Patient and staff doses in paediatric interventional cardiology derived from experimental measurements with phantoms.

The aim of this paper was to determine experimentally the entrance surface air kerma (ESAK) and kerma-area product (KAP) levels to patients and scatter doses at the cardiologist's eyes during paediatric interventional cardiology (IC) procedures for Chile, on the basis of measurements taken from X-ray systems characterization for different thicknesses of polymethyl methacrylate, together with the average values of fluoroscopy time and number of cine frames for ten paediatric IC procedures. The range of cumulative ESAK values when the different clinical procedures were simulated was from 2 to 1100 mGy. KAP values ranged from 0.30 to 150 Gy cm(2). Scatter doses at cardiologist's eyes for the simulated procedures ranged from 0.20 to 116 µSv per procedure. Large differences between the X-ray systems were found in our study. Standardized guidelines in terms of X-ray system setting and protocols should be developed for hospitals that perform paediatric IC procedures in Chile.

Oncogenic H-Ras and PI3K signaling can inhibit E-cadherin-dependent apoptosis and promote cell survival after photodynamic therapy in mouse keratinocytes.

Maintenance of E-cadherin mediated cell-cell contacts is often required for the survival of epithelial cells and tissues. Here we report that oncogenic activation of H-Ras in murine keratinocytes can prevent cell death induced by immunological disruption of E-cadherin adhesion. A similar situation was observed in cells showing constitutive activation of the p110 alpha catalytic subunit of class IA PI3K. This protective effect is associated with beta-catenin-dependent transcription and with activation of survival factor Akt/PKB. In addition, we induced cell death by employing photodynamic therapy, using Zn-phthalocyanine as a photosensitizer that targets E-cadherin adhesion complexes. We have found that cell death based on this photodynamic action is also bypassed in cells showing constitutive activation of H-Ras and p110 alpha. Taken together, these results indicate that H-Ras/PI3K/Akt signaling plays a key role in cell survival mediated by E-cadherin cell-cell contacts.

A mechanism for the fluorogenic reaction of amino groups with fluorescamine and MDPF.

Fluorescamine and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) react with primary amines at alkaline pH to yield highly fluorescent products, claimed to be diaryl-2-hydroxy-pyrrolinones. However, it seems to have been overlooked that these products appear as the pseudobase of coplanar and cationic diarylpyrrolones. Horse blood smears subjected to fluorogenic MDPF and fluorescamine reactions showed eosinophil granules with bright blue-white fluorescence, which required washing at neutral pH and was dependent on the presence of amino groups. The fluorescence of MDPF-butylamine product was abolished at alkaline pH and by bisulphite, suggesting that nucleophilic attacks to the pyrrolone ring (with formation of carbinol (pseudobase) and sulfonate derivatives, respectively), destroy the planarity and conjugation of the whole molecule, thus abolishing the emission at long wavelength. Analysis of the correlation between the largest conjugated fragment (LCF) values and the emission peaks of several fluorophores (fluorescamine- and MDPF-butylamino products, non-rigid fluorochromes) showed the best-fit when the cationic pyrrolones were considered. Although the pseudobases of fluorescamine- and MDPF-amino derivatives are formed at alkaline pH, a full conjugated, coplanar and cationic molecule is suggested to be the true fluorescent product.

Loss of E-cadherin mediated cell-cell adhesion as an early trigger of apoptosis induced by photodynamic treatment.

Photodynamic treatment with different photosensitizers (PSs) can result in the specific induction of apoptosis in many cell types. It is commonly accepted that this apoptotic response depends on the mitochondrial accumulation of the PS. Accumulation in other cellular organelles, such as lysosomes or the Golgi complex, and subsequent photodamage resulting in an apoptotic process has been also described. However, the role played by cell adhesion in apoptosis induced in epithelial cells after photodynamic treatment is not well characterized. Here, we have used a murine keratinocyte line, showing a strong dependence on E-cadherin for cell-cell adhesion and survival, to analyze the relevance of this adhesion complex in the context of zinc(II)-phthalocyanine (ZnPc) photodynamic treatment. We report that under apoptotic conditions, ZnPc phototreatment induces a rapid disorganization of the E-cadherin mediated cell-cell adhesion, which largely preceded both the detachment of cells from the substrate, via beta-1 integrins and the induction of apoptotic mitochondrial markers. Therefore, the alteration in E-cadherin, alpha- and beta-catenins adhesion proteins preceded the release of cytochrome c (cyt c) from mitochondria to the cytosol and the activation of caspase 3. In addition, blocking E-cadherin function with a specific antibody (Decma-1) induced apoptosis in this cell system. These results strongly suggest that the E-cadherin adhesion complex could be the primary target of ZnPc phototreatment, and that loss of E-cadherin mediated cell adhesion after early photodamage triggers an apoptotic response.

Non-aqueous permanent mounting for immunofluorescence microscopy.

It is generally assumed that an aqueous mounting medium is necessary for the preservation of immunofluorescent-labelled microscopical preparations and polyvinyl alcohol-based solutions (e.g. Mowiol) being the most frequently used mounting media; however, both the quality and intensity of the fluorescence signal in most immunolabelled preparations after aqueous mounting slowly diminish with time, and finally, samples become unsuitable for examination. In the present work, we describe a very simple and rapid non-aqueous mounting procedure for cultured cells and tissue sections, which preserves the fluorescent signal in an excellent way after immunodetection or use of other specific labelling methods. It is based on the current histological protocol in which, after fluorescence labelling, preparations are dehydrated in ethanol, cleared in xylene and mounted in DePeX. Using this non-aqueous mounting medium, the fluorescent signal remains high and stable, allowing a suitable and permanent preservation of labelled and counterstained microscopical preparations.