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Phagocytosis, melanization and nodulation in insects depend on phenoloxidase (PO) activity. In this report, we demonstrated that these three processes appear to be also dependent on dopa decarboxylase (Ddc) activity. Using flow cytometry, RNA interference, immunoprecipitation and immunofluorescence, we demonstrated the constitutive expression of Ddc and its strong association with the haemocyte surface, in the medfly Ceratitis capitata. In addition, we showed that Escherichia coli phagocytosis is markedly blocked by small interfering RNA (siRNA) for Ddc, antibodies against Ddc, as well as by inhibitors of Ddc activity, namely carbidopa and benzerazide, convincingly revealing the involvement of Ddc activity in phagocytosis. By contrast, latex beads and lipopolysaccharide (LPS) did not require Ddc activity for their uptake. It was also shown that nodulation and melanization processes depend on Ddc activation, because antibodies against Ddc and inhibitors of Ddc activity prevent haemocyte aggregation and melanization in the presence of excess E. coli. Therefore, phagocytosis, melanization and nodulation depend on haemocyte-surface-associated PO and Ddc. These three unrelated mechanisms are based on tyrosine metabolism and share a number of substrates and enzymes; however, they appear to be distinct. Phagocytosis and nodulation depend on dopamine-derived metabolite(s), not including the eumelanin pathway, whereas melanization depends exclusively on the eumelanin pathway. It must also be underlined that melanization is not a prerequisite for phagocytosis or nodulation. To our knowledge, the involvement of Ddc, as well as dopa and its metabolites, are novel aspects in the phagocytosis of medfly haemocytes. [ABSTRACT FROM AUTHOR]

Abstract: E. coli phagocytosis by medfly hemocytes, in contrast to mammalian macrophages, associates with E. coli-challenged hemocyte secretion by mitogen activating protein (MAP) kinases. In the present work, we examined whether this system links with the proteolytic activation of prophenoloxidase (proPO). ProPO and prophenoloxidase-activating proteinases (PAPs) were initially identified within freshly isolated medfly hemocytes. Moreover, flow cytometry and immunocytochemical analysis revealed the constitutive expression of proPO and its stable association with hemocyte surface. The expression level of hemocyte surface proPO is not affected by E. coli infection. In addition, flow cytometry analysis in freshly isolated hemocytes showed that E. coli phagocytosis is markedly blocked by antibodies against proPO or PAPs, as well as by several serine protease inhibitors, strongly supporting the involvement of proPO cascade in the phagocytosis process. Similarly, it was shown that melanization process depends on proPO activation. MAP kinases appeared to control both phagocytosis and melanization, since they regulate PAPs secretion, a prerequisite for the conversion of proPO to active PO. From this and previous studies, hemocytes appear to be central to immune response in medfly. [Copyright &y& Elsevier]

Abstract: In response to LPS/E. coli treatment, extracellular signal-regulated kinase (ERK) is activated in medfly hemocytes. To explore the molecular mechanisms underlying LPS/E. coli/latex beads endo- and phagocytosis, we studied the signalling pathways leading to p38 and c-jun N-terminal kinase (JNK) activation. JNK and p38-like proteins were initially identified within medfly hemocytes. Flow cytometry analysis revealed that mitogen-activated protein kinases (MAPK) are required for phagocytosis. Inhibition of specific MAPK signalling pathways, with manumycin A, toxin A, cytochalasin D and latrunculin A, revealed activation of p38 via Ras/Rho/actin remodelling pathway and activation of JNK that was independent of actin cytoskeleton reorganization. ERK and p38 pathways, but not JNK, appeared to be involved in LPS-dependent hemocyte secretion, whereas all MAPK subfamilies seemed to participate in E. coli-dependent secretion. In addition, flow cytometry experiments in hemocytes showed that the LPS/E. coli-induced release was a prerequisite for LPS/E. coli uptake, whereas latex bead phagocytosis did not depend on hemocyte secretion. This is a novel aspect, as in mammalian monocytes/macrophages LPS/E. coli-triggered release has not been yet correlated with phagocytosis. It is of interest that these data suggest distinct mechanisms for the phagocytosis of E. coli and latex beads in medfly hemocytes. [Copyright &y& Elsevier]

Background. Transforming growth factor-β1 (TGF-β1), the major fibrogenic growth factor, is implicated in the pathogenesis of renal scarring in experimental and clinical nephropathies as well as in chronic allograft nephropathy. In this study we examined the pattern of changes of TGF-β1 excretion in the urine and the sites of TGF-β1 expression in the kidney of transplanted patients during the early post-transplantation period. METHODS: Eighteen renal allograft recipients were included in the study. In all patients urinary TGF-β1 levels were determined by ELISA in sequential measurements during the first two postoperative months and compared to that of 14 healthy subjects. The renal expression of TGF-β1 protein was studied in 4 patients that underwent a biopsy of the transplanted kidney at the same period. All patients were treated with prednisolone, cyclosporin, and mycophenolate mofetil. RESULTS: Urinary TGF-β1 levels were increased during the first postoperative days. Although they were gradually reduced during the first two post-operative months, they remained significantly higher compared to those of normal subjects (580 ± 148 ng/24 h vs. 310 ± 140 ng/24 h p<0.01). The decline of urinary TGF-β1 excretion followed that of serum creatinine. TGF-β1 protein expression was identified within the cytoplasm of tubular epithelial cells of transplanted patients. CONCLUSIONS: Elevated urinary TGF-β1 levels are observed during the early post-transplantation period in renal allograft recipients and are maintained high even after restoration of renal function to normal. [ABSTRACT FROM AUTHOR]

Alcohol-related health and social problems are prevalent in almost all societies that consume alcohol and the presence of alcohol use in the movies is a known issue. The same holds true for smoking prevalence in film-making. The aim of this study is to assess tobacco-related content and alcohol consumption in "The Saint" series and movies. Five episodes from each "The Saint" TV series, from the '60s, were randomly selected. A predefined template was used for data collection and multiple variables were recorded and then analyzed. The main character was reported to smoke in 81.9% of episodes and consume alcohol in 87.1% episodes and similar were the results for supporting actors. Mean time to first cigarette and first drink ranged from 0.5 to 40 minutes and from 0.5 to 40.5 minutes, respectively. The prevalence of smoking and drinking in "The Saint" movies is high on average; however, the main character has ceased smoking and reduced alcohol consumption in the two contemporary movies, probably following the changes in the era and respecting the law. [ABSTRACT FROM AUTHOR]

Exposure of tobacco and alcohol consumption in media and filmmaking has been related to promotion of smoking and drinking in adults. Current regulation aims to restrict tobacco and alcohol advertising in order to avoid alcohol consumption and smoking habits. We aimed to assess the impact of smoking and drinking habits in video-on-demand services. Three independent investigators watched the 50 most popular movies available in Netflix streaming platform, according to York Times and recorded incidence of smoking and drinking scenes for both primary and secondary actors. 45 movies were included in our analysis. Main characters appeared to smoke in 19 movies and to consume alcohol in 33 movies, while secondary characters in 32 movies with 121 scenes and consumed alcohol in 38 movies, respectively. First actors were males in 22 movies, females in 7 movies and both males and females in 6 movies. Movies' directors were males in 29 movies and females in 6 movies. Our analysis found increased content of smoking and drinking scenes in online movies and showed that recently released movies presented with increased incidence of drinking and smoking scenes, while era depicted in movies also affects smoking and drinking content. [ABSTRACT FROM AUTHOR]

Abstract: Background: Plasma levels of endothelin-1 (ET-1) increase after coronary angioplasty (PTCA) due to endothelial injury during the procedure. ET-1 has been found in human endocardial and myocardial cells. It is not known whether ET-1 increases after thermal injury induced by radiofrequency catheter ablation (RFA). Methods: We determined plasma ET-1 levels at baseline, immediately after, and at 2 and 6 h post-procedure in 31 patients undergoing PTCA and 16 patients undergoing RFA. Patients subjected to diagnostic coronary angiography (n=15) or electrophysiology study (n=13) served as controls. Results: Compared to baseline, ET-1 levels increased significantly immediately post-PTCA (55.1±20.1 vs. 42.7±14.9 pg/ml, p&#60;0.01) and at 2 h post-RFA (98.0±11.7 vs. 53.0±17.4 pg/ml, p&#60;0.01) and returned to baseline measurements at 2 h post-PTCA and 6 h post-RFA. There was no change of ET-1 levels in the control groups. ET-1 kinetics curve was significantly higher post-RFA compared to post-PTCA (p&#60;0.001). ET-1 immediately post-PTCA correlated with total pressure–time product applied for balloon inflation during the procedure (r=0.56, p&#60;0.01). There was no correlation between ET-1 levels and the number of RFA applications. No patient developed ischemia post-PTCA. There were no complications or arrhythmia recurrences post-RFA. Conclusion: Endocardial thermal injury incurred during RFA is another mechanism of endothelin increase apart from mechanical injury of the coronary endothelium during PTCA and represents further evidence for the existence of the peptide in human endocardial endothelial and myocardial cells. ET-1 increase is delayed and more pronounced post-RFA compared to post-PTCA. Despite that, it does not seem to have any clinical impact in the immediate post-RFA period. [Copyright &y& Elsevier]

[Display omitted] • TiO 2 nanoparticle photo (370–410 nm)-excitation system (TiO 2-NP) • TiO 2-NP generates •OH, O 2 •− or both. • TiO 2-NP substitutes the non-specifically •OH-producing standard Fenton system. • TiO 2-NP is used as generator of •OH/O 2 •− for oxidative biological simulations. The present study introduces a TiO 2 nanoparticle-based (TiO 2-NP) system for the generation of •OΗ and O 2 •− upon visible photo-excitation, in order to be used for high oxidative stress biological simulations in vitro. The main novelties of TiO 2-NP system are: It is set to produce •OΗ and O 2 •− alone or both (in contrast to •OΗ-based common use of TiO 2), as these options cover all possible generation means of these radicals in biological systems in vivo. Moreover, the known non-specific electrostatic interactions of TiO 2-NP with H 2 O and various biological systems (e.g., cells, membrane proteins, even drugs) simulate direct/distant interactions of any in vivo •OΗ, O 2 •− source with extra/intracellular biological targets taking place in a densely packed biomolecular environment. The TiO 2-NP system can use any commercially available TiO 2-NP source (dispersion, nanopowder, crystal type), as long as TiO 2-NP s ' concentrations in use meet the critical criterion to produce •OΗ levels linearly proportional to irradiation time, set for a given simulation study. The TiO 2-NP system is calibrated by a standardized protocol developed to be applicable to most biological systems, offering the option of TiO 2-NP removal via coagulation when needed. The production rates of •OΗ and O 2 •− by the TiO 2-NP system are specifically calibrated with the respective specific probes terephthalic acid (TPA) and hydroethidine (HE), and tested in comparison to the •OΗ-producing Fenton system. The reaction kinetics of •OΗ and O 2 •− with TPA and HE is found to be in competition with their generating source, the TiO 2-NP system. Similar ROS source competition phenomena with biological targets (simulated by TPA/HE) are very common in biological systems. In contrast, the Fenton system is shown not to exhibit such competition kinetics. The TiO 2-NP system can be used to study •OΗ/O 2 •− dose–response-depended oxidative modifications in biological simulations. This stems from the fact that •OΗ and O 2 •− linear production rates (60 min and up to 8 min, respectively) can be controlled by varying (i) TiO 2 concentration, (ii) light-source photon emission energy (decreasing from 370 to 410 nm), and (iii) light intensity (as a function of the inverse of squared distance from the irradiated sample). In contrast, •OΗ production by the Fenton system reaches steady state in ∼5 s regardless of varying Fe-II concentration, rendering it inappropriate for •OΗ simulation studies on biological systems. The biological simulating potential of the TiO 2-NP system, as producer of both •OΗ and O 2 •−, is also experimentally verified on indicative biological examples selected to structurally represent most biological systems: BSA, a model hydrophilic protein; LDL, structurally resembling most of the biological systems (cells, membranes, organelles, lipoproteins, proteins, lipids). The TiO 2-NP system causes a linear increase of all the tested oxidative modifications on both BSA and LDL for irradiation exposure 20 to 40 min, which strongly suggests that they are mainly •OΗ dose-proportional. In contrast, the Fenton system does not display •OΗ dose-associated oxidative modifications on BSA. [ABSTRACT FROM AUTHOR]

Abstract: Hydrogen peroxide (H2O2) participates as a second messenger in cell signaling. In this paper, the role of H2O2 was investigated, in Escherichia coli phagocytosis by the haemocytes of the medfly Ceratitis capitata. Block of H2O2 synthesis by specific enzymic inhibitors, namely N-ethylmaleimide (NEM) for NADPH oxidase and diethyldithiocarbamate (DDC) for SOD, resulted in the increase of E. coli phagocytosis. Immunoblot analysis, flow cytometry and confocal microscopy, revealed the constitutive expression of SOD, in the medfly haemocytes. Phagocytosis increased by small interfering RNA (siRNA) for SOD, revealing the active involvement of SOD and H2O2. Immunoblot analysis showed an increase of the ERK1/2 phosphorylation, in the presence of the above H2O2 synthesis enzymic inhibitors. In addition, confocal microscopy showed no co-localization of SOD with β integrin subunit. It appears that SOD participates in the regulation of bacterial phagocytosis, due to involvement of the produced H2O2 in the differential phosphorylation of MAP kinases. [Copyright &y& Elsevier]

Abstract: Objective: The purpose of this prospective study was to record endothelin-1 (ET-1) concentrations in the second trimester amniotic fluid and compare these values in women who developed intrauterine growth restriction (IUGR) later in pregnancy with those with uneventful pregnancies. Method: Amniotic fluid was retrieved by amniocentesis from 125 women in the second trimester of pregnancy. The levels of ET1 were measured by a sensitive and specific radioimmunoassay. Results: From the 125 women included in the study 12 had pregnancies that later developed IUGR and 88 had uneventful pregnancies. The ET1 concentration was significantly higher (P <0.005) in women who later developed IUGR than in normal pregnancy (106pg/ml versus 64.7pg/ml). Conclusion: The amniotic fluid concentration of ET1 is elevated by the second trimester in women who later develop IUGR. [Copyright &y& Elsevier]

Background . Endothelin-1 (ET-1), which acts via the specific receptors ET-A and ET-B, has been implicated in the development of renal scarring. The activation of the endothelin system was observed in experimental models of glomerular diseases and was attributed to the toxic action of proteinuria on the tubular epithelial cells. The aim of this study was to investigate whether the endothelin system in the kidney is altered in glomerular diseases and possibly related to proteinuria. Methods . Thirty-seven patients with different types of glomerulonephritis and 14 controls were included. Patients presented either nephrotic syndrome (n=25) or mild proteinuria (<1g/24h, n=12). The expression of ET-A and ET-B receptors in the renal tissue was examined immunohistochemically. At the time of biopsy, urinary ET-1 was determined. Results. Both receptors were mainly localized within tubular epithelial cells, and their expression was significantly higher in patients with glomerulonephritis compared to controls. The expression of ET-B was higher in nephrotic compared to non-nephrotic patients, while no difference was observed in the expression of ET-A receptors. A significant positive correlation of the degree of proteinuria with the excreted ET-1 (r= 0.487, p&l;t0.05) and the extent of immunostaining for ET-B receptors (r=0.420, p<0.05) was observed. The expression of ET-B receptors and the excretion of ET-1 decreased significantly in patients with remission of nephrotic syndrome after therapy. Conclusion. This study provides evidence that the endothelin system is activated in human glomerular disease, confirming data from experimental studies. Proteinuria seems to be related to the activation of endothelin system, though further investigation is necessary to clarify this issue. [ABSTRACT FROM AUTHOR]

Background. Apoptosis, a gene-directed form of cell death, has been involved in the resolution of renal injury but also in the development of scarring. Bcl-2 and bax are proteins related to apoptotic process that either provides a survival advantage to rapidly proliferating cells(bcl-2) or promote cell death by apoptosis(bax). Various cytokines and growth factors are involved in this process. This study investigates the expression of bcl-2 and bax and the presence of apoptotic bodies in relation to the TGF-β1 expression at the time of diagnosis in the renal biopsies of patients with glomerulonephritis(GN). METHODS: Fifty patients with various types of GN and ten controls were included in the study. Bcl-2, bax and Transforming Growth Factor(TGF-β1) positive cells were detected in kidney biopsies by immunohistochemistry, while apoptotic cells were detected by in situ end labeling of fragmented DNA(ISEL). Morphometric analysis was used for quantitation of immunostaining. RESULTS: The intensity of bcl-2, bax and TGF-β1 immunostaining in the renal tissue of patients with GN was significantly more to the observed in the control biopsies. Bcl-2 and bax were expressed within the epithelial cells of proximal, distal and collecting tubules and in the renal interstitium. Bax and bcl-2 proteins were also identified within the glomeruli in a few patients but their distribution was not related to the type of GN. TGF-β1 was expressed in the cytoplasm of tubular epithelial cells and to a lesser extent in the renal interstitium and glomeruli. A positive correlation of TGF-β1 with the extent of bax immunostaining(r = 0.498, p < 0.05) and an inverse correlation with that of bcl-2(r = − 0.490, p < 0.05) were identified. Apoptotic bodies were identified only in the renal tissue of patients with GN and were mainly localized among tubular epithelial and interstitial cells. CONCLUSION: The intensity of bcl-2 and bax proteins expression and the presence of apoptotic bodies in the renal tissue of patients with GN suggest that apoptotic process is ongoing during the evolution of renal disease. The correlation of TGF-β1 expression with that of apoptosis-related proteins might represent an implication of this growth factor with apoptotic process in the human diseased kidney. [ABSTRACT FROM AUTHOR]