Reevaluation of the proposed autocrine proliferative function of prolactin in breast cancer

The pituitary hormone prolactin (PRL) has been implicated in tumourigenesis. Expression of PRL and its receptor (PRLR) was reported in human breast epithelium and breast cancer cells. It was suggested that PRL may act as an autocrine/paracrine growth factor. Here, we addressed the role of locally synthesised PRL in breast cancer. We analysed the expression of PRL in human breast cancer tumours using qPCR analysis and in situ hybridization (ISH). PRL mRNA expression was very low or undetectable in the majority of samples in three cDNA arrays representing samples from 144 breast cancer patients and in 13 of 14 breast cancer cell lines when analysed by qPCR. In accordance, PRL expression did not reach detectable levels in any of the 19 human breast carcinomas or 5 cell lines, which were analysed using a validated ISH protocol. Two T47D-derived breast cancer cell lines were stably transfected with PRL-expressing constructs. Conditioned medium from the T47D/PRL clones promoted proliferation of lactogen-dependent Nb2 cells and control T47D cells. Surprisingly, the PRL-producing clones themselves displayed a lower proliferation rate as compared to the control cells. Their PRLR protein level was reduced and the cells were no longer responsive to exogenous recombinant PRL. Taken together, these data strongly indicate that autocrine PRL signalling is unlikely to be a general mechanism promoting tumour growth in breast cancer patients. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10549-013-2731-7) contains supplementary material, which is available to authorized users

Comparative analysis of inflamed and non-inflamed colon biopsies reveals strong proteomic inflammation profile in patients with ulcerative colitis

<p>Abstract</p> <p>Background</p> <p>Accurate diagnostic and monitoring tools for ulcerative colitis (UC) are missing. Our aim was to describe the proteomic profile of UC and search for markers associated with disease exacerbation. Therefore, we aimed to characterize specific proteins associated with inflamed colon mucosa from patients with acute UC using mass spectrometry-based proteomic analysis.</p> <p>Methods</p> <p>Biopsies were sampled from rectum, sigmoid colon and left colonic flexure from twenty patients with active proctosigmoiditis and from four healthy controls for proteomics and histology. Proteomic profiles of whole colonic biopsies were characterized using 2D-gel electrophoresis, and peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for identification of differently expressed protein spots.</p> <p>Results</p> <p>A total of 597 spots were annotated by image analysis and 222 of these had a statistically different protein level between inflamed and non-inflamed tissue in the patient group. Principal component analysis clearly grouped non-inflamed samples separately from the inflamed samples indicating that the proteomic signature of colon mucosa with acute UC is strong. Totally, 43 individual protein spots were identified, including proteins involved in energy metabolism (triosephosphate isomerase, glycerol-3-phosphate-dehydrogenase, alpha enolase and L-lactate dehydrogenase B-chain) and in oxidative stress (superoxide dismutase, thioredoxins and selenium binding protein).</p> <p>Conclusions</p> <p>A distinct proteomic profile of inflamed tissue in UC patients was found. Specific proteins involved in energy metabolism and oxidative stress were identified as potential candidate markers for UC.</p

Applied Element Method Analysis of Porous GFRP Barrier Subjected to Blast

Numerical analysis of highly dynamic phenomena represents a critical field of study and application for structural engineering as it addresses extreme loading conditions on buildings and the civil infrastructure. In fact, large deformations and material characteristics of elements and structures different from those exhibited under static loading conditions are important phenomena to be accounted for in numerical analysis. The present paper describes the results of detailed numerical analyses simulating blast tests conducted on a porous (i.e. discontinuous) glass fiber reinforced polymer (GFRP) barrier aimed at the conception, validation and deployment of a protection system for airport infrastructures against malicious disruptions. The numerical analyses herein presented were conducted employing the applied element method (AEM). This method adopts a discrete crack approach that allows auto cracking, separation and collision of different elements in a dynamic scheme, where fully nonlinear path-dependant constitutive material models are adopted. A comparison with experimental results is presented and the prediction capabilities of the software are demonstrated

Posttranslational processing of barley beta-glucan endohydrolases in the baculovirus insect cell expression system

Two cDNAs encoding barley (1→3,1→4)-β-glucanase (EC 3.2.1.73) isoenzymes EI and EII have been expressed in Spodoptera frugiperda (Sf9) cell cultures using the baculovirus AcNPV vector. Modifications to both the 5′ and 3′ ends of the cDNAs were required before satisfactory levels of expression were obtained. The modified cDNAs directed high levels of (1→3,1→4)-β-glucanase expression in the Sf9 insect cell cultures, with yields of 10 mg/liter of isoenzyme EI (expEI) and 15 mg/liter of isoenzyme EII (expEII). Amino acid sequence analyses showed that the expressed enzymes were processed correctly at their amino termini. However, affinity chromatography of the isoenzyme expEII on concanavalin-A (conA)-Sepharose indicated that, although the enzyme is glycosylated, the structures of the carbohydrate chains differ from those of the native enzyme. When a cDNA encoding the homologous barley (1→3)-β-glucanase (EC 3.2.1.39) isoenzyme GII was expressed in insect cells, aberrant amino-terminal processing of the nascent polypeptide was sometimes observed. The forms with incompletely removed signal peptides retained their substrate specificity, but exhibited slightly reduced catalytic efficiency, altered Chromatographic behavior, and reduced stability at elevated temperatures. The results show that high levels of expression of recombinant plant proteins can be obtained in insect cells, but they emphasize the need to characterize thoroughly the products that are expressed in the heterologous insect cell system before comparisons are made with the native enzyme or with engineered enzyme mutants