The loss of muscle mass leading to cachexia is rarely identified in early lung cancer. Fasting blood and muscle biopsy were collected in 59 non-small cell lung cancer (NSCLC) and 16 non-cancer patients, at the beginning of thoracic surgery. Serum C-reactive protein (CRP), and IL-6 were higher in NSCLC. In weight-losing NSCLC, food intake and serum albumin were lower, CRP, and TNF-alpha were higher. Although the expression of genes of the ubiquitin-proteasome system was not different, ubiquitinated-protein levels were lower and negatively correlated with ph-FOX01 in weight-losing patients. This would suggest lower muscle proteolytic rates in the early stages of NSCLC. Ph-FOXO1 also related to the degree of weight loss and stage of NSCLC. These data suggest that in early stages of the disease, weight and muscle loss could be mainly due to reduced food intake, rather than accelerated proteolysis, which reinforces the potential for successful dietary interventions to prevent or delay the onset of cachexia.

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The liver is a vital organ, playing numerous critical roles in the body. The liver's ability to perform essential functions is disturbed by injuries that are often associated with many complications such as calcification. Although many reports in the literature document observations of liver calcification, the mechanisms regulating this phenomenon remain unclear. Herein, we aim to investigate the cellular and molecular events that occur during pathological calcification of the liver. / To study the mechanisms of calcification, assessments included histological-staining, immunolabeling, and biochemical and electron microscopy analyses. The findings suggest that calcification may result from hydroxyapatite precipitation in necrotic or apoptotic hepatocytes. Similarly, calcification may be associated with differentiated myofibroblasts expressing bone matrix proteins downstream of TGFbeta signalling. / To identify specific protein regulators linked to the various stages in calcification, and to assess the protein composition of the tissue, a proteomic analysis was used. This analysis identified IQGAP1, an effector of the Rho-GTPases and a master regulator of cell adhesion and migration. IQGAP1 is strongly expressed in myofibroblasts, suggesting that IQGAP1 may be implicated in myofibroblasts migrating towards calcification. Studies on IQGAP1 interactions with its binding partners reveal that it is part of a protein complex that includes beta-catenin, an adhesion protein, and Rac1, a cytoskeletal regulator. These results suggest that IQGAP1 may play an important role in myofibroblast migration upon liver injury. / Having identified that activin and TGFbeta signalling are activated in myofibroblasts, we hypothesised that they may stimulate myofibroblast differentiation and proliferation. Studies using a C3H/10T1/2 cell model reveal that both activin and TGFbeta stimulate differentiation, but only activin induces cell proliferation in a Smad-independent fashion, which requires activation of the ERK/MAPK pathway. / In summary, this work provides new mechanistic insights on the global regulation of liver calcification. The various phases of this work collectively cover the central role of myofibroblasts in liver injury: association with calcification, rapid proliferation, differentiation to an activated form, and migration toward the injured area. The findings allow us to better understand the mechanisms by which liver myofibroblasts are regulated in a specific pathological context.

Postprandial urea production in subjects with insulin dependent diabetes mellitus (IDDM) on conventional insulin therapy is normal when the previous diet is high in protein, but there is an incomplete adaptive reduction in urea production following protein restriction. To evaluate the nutritional implications of restricted protein intake in human diabetes mellitus, it is first necessary to establish a reliable method to measure changes in urea production and amino acid catabolism in response to changes in dietary protein intake. We therefore tested (1) the accuracy of the urea production rate (Ra) to depict changes in urea production, (2) whether sulfate production can be accurately depicted using tracer or nontracer approaches, after establishing the use of electrospray tandem mass spectrometry to measure sulfate concentrations and 34SO4 enrichments following administration of the stable isotope tracer sodium [34S]sulfate, (3) the reproducibility of urea and sulfate measurements following a test meal low in protein (0.25 g/kg) in subjects previously adapted to high (1.5 g/kg.d) and low (0.3 g/kg.d) protein intakes, and compared the metabolic fate of [ 15N]alanine added to the test meal with that of [15N] Spirulina platensis, a 15N-labeled intact protein, and (4) whether we could identify the differences in postprandial urea and sulfate productions between normal subjects and persons with IDDM receiving conventional insulin therapy previously adapted to high protein intake, when the test meal was limiting in protein. Under basal conditions, steady state urea Ra is an accurate measure of urea production. Following changes in urea production, both the tracer and nontracer methods seriously underestimated total urea Ra. The tracer method overestimated sulfate production by 20%, but the nontracer method provided an accurate measure of sulfate production and, hence, sulfur amino acid catabolism. Postprandial changes in urea and sulfate productions following normal ada

Muscle tissue development (myogenesis) involves the formation of specific fibers (myotubes) from muscle cells (myoblasts). For this to occur, the sequential expression of Myogenic Regulatory Factors (MRFs), such as MyoD and myogenin, is required. The expression of these MRFs is regulated posttranscriptionally by the RNA-binding protein HuR, whereby HuR associates with the 3'-untranslated regions of MyoD and myogenin mRNA, leading to a significant increase in their half-lives. Here we show that the cleavage of HuR by caspases at the aspartate (D) 226 residue is one of the main regulators of its pro-myogenic function. This proteolytic activity generates two cleavage products (CPs), HuR-CP1 and HuR-CP2, that differentially affect the myogenic process. Myoblasts overexpressing HuR-CP1 or the non-cleavable mutant of HuR, HuRD226A, are not able to engage myogenesis, while overexpressing HuR-CP2 enhances myotube formation. HuR-CP2 but not -CP1 promotes myogenesis by stabilizing the MyoD and myogenin mRNAs to the same levels as wt-HuR. Conversely, the inhibitory effects of HuR-CP1 and HuRD226A depend on their abilities to associate during myogenesis with the HuR import receptor, Trn2, leading to HuR accumulation in the cytoplasm. Therefore, we propose a model whereby the caspase-mediated cleavage of HuR generates two CPs that collaborate to regulate myogenesis; HuR-CP1 by interfering with the Trn2-mediated import of HuR and HuR-CP2 by participating in the stabilization of mRNAs encoding key MRFs.

Cyclic guanosine 3', 5'-monophosphate (cGMP) has recently emerged as an endogenous regulator for controlling or reversing cardiac hypertrophy. Increased protein accumulation is a key feature of cardiac hypertrophy; thus, our study investigates the effects of a cGMP analog on protein accumulation in primary culture of adult rat cardiomyocytes and dissects out the mechanisms involved. We confirmed that a cGMP analog, 8-bromo-cGMP, inhibits phenylephrine (PE)-increased accumulation of newly synthesized proteins in cultured adult rat ventricular cardiomyocytes. Firstly, we have obtained data showing that 8-bromo-cGMP does not inhibit phosphorylation of S6K1 by PE during short time treatment (10 min to 2 h), but blocks phosphorylation of S6K1 by PE at 6 h; moreover this blocking effect is completely abolished by phosphatase inhibitor Tautomycin. Then, we have demonstrated that PE and cGMP induce sustained and transient increased phosphorylation of ERK, respectively. Moreover, cGMP inhibits PE-induced phosphorylation of ERK during long term treatment (3 and 6h). We have also shown that 8-bromo-cGMP inhibits ROS generation induced by PE. Other effects of PE that could be related to hypertrophy (i.e. increased concentration of upstream binding factor mRNA and decreased concentration of the mRNAs of Atrogin and muscle specific RING finger) were not abolished by 8-bromo-cGMP. We conclude that cGMP analog blocks protein accumulation by inhibiting the sustained phosphorylation of S6K1 via the activation of phosphatases.

Aggrecan is one of the most important structural components of the extracellular matrix (ECM) of articular cartilage, where it contributes to the hydration of the tissue and its ability to resist compressive loads during joint movement. Increased aggrecan degradation and loss occurs in joint diseases and is thought to be mediated by enzymes such as the matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS). It has also been proposed that aggrecan release from the cartilage can be mediated by a non-proteolytic mechanism which involves the degradation of hyaluronan (RA) to which the aggrecan is bound. As aggrecan degradation and loss is known to be induced by pro-inflammatory cytokines, IL-1, TNFalpha, IL-6, IL-17 and OSM were used to investigate the mechanisms involved in proteoglycan catabolism in organ cultures of bovine articular cartilage. Irrespective of the cytokine, all aggrecan fragments generated were characteristic of aggrecanase action, and no additional aggrecan-degrading enzymatic activity was detected. In the presence of OSM, more rapid aggrecan release was observed, due to both proteolysis and fragmentation of HA by hyaluronidase activity. Moreover, addition of OSM resulted in the cleavage of aggrecan at a non-canonical aggrecanase site near its carboxy-terminal globular domain. Such cleavage could be reproduced in vitro by the action of either ADAMTS-4 or ADAMTS-5. Gene expression analysis revealed that both aggrecanases were highly induced by the cytokines, and while ADAMTS-4 was the major aggrecanase to be stimulated in all conditions, ADAMTS-5 remains the predominant aggrecanase to be expressed in cartilage. Thus, the present study shows that aggrecanase activity is primarily responsible for aggrecan degradation in the early stages of cytokine stimulation, and that in the presence of OSM, aggrecanase substrate specificity can be differentially modulated and hyaluronidase-mediated RA degradation can be induced.

Similar to other non-enveloped viruses, the mechanism of cell entry for polyomaviruses is poorly understood. The polyomavirus capsid is an icosahedron composed of 72 pentamers of the major capsid protein VP1. There is one copy of minor capsid proteins, VP2 or VP3, at the center of each pentamer. According to previous studies, polyomavirus cell entry is a multi-step process which includes: 1) VP1 binding to sialic acid (SA) on the surface of host cells, 2) interaction of VP1 with alpha4beta1 integrin and 3) subsequent cell penetration. Biochemical studies have shown that SA alters polyomavirus protease sensitivity, suggesting a conformational change. The aim of this study was to determine these conformational changes at the molecular level. Therefore, we used single particle cryo-electron microscopy to construct 3D maps of wild type (WT) murine polyomavirus, WT bound to SA, a mutant with a disrupted integrin binding site, and the mutant bound to SA. Our results reveal that in both WT and mutant viruses, a significant conformational change happens after binding with SA which is seen as an additional ring of density inside the virus. Moreover some negative densities are seen in the difference map of WT and WT bound with SA, which suggests movement of some viral proteins after binding with SA.

The Ras signaling cascade is a vital component in the processes that mediate cell survival, growth, differentiation and transformation through activation of MAP kinase (mitogen-activated protein kinase). The recent discovery of a new scaffold of the Ras signaling pathway, Kinase Suppressor of Ras (KSR), is found to be a positive effector of Ras signaling which further contributes to proliferation and transformation in the ERK/MAPK pathway. This thesis describes the roles of Ras and Kinase Suppressor of Ras 1 (KSR-1) in regulating the expression of tumor promoting genes such as urokinase plasminogen activator (uPA) in the development and progression of breast cancer in vitro and in vivo. Ras and KSR increase the proliferative capacity and migration of MDAMB-231 human breast cancer cells in vitro. In contrast, Ras and KSR decrease the invasiveness of MDA-MB-231 human breast cancer cells in vitro. Furthermore, uPA gene expression levels do not correlate with uPA protein expression levels suggesting a possible mutation induced by KSR and/or Ras. In vivo studies reveal that Ras and KSR increase tumor volume in mice, as well as more advanced osteolytic bone metastases. Collectively, these results indicate that Ras and KSR play significant roles in breast cancer development and metastasis.

Saccharomyces cerevisiae has developed mechanisms in order to survive harsh environmental conditions. This species responds to stresses such as ethanol, heat, and weak acid exposure via two well-characterized stress response pathways. These typically involve either the Hsf1p or the Msn2/4p transcriptional regulators. Recently, our lab has begun to characterize a member of the zinc cluster protein family: Asg1p (Activator of Stress Genes, systematic name: YIL130W), which is presumed to stimulate stress response genes independently of the Hsflp and Msn2/4p pathways. Previous work has revealed five target genes of Asg1p (HSP30, STP4, YER130C, TPO2, YRO2) thought to be involved in this novel stress response pathway. In this study, we attempted to better characterize the role of Asg1p and its target genes during stress induction. We first determined if the induction of certain Asg1p target genes by stress is strain specific. HSP30 induction by heat shock is specific to the W303 strain as shown by primer extension analysis. We then generated the deletion strains Deltaasg1 and Astp4 in W303. We observed a loss of induction of HSP30 in the Deltaasg1 deletion strain when cells were exposed to ethanol. This led us to believe that Asg1p does play a role in the stress response pathway. Also, we attempted to globally define the target sites of Asg1p in vivo on a genome-wide scale by combining Chromatin Immuno Precipitation with microarrays (ChIP-chip). We identified eight putative Asg1p target genes: YRO2, HSP78, ZRT2, ZRT1, MSN4, STP4, TPO2, and HSP30.