A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin

In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin-Darby canine kidney epithelial cells. A simple 2D IEF/SDS/PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin-Darby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unknown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS/MS analysis. Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively.

Metalloprotease meprin beta in rat kidney: glomerular localization and differential expression in glomerulonephritis

Meprin (EC 3.4.24.18) is an oligomeric metalloendopeptidase found in microvillar membranes of kidney proximal tubular epithelial cells. Here, we present the first report on the expression of meprin beta in rat glomerular epithelial cells and suggest a potential involvement in experimental glomerular disease. We detected meprin beta in glomeruli of immunostained rat kidney sections on the protein level and by quantitative RT-PCR of laser-capture microdissected glomeruli on the mRNA level. Using immuno-gold staining we identified the membrane of podocyte foot processes as the main site of meprin beta expression. The glomerular meprin beta expression pattern was altered in anti-Thy 1.1 and passive Heymann nephritis (PHN). In addition, the meprin beta staining pattern in the latter was reminiscent of immunostaining with the sheep anti-Fx1A antiserum, commonly used in PHN induction. Using Western blot and immunoprecipitation assays we demonstrated that meprin beta is recognized by Fx1A antiserum and may therefore represent an auto-antigen in PHN. In anti-Thy 1.1 glomerulonephritis we observed a striking redistribution of meprin beta in tubular epithelial cells from the apical to the basolateral side and the cytosol. This might point to an involvement of meprin beta in this form of glomerulonephritis.

The metalloprotease meprinbeta processes E-cadherin and weakens intercellular adhesion

BACKGROUND: Meprin (EC 3.4.24.18), an astacin-like metalloprotease, is expressed in the epithelium of the intestine and kidney tubules and has been related to cancer, but the mechanistic links are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We used MDCK and Caco-2 cells stably transfected with meprin alpha and or meprin beta to establish models of renal and intestinal epithelial cells expressing this protease at physiological levels. In both models E-cadherin was cleaved, producing a cell-associated 97-kDa E-cadherin fragment, which was enhanced upon activation of the meprin zymogen and reduced in the presence of a meprin inhibitor. The cleavage site was localized in the extracellular domain adjacent to the plasma membrane. In vitro assays with purified components showed that the 97-kDa fragment was specifically generated by meprin beta, but not by ADAM-10 or MMP-7. Concomitantly with E-cadherin cleavage and degradation of the E-cadherin cytoplasmic tail, the plaque proteins beta-catenin and plakoglobin were processed by an intracellular protease, whereas alpha-catenin, which does not bind directly to E-cadherin, remained intact. Using confocal microscopy, we observed a partial colocalization of meprin beta and E-cadherin at lateral membranes of incompletely polarized cells at preconfluent or early confluent stages. Meprin beta-expressing cells displayed a reduced strength of cell-cell contacts and a significantly lower tendency to form multicellular aggregates. CONCLUSIONS/SIGNIFICANCE: By identifying E-cadherin as a substrate for meprin beta in a cellular context, this study reveals a novel biological role of this protease in epithelial cells. Our results suggest a crucial role for meprin beta in the control of adhesiveness via cleavage of E-cadherin with potential implications in a wide range of biological processes including epithelial barrier function and cancer progression.

[Swiss Research Agenda for Nursing (SRAN): the development of an agenda for clinical nursing research in Switzerland]

In many Anglo-Saxon and North European countries nursing research agendas have been developed to address priorities in nursing research in accordance with a nationally defined health policy. In Switzerland, due to lack of a nationwide governmental health policy, co-ordination of nursing research so far was scarce. The "Swiss Research Agenda for Nursing (SRAN)" project developed an agenda for clinical nursing research between 2005 and 2007. Based on literature reviews, expert panels and a national survey a project team formulated an agenda which passed a consensus conference. The agenda recommends aspects that should lead research and defines seven research priorities for nursing in Switzerland for the time between 2007 and 2017. Nursing research should prioritize to investigate 1) the effectiveness of nursing interventions; 2) the influences of service adaptations in a changing health care system; 3) the phenomena in patients requiring nursing care; 4) the influence of the work environment on the quality of nursing care; 5) the functioning of family and social systems; 6) varieties of life circumstances and their integration; and 7) the implementation of ethical principles in nursing. Written in German and French, the Swiss Research Agenda for Nursing for the first time formulates priorities for nursing research in Switzerland and can be used for strategic discussions. As a next step, the development of an action plan to enhance nursing research will take place in Switzerland.

MCAM/MUC18 REGULATES MELANOMA PROGRESSION BY MODULATING THE EXPRESSION OF ID-1

The acquisition of the metastatic melanoma phenotype is associated with increased expression of the melanoma cell adhesion molecule MCAM/MUC18 (CD146). However, the mechanism by which MUC18 contributes to melanoma metastasis remains unclear. Herein, we stably silenced MUC18 expression utilizing lentivirus-incorporated small hairpin RNA, in two metastatic melanoma cell lines, A375SM and C8161, and conducted cDNA microarray analysis. We identified and validated that the transcriptional regulator, Inhibitor of DNA Binding-1 (Id-1), previously shown to function as an oncogene in several malignancies, was downregulated by 5.6-fold following MUC18 silencing. Additionally, we found that MUC18 regulated Id-1 expression at the transcriptional level via ATF-3. Interestingly, ATF-3 was upregulated by 6.9 fold in our cDNA microarray analysis following MUC18 silencing. ChIP analysis showed increased binding of ATF-3 to the Id-1 promoter after MUC18 silencing, while mutation of the ATF-3 binding site on the Id-1 promoter increased Id-1 promoter activity in MUC18-silenced cells. These Data suggest that MUC18 silencing promotes inhibition of Id-1 expression by increasing ATF-3 expression and binding to the Id-1 promoter. Rescue of MUC18 reverted the expression of Id-1 and ATF-3, thus validating that they are not off-target effects of MUC18. To further assess the role of Id-1 in melanoma invasion and metastasis, we overexpressed Id-1 in MUC18-silenced cells. Overexpression of Id-1 in MUC18-silenced cells resulted in increased cell invasion, as well as increased expression and activity of MMP-2. Our data further reveal that Id-1 regulates MMP-2 at the transcriptional level through Sp1 and Ets-1. This is the first report to demonstrate that MUC18 does not act exclusively in cell adherence, but is also involved in cell signaling that regulates the expression of genes, such as Id-1 and ATF-3, thus contributing to the metastatic melanoma phenotype.

Ab 50 Jahr - Frau unsichtbar?

Ab einem gewissen Alter fühlen sich viele Frauen von Männern nicht mehr wahrgenommen. Gleichzeitig stehen sie mitten im Leben und wissen, was sie wollen. Mona Vetsch diskutiert, was Frauen attraktiv macht, ob sie ab 50 weg vom Fenster sind und wie Männer mit den Bedürfnissen reifer Frauen umgehen.

Home artificial nutrition in Switzerland: an epidemiological survey from 2005 to 2009.

BACKGROUND AND AIMS Home artificial nutrition (HAN), including oral nutritional supplements (ONS) and enteral (HEN) and parenteral (HPN) nutrition, is an established, important treatment for malnourished patients. The aim of this study was to analyze the epidemiological data of patients on HAN in Switzerland. METHODS This retrospective study recorded all new cases of HAN in Switzerland from January 2005 to December 2009. RESULTS A total of 12,917 cases were recorded: 6,731 (52%) males and 6,186 (48%) females, with a mean age of 65.0 ± 17.6 years. The number of patients on ONS was 7,827 (57.4%), on HEN 3,966 (39.4%) and on HPN 433 (3.2%). The most common underlying disease category was neoplasms (6,519, 50.7%). The number of patients on ONS increased from 57.0% (n = 1,252) to 60.8% (n = 2,039), and on HPN from 2.1% (n = 45) to 4.0% (n = 134) between 2005 and 2009. CONCLUSIONS This first analysis of the large-scale Swiss registry of HAN shows that approximately half of the patients received ONS, whereas HPN was rarely delivered. The frequency of ONS and HPN increased from the year 2005 to 2009. In accordance with previous European studies, malignant tumors were by far the most frequent indication for HAN.

[Sausage or carrot--spoilt for choice].

As far as healthy food is concerned, it cannot be categorized simply as "good" or "bad" since its effect on health depends mainly on the amount and method consumed. Today's recommendations include a diversified diet, a diet which targets energy-balance and provides all nutrients necessary. Living in an affluent society aggravates healthy choices because of a constantly available, large assortment of food items. In general, the way food is prepared these days has changed a lot: mainly, the energy content has constantly increased, while the fiber and natural micronutrient concentrations decreased. Food items with a high energy yield, containing a lot of fat and sugar, affects our energy balance, which may lead to diseases of affluence such as type 2 diabetes, cardiovascular diseases, and some kinds of tumors.

Observational Research on NCDs in HIV-Positive Populations: Conceptual and Methodological Considerations

: Noncommunicable diseases (NCDs) account for a growing burden of morbidity and mortality among people living with HIV in low- and middle-income countries (LMICs). HIV infection and antiretroviral therapy interact with NCD risk factors in complex ways, and research into this "web of causation" has so far been largely based on data from high-income countries. However, improving the understanding, treatment, and prevention of NCDs in LMICs requires region-specific evidence. Priority research areas include: (1) defining the burden of NCDs among people living with HIV, (2) understanding the impact of modifiable risk factors, (3) evaluating effective and efficient care strategies at individual and health systems levels, and (4) evaluating cost-effective prevention strategies. Meeting these needs will require observational data, both to inform the design of randomized trials and to replace trials that would be unethical or infeasible. Focusing on Sub-Saharan Africa, we discuss data resources currently available to inform this effort and consider key limitations and methodological challenges. Existing data resources often lack population-based samples; HIV-negative, HIV-positive, and antiretroviral therapy-naive comparison groups; and measurements of key NCD risk factors and outcomes. Other challenges include loss to follow-up, competing risk of death, incomplete outcome ascertainment and measurement of factors affecting clinical decision making, and the need to control for (time-dependent) confounding. We review these challenges and discuss strategies for overcoming them through augmented data collection and appropriate analysis. We conclude with recommendations to improve the quality of data and analyses available to inform the response to HIV and NCD comorbidity in LMICs.