearly elucidated. We hypothesize that the effect of fractional CO₂ laser on human adipose-derived stem cells (ADSC) could be due to changes in redox homeostasis and secretion of factors contributing to cellular proliferation and angiogenic potential. ADSC incubated in medium containing 0.5 or 10 % FBS were exposed to a single irradiation of a 10,600-nm fractional CO₂ laser; non-irradiated ADSC were used as control. Viability/proliferation of ADSC was assessed by MTT assay; the intracellular reactive oxygen species (ROS) levels and the mitochondrial membrane potential (?Ψ_m) were determined with DCFH-DA and JC-1 fluorescent probes, respectively. Molecules secreted by ADSC in the medium were determined by ELISA assay, and their capacity to support endothelial tube-like formation by the Matrigel assay. The results showed that compared to controls, ADSC kept in low FBS medium and irradiated with CO₂ laser at 9 W exhibited: (a) increased proliferation (～20 %), (b) transient increase of mitochondrial ROS and the capacity to restore Δψ_m after rotenone induced depolarization, and (c) augmented secretion in the conditioned medium of MMP-2 (twofold), MMP-9 (eightfold), VEGF (twofold), and adiponectin (～50 %) that have the capacity to support angiogenesis of endothelial progenitor cells. In conclusion, the mechanisms underlying the benefic effect of CO₂ laser on ADSC are the activation of the redox pathways which increases cell proliferation and enhances secretion of angiogenic molecules. These results explain, in part, the mechanisms involved in the increased regenerative potential of CO₂ laser-exposed ADSC that could be exploited for clinical applications.     

Elevated tear fluid levels of MIP-1alpha in patients with cystic fibrosis.

Cystic fibrosis (CF) is the commonest multisystem genetic disease of white races, caused by mutations in the cystic fibrosis transmembrane regulator (CFTR), encoded on the long arm of chromosome 7. Mutations in the CFTR gene result in defective sodium, chloride, and water transport in the epithelial cells of the respiratory, hepatobiliary, gastrointestinal, and reproductive tracts, the pancreas, and the eye. The pathogenesis of ocular changes in CF is still unknown, but CF belongs to the large pathologic group of ocular surface epithelial diseases, termed keratoconjunctivitis sicca (KCS), that develop in dry eye syndrome. The aim of this study was to evaluate the levels of macrophage inflammatory protein-1alpha (MIP-1alpha) in the tear fluid of CF patients. We also investigated the correlation between the tear levels of this chemokine and clinical severity of CF and ocular surface disease. We studied 25 patients with CF with a mean age of 14 years. Chemokine levels were determined by ELISA. Complete ophthalmic examination, including dry eye tests, were used to study the ocular surface. The tear levels of MIP-1alpha in the CF patients were significantly higher when compared with healthy controls. We found a negative correlation between the tear levels of MIP-1alpha and clinical severity in CF patients and a positive correlation between the tear levels of MIP-1alpha and the presence of dry eye findings in CF patients. This current study indicates that chemokines play an important role in the ongoing inflammatory response. Our findings may help to explain one of the key factors contributing to the pathogenesis of ocular surface changes in CF patients.     

Role of superior colliculus in adaptive eye-head coordination during gaze shifts

The goal of this study was to determine which aspects of adaptive eye-head coordination are implemented upstream or downstream from the motor output layers of the superior colliculus (SC). Two monkeys were trained to perform head-free gaze shifts while looking through a 10 degrees aperture in opaque, head-fixed goggles. This training produced context-dependent alterations in eye-head coordination, including a coordinated pattern of saccade-vestibuloocular reflex (VOR) eye movements that caused eye position to converge toward the aperture, and an increased contribution of head movement to the gaze shift. One would expect the adaptations that were implemented downstream from the SC to be preserved in gaze shifts evoked by SC stimulation. To test this, we analyzed gaze shifts evoked from 19 SC sites in monkey 1 and 38 sites in monkey 2, both with and without goggles. We found no evidence that the goggle paradigm altered the basic gaze position-dependent spatial coding of the evoked movements (i.e., gaze was still coded in an eye-centered frame). However, several aspects of the context-dependent coordination strategy were preserved during stimulation, including the adaptive convergence of final eye position toward the goggles aperture, and the position-dependent patterns of eye and head movement required to achieve this. For example, when initial eye position was offset from the learned aperture location at the time of stimulation, a coordinated saccade-VOR eye movement drove it back to the original aperture, and the head compensated to preserve gaze kinematics. Some adapted amplitude-velocity relationships in eye, gaze, and head movement also may have been preserved. In contrast, context-dependent changes in overall eye and head contribution to gaze amplitude were not preserved during SC stimulation. We conclude that 1) the motor output command from the SC to the brain stem can be adapted to produce different position-dependent coordination strategies for different behavioral contexts, particularly for eye-in-head position, but 2) these brain stem coordination mechanisms implement only the default (normal) level of head amplitude contribution to the gaze shift. We propose that a parallel cortical drive, absent during SC stimulation, is required to adjust the overall head contribution for different behavioral contexts.