Analysis of the epidermal growth factor receptor specific transcriptome: effect of receptor expression level and an activating mutation

Overexpression or expression of activating mutations of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. The present study employed Affymetrix oligonucleotide arrays to profile genes induced by ligand-activated EGFR with the receptor either moderately expressed or overexpressed at an in-itself transforming level. These changes were compared to those induced by the naturally occurring constitutively active variant EGFRvIII. This study provides novel insight on the activities and mechanisms of EGFRvIII and EGFR mediated transformation, as genes encoding proteins with functions in promoting cell proliferation, invasion, antiapoptosis, and angiogenesis featured prominently in the EGFRvIII- and EGFR-expressing cells. Surprisingly, it was found that ligand-activated EGFR induced the expression of a large group of genes known to be inducible by interferons. Expression of this module was absent in the EGFRvIII-expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor-mediated events. Furthermore, activation of this module correlated with activation of STAT1 and STAT3. The results thus demonstrate that ligand-activated EGFR at different expression levels results in different kinetics of signaling and induction of gene expression. In addition, the constitutively active variant EGFRvIII seems to activate only a subset of signal pathways and induce a subset of genes as compared to the ligand-activated EGFR.     

Homologous recombination is facilitated in starving populations of Pseudomonas putida by phenol stress and affected by chromosomal location of the recombination target

Homologous recombination (HR) has a major impact in bacterial evolution. Most of the knowledge about the mechanisms and control of HR in bacteria has been obtained in fast growing bacteria. However, in their natural environment bacteria frequently meet adverse conditions which restrict the growth of cells. We have constructed a test system to investigate HR between a plasmid and a chromosome in carbon-starved populations of the soil bacterium Pseudomonas putida restoring the expression of phenol monooxygenase gene pheA. Our results show that prolonged starvation of P. putida in the presence of phenol stimulates HR. The emergence of recombinants on selective plates containing phenol as an only carbon source for the growth of recombinants is facilitated by reactive oxygen species and suppressed by DNA mismatch repair enzymes. Importantly, the chromosomal location of the HR target influences the frequency and dynamics of HR events. In silico analysis of binding sites of nucleoid-associated proteins (NAPs) revealed that chromosomal DNA regions which flank the test system in bacteria exhibiting a lower HR frequency are enriched in binding sites for a subset of NAPs compared to those which express a higher frequency of HR. We hypothesize that the binding of these proteins imposes differences in local structural organization of the genome that could affect the accessibility of the chromosomal DNA to HR processes and thereby the frequency of HR.     

Comparison of acute cardiovascular responses to water immersion and head-down tilt in humans.

The hypothesis was tested that acute water immersion to the neck (WI) compared with 6 degrees head-down tilt (HDT) induces a more pronounced distension of the heart and lower plasma levels of vasoconstrictor hormones. Ten healthy males underwent 30 min of HDT, WI, and a seated control (randomized). During WI, left atrial diameter and stroke volume increased to the same extent as during HDT. Cardiac output increased by 1 l/min more during WI than during HDT. (P < 0.05). Plasma atrial natriuretic peptide increased during WI (P < 0.05) but not during HDT, whereas plasma norepinephrine, vasopressin, and renin activity were suppressed similarly. Mean arterial pressure decreased by 9 mmHg (P < 0.05) during HDT and was unchanged during WI, and heart rate decreased more during HDT (P < 0.05). Arterial pulse pressure increased considerably more during HDT than during WI. In conclusion, the hypothesis was not confirmed because the cardiac atria were similarly distended by acute HDT and WI and the release of vasoconstrictor hormones were suppressed to the same extent.     

Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

In all vertebrate animals, CD8⁺ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC effectively individualizes the immune response of each member of the species. We have recently developed efficient methods to generate recombinant human MHC-I (also known as human leukocyte antigen class I, HLA-I) molecules, accompanying peptide-binding assays and predictors, and HLA tetramers for specific CTL staining and manipulation. This has enabled a complete mapping of all HLA-I specificities (“the Human MHC Project”). Here, we demonstrate that these approaches can be applied to other species. We systematically transferred domains of the frequently expressed swine MHC-I molecule, SLA-1*0401, onto a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules. A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data indicate that it is possible to extend the biochemical and bioinformatics tools of the Human MHC Project to other vertebrate species.     

Sucrose and IQ induced mutations in rat colon by independent mechanism

Sucrose-rich diets have repeatedly been observed to have co-carcinogenic actions in colon and liver of rats and to increase the number of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) induced aberrant crypt foci in rat colon. To investigate a possible interaction between sucrose and IQ on the genotoxicity in rat liver and colon, we gave Big Blue rats a diet containing sucrose (0%, 3.45% or 13.4% w/w) and/or IQ (70 ppm) for a period of 3 weeks. Sucrose and IQ increased the mutation frequency in the colon. The effect of combined treatments with IQ and sucrose on the mutation frequencies was additive indicating that sucrose and IQ act independently. This was supported by the mutation spectra where sucrose expands the background mutations in the colon, whereas IQ, in other studies, more specifically has induced G:C --> T:A transversions. In the liver IQ increased the mutation frequency, whereas addition of sucrose reduced the effect of IQ in a dose-dependent manner. The level of bulky DNA adducts in liver and colon was increased in animals exposed to either sucrose or IQ. In animals exposed to IQ, addition of sucrose had marginal effects on the level of bulky DNA adducts. Markers of oxidative damage and DNA repair were generally unaffected by the treatments. In conclusion, sucrose and IQ in the diet induced mutations in the colon by independent mechanisms, whereas an interaction was observed in liver leading to a decrease in mutations by the combined treatment.     

Cardiac Dysfunction in a Porcine Model of Pediatric Malnutrition

Background

Half a million children die annually of severe acute malnutrition and cardiac dysfunction may contribute to the mortality. However, cardiac function remains poorly examined in cases of severe acute malnutrition.

Objective

To determine malnutrition-induced echocardiographic disturbances and longitudinal changes in plasma pro-atrial natriuretic peptide and cardiac troponin-T in a pediatric porcine model.

Methods and Results

Five-week old piglets (Duroc-x-Danish Landrace-x-Yorkshire) were fed a nutritionally inadequate maize-flour diet to induce malnutrition (MAIZE, n = 12) or a reference diet (AGE-REF, n = 12) for 7 weeks. Outcomes were compared to a weight-matched reference group (WEIGHT-REF, n = 8). Pro-atrial natriuretic peptide and cardiac troponin-T were measured weekly. Plasma pro-atrial natriuretic peptide decreased in both MAIZE and AGE-REF during the first 3 weeks but increased markedly in MAIZE relative to AGE-REF during week 5–7 (p≤0.001). There was overall no difference in plasma cardiac troponin-T between groups. However, further analysis revealed that release of cardiac troponin-T in plasma was more frequent in AGE-REF compared with MAIZE (OR: 4.8; 95%CI: 1.2–19.7; p = 0.03). However, when release occurred, cardiac troponin-T concentration was 6.9-fold higher (95%CI: 3.0–15.9; p<0.001) in MAIZE compared to AGE-REF. At week 7, the mean body weight in MAIZE was lower than AGE-REF (8.3 vs 32.4 kg, p<0.001), whereas heart-weight relative to body-weight was similar across the three groups. The myocardial performance index was 86% higher in MAIZE vs AGE-REF (p<0.001) and 27% higher in MAIZE vs WEIGHT-REF (p = 0.025).

Conclusions

Malnutrition associates with cardiac dysfunction in a pediatric porcine model by increased myocardial performance index and pro-atrial natriuretic peptide and it associates with cardiac injury by elevated cardiac troponin-T. Clinical studies are needed to see if the same applies for children suffering from malnutrition.     

A magnetic adsorbent-based process for semi-continuous PEGylation of proteins

A semi-continuous magnetic particle-based process for the controlled attachment of PEG (PEGylation) to proteins is described for the first time. Trypsin and 2 kDa mono-activated PEG were used to systematically develop the steps in the process. Proof of concept was shown in a microfluidics system to minimize reagent consumption. Two streams containing (i) 1.2 g/L trypsin and (ii) 4 g/L magnetic adsorbents derivatized with the reversible affinity ligand benzamidine were pumped into a pipe reactor. At the exit, a third solution of activated PEG (0-40 g/L) was introduced and the solutions immediately fed into a second reactor. Upon exiting, the mixture was combined in a third reactor with a fourth stream of free amine groups to stop the reaction (50 mM lysine). The mixture continued into a high-gradient magnetic separator where magnetic supports, with PEGylated trypsin still attached, were captured and washing and elution steps were subsequently carried out. Analysis of the conjugates (with SDS-PAGE & LC-MS) showed that the extent of PEGylation could be controlled by varying the reaction time or PEG concentration. Furthermore, the PEG-conjugates had higher enzyme activity compared to PEGylation of non-immobilized trypsin.     

Impacts of predator-mediated interactions along a climatic gradient on the population dynamics of an alpine bird

According to classic theory, species'' population dynamics and distributions are less influenced by species interactions under harsh climatic conditions compared to under more benign climatic conditions. In alpine and boreal ecosystems in Fennoscandia, the cyclic dynamics of rodents strongly affect many other species, including ground-nesting birds such as ptarmigan. According to the ‘alternative prey hypothesis’ (APH), the densities of ground-nesting birds and rodents are positively associated due to predator–prey dynamics and prey-switching. However, it remains unclear how the strength of these predator-mediated interactions change along a climatic harshness gradient in comparison with the effects of climatic variation. We built a hierarchical Bayesian model to estimate the sensitivity of ptarmigan populations to interannual variation in climate and rodent occurrence across Norway during 2007–2017. Ptarmigan abundance was positively linked with rodent occurrence, consistent with the APH. Moreover, we found that the link between ptarmigan abundance and rodent dynamics was strongest in colder regions. Our study highlights how species interactions play an important role in population dynamics of species at high latitudes and suggests that they can become even more important in the most climatically harsh regions.     

The Focal Adhesion: A Regulated Component of Aortic Stiffness

Increased aortic stiffness is an acknowledged predictor and cause of cardiovascular disease. The sources and mechanisms of vascular stiffness are not well understood, although the extracellular matrix (ECM) has been assumed to be a major component. We tested here the hypothesis that the focal adhesions (FAs) connecting the cortical cytoskeleton of vascular smooth muscle cells (VSMCs) to the matrix in the aortic wall are a component of aortic stiffness and that this component is dynamically regulated. First, we examined a model system in which magnetic tweezers could be used to monitor cellular cortical stiffness, serum-starved A7r5 aortic smooth muscle cells. Lysophosphatidic acid (LPA), an activator of myosin that increases cell contractility, increased cortical stiffness. A small molecule inhibitor of Src-dependent FA recycling, PP2, was found to significantly inhibit LPA-induced increases in cortical stiffness, as well as tension-induced increases in FA size. To directly test the applicability of these results to force and stiffness development at the level of vascular tissue, we monitored mouse aorta ring stiffness with small sinusoidal length oscillations during agonist-induced contraction. The alpha-agonist phenylephrine, which also increases myosin activation and contractility, increased tissue stress and stiffness in a PP2- and FAK inhibitor 14-attenuated manner. Subsequent phosphotyrosine screening and follow-up with phosphosite-specific antibodies confirmed that the effects of PP2 and FAK inhibitor 14 in vascular tissue involve FA proteins, including FAK, CAS, and paxillin. Thus, in the present study we identify, for the first time, the FA of the VSMC, in particular the FAK-Src signaling complex, as a significant subcellular regulator of aortic stiffness and stress.