Signal transmission by epidermal growth factor receptor: coincidence of activation and dimerization.

Dimerization of epidermal growth factor receptor dissolved in a solution of nonionic detergent was followed with a resolution of 1 min by quantitative cross-linking with glutaraldehyde. Upon addition of epidermal growth factor to the solution, the initially monomeric protein dimerized in a reaction that was second-order in the concentration of receptor. A second-order rate constant, on the basis of enzymatic activity as a measure of the concentration of functional receptor, was calculated from time courses of dimerization at various initial concentrations of receptor. The activation of the protein tyrosine kinase of the receptor was monitored directly under the same conditions with an exogenous substrate. The increase in tyrosine kinase activity displayed kinetics that were also second-order in the concentration of receptor. A second-order rate constant for the activation of the tyrosine kinase could be calculated from the time courses. The second-order rate constant for the activation of the tyrosine kinase by epidermal growth factor was indistinguishable from the second-order rate constant for the dimerization induced by epidermal growth factor. Therefore, dimerization of epidermal growth factor receptor and activation of its tyrosine kinase are coincident events, both initiated by the binding of epidermal growth factor.     

Clinical Audit of the Radiotherapy Process in Rectal Cancer: Clinical Practice Guidelines and Quality Certification Do Not Avert Variability in Clinical Practice

Background: The therapeutic approach to cancer is complex and multidisciplinary. Radiotherapy is among the essential treatments, whether used alone or in conjunction with other therapies. This study reports a clinical audit of the radiotherapy process to assess the process of care, evaluate adherence to agreed protocols and measure the variability to improve therapeutic quality for rectal cancer. Methods: Multicentre retrospective cohort study in a representative sample of patients diagnosed with rectal cancer in the Institut Català d’Oncologia, a comprehensive cancer centre with three different settings. We developed a set of indicators to assess the key areas of the radiotherapy process. The clinical audit consisted of a review of a random sample of 40 clinical histories for each centre. Results: The demographic profile, histology and staging of patients were similar between centres. The MRI reports did not include the distance from tumour to mesorectal fascia (rCRM) in 38.3% of the cases. 96.7% of patients received the planned dose, and 57.4% received it at the planned time. Surgery followed neoadjuvant treatment in 96.7% of the patients. Among this group, postoperative CRM was recorded in 65.5% of the cases and was negative in 93.4% of these. With regard to the 34.5% (n = 40) of cases where no CRM value was stated, there were differences between the centres. Mean follow-up was 3.4 (SD 0.6) years, and overall survival at four years was 81.7%. Conclusions: The audit revealed a suboptimal degree of adherence to clinical practice guidelines. Significant variability between centres exists from a clinical perspective but especially with regard to organization and process.     

Serum depletion induces changes in protein expression in the trophoblast-derived cell line HTR-8/SVneo

Background

How nutrition and growth factor restriction due to serum depletion affect trophoblast function remains poorly understood. We performed a proteomic differential study of the effects of serum depletion on a first trimester human immortalized trophoblast cell line.

Methods

The viability of HTR-8/SVneo trophoblast cells in culture with 0, 0.5 and 10 % fetal bovine serum (FBS) were assayed via MTT at 24, 48 and 64 h. A comparative proteomic analysis of the cells grown with those FBS levels for 24 h was performed using two-dimensional electrophoresis (2DE), followed by mass spectrometry for protein spot identification, and a database search and bioinformatics analysis of the expressed proteins. Differential spots were identified using the Kolmogorov-Smirnov test (n?=?3, significance level 0.10, D?>?0.642) and/or ANOVA (n?=?3, p?<?0.05).

Results

The results showed that low serum doses or serum depletion differentially affect cell growth and protein expression. Differential expression was seen in 25 % of the protein spots grown with 0.5 % FBS and in 84 % of those grown with 0 % FBS, using 10 % serum as the physiological control. In 0.5 % FBS, this difference was related with biological processes typically affected by the serum, such as cell cycle, regulation of apoptosis and proliferation. In addition to these changes, in the serum-depleted proteome we observed downregulation of keratin 8, and upregulation of vimentin, the glycolytic enzymes enolase and pyruvate kinase (PKM2) and tumor progression-related inosine-5’-monophosphate dehydrogenase 2 (IMPDH2) enzyme. The proteins regulated by total serum depletion, but not affected by growth in 0.5 % serum, are members of the glycolytic and nucleotide metabolic pathways and the epithelial-to-mesenchymal transition (EMT), suggesting an adaptive switch characteristic of malignant cells.

Conclusions

This comparative proteomic analysis and the identified proteins are the first evidence of a protein expression response to serum depletion in a trophoblast cell model. Our results show that serum depletion induces specific changes in protein expression concordant with main cell metabolic adaptations and EMT, resembling the progression to a malignant phenotype.

     

Emerging conflicts for the environmental use of water in high-valuable rangelands. Can livestock water ponds be managed as artificial wetlands for amphibians?

Continental freshwater, irrespective of its origin, natural or artificial, may contribute significantly to biodiversity conservation. Because of the decline of natural aquatic habitats, an increasing concern exists about the role of water ponds as spots of biological richness. Amphibians are strongly at risk since the loss of aquatic habitats, among other factors, causes the isolation of their populations. The implementation of livestock ponds as artificial wetlands may be an effective measure for enhancing amphibian decaying communities. This policy assumes that managing ponds for wildlife conservation purposes joins livestock welfare requirements, but this hypothesis has not been specifically studied. The purpose of this research is to evaluate this premise in the Urbasa-Andia Natural Park, a high-valuable environmental area that holds a relevant amphibian community and has an extended grazing history. We analyse the relationship between the amphibian assemblages present and the design and attributes of a variety of drinking points previously chosen by embodying a high environmental heterogeneity of water resources. The results of this study indicate that the quality of the water stored varies largely along the season, degrading severely in summer because of the wading of animals (in unfenced ponds) and the low water recharge. The contamination, caused by increased enteric microorganisms and dissolved N, is likely to affect livestock more severely than amphibian populations, since the sensitive breeding stage of many amphibians occurs before the loss of water quality. Although the quality of the water is essential, and mammals (wild and domestic) have an influence on it, other factors that are less considered by environmental managers emerge as main drivers of amphibian assemblages, such as hydroperiod, predator occurrence and the environmental quality of the surrounding habitat.     

Functional MRI of long-term potentiation: imaging network plasticity

Neurons are able to express long-lasting and activity-dependent modulations of their synapses. This plastic property supports memory and conveys an extraordinary adaptive value, because it allows an individual to learn from, and respond to, changes in the environment. Molecular and physiological changes at the cellular level as well as network interactions are required in order to encode a pattern of synaptic activity into a long-term memory. While the cellular mechanisms linking synaptic plasticity to memory have been intensively studied, those regulating network interactions have received less attention. Combining high-resolution fMRI and in vivo electrophysiology in rats, we have previously reported a functional remodelling of long-range hippocampal networks induced by long-term potentiation (LTP) of synaptic plasticity in the perforant pathway. Here, we present new results demonstrating an increased bilateral coupling in the hippocampus specifically supported by the mossy cell commissural/associational pathway in response to LTP. This fMRI-measured increase in bilateral connectivity is accompanied by potentiation of the corresponding polysynaptically evoked commissural potential in the contralateral dentate gyrus and depression of the inactive convergent commissural pathway to the ipsilateral dentate. We review these and previous findings in the broader context of memory consolidation.     

Endothelin-converting Enzyme 1 and β-Arrestins Exert Spatiotemporal Control of Substance P-induced Inflammatory Signals

Although the intracellular trafficking of G protein-coupled receptors controls specific signaling events, it is unclear how the spatiotemporal control of signaling contributes to complex pathophysiological processes such as inflammation. By using bioluminescence resonance energy transfer and superresolution microscopy, we found that substance P (SP) induces the association of the neurokinin 1 receptor (NK₁R) with two classes of proteins that regulate SP signaling from plasma and endosomal membranes: the scaffolding proteins β-arrestin (βARRs) 1 and 2 and the transmembrane metallopeptidases ECE-1c and ECE-1d. In HEK293 cells and non-transformed human colonocytes, we observed that G protein-coupled receptor kinase 2 and βARR1/2 terminate plasma membrane Ca²⁺ signaling and initiate receptor trafficking to endosomes that is necessary for sustained activation of ERKs in the nucleus. βARRs deliver the SP-NK₁R endosomes, where ECE-1 associates with the complex, degrades SP, and allows the NK₁R, freed from βARRs, to recycle. Thus, both ECE-1 and βARRs mediate the resensitization of NK₁R Ca²⁺ signaling at the plasma membrane. Sustained exposure of colonocytes to SP activates NF-κB and stimulates IL-8 secretion. This proinflammatory signaling is unaffected by inhibition of the endosomal ERK pathway but is suppressed by ECE-1 inhibition or βARR2 knockdown. Inhibition of protein phosphatase 2A, which also contributes to sustained NK₁R signaling at the plasma membrane, similarly attenuates IL-8 secretion. Thus, the primary function of βARRs and ECE-1 in SP-dependent inflammatory signaling is to promote resensitization, which allows the sustained NK₁R signaling from the plasma membrane that drives inflammation.     

Intraspecific basal metabolic rate varies with trophic level in rufous-collared sparrows

One of the most controversial hypotheses that associate basal metabolic rate (BMR) with food habits and habitat productivity is the food habit hypothesis (FHH). Here we examined the relationship between BMR, diet, and climate among populations of the omnivorous passerine, Zonotrichia capensis (Emberizidae). We used nitrogen stable isotopes to estimate each individual's relative trophic level. To tease apart the effect of climatic variables and diet on BMR, we also used structural equation modeling. After the effect of body mass and climatic variables was taken into account, a significant effect of trophic level as estimated by δ¹⁵N on BMR was found. Our result seems to support the FHH at the intraspecific level, i.e., birds from the lower trophic levels – feeding on seeds and bud – had higher BMR than individuals from higher trophic levels.     

Identification of substrates of the extracellular protease ADAMTS1 by DIGE proteomic analysis

Proteolytic modification of components of the extracellular milieu by metalloproteinases plays important roles in the regulation of multiple cellular and physiological processes and pathological conditions. ADAMTS1 is a secreted enzyme of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases, which is related to angiogenesis and inflammation processes. Here, we describe a proteomic screening for putative ADAMTS1 substrates by analyzing the protein profiles obtained from cultures of transfected cells overexpressing the protease as compared to parental cells. Conditioned medium proteins of cultures of the two cell lines have been quantitatively compared by DIGE. Proteins showing differential levels have been identified by MS techniques leading to the finding of five potential new substrates of ADAMTS1: the basement membrane proteins nidogen-1 and -2, the desmosomal protein desmocollin-3, and the extracellular glycoproteins dystroglycan 1 and Mac-2-binding protein. Nidogen-1 and -2 have been further validated as substrates by immunochemical analysis. Our results demonstrate the utility of the DIGE proteomic technique for the discovery of specific substrates of matrix proteases.     

Agonist-biased Trafficking of Somatostatin Receptor 2A in Enteric Neurons

Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with β-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and β-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/β-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from β-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained β-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.