Modulatory role of phospholipase D in the activation of signal transducer and activator of transcription (STAT)-3 by thyroid oncogenic kinase RET/PTC

<p>Abstract</p> <p>Background</p> <p>RET/PTC (rearranged in transformation/papillary thyroid carcinomas) gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although it has been established that RET/PTC kinase plays a crucial role in intracellular signaling pathways that regulate cellular transformation, growth, and proliferation in thyroid epithelial cells, the upstream signaling that leads to the activation of RET/PTC is largely unknown. Based on the observation of high levels of PLD expression in human papillary thyroid cancer tissues, we investigated whether PLD plays a role in the regulating the RET/PTC-induced STAT3 activation.</p> <p>Methods</p> <p>Cancer tissue samples were obtained from papillary thyroid cancer patients (n = 6). The expression level of PLD was examined using immunohistochemistry and western blotting. Direct interaction between RET/PTC and PLD was analyzed by co-immunoprecipitation assay. PLD activity was assessed by measuring the formation of [³H]phosphatidylbutanol, the product of PLD-mediated transphosphatidylation, in the presence of <it>n</it>-butanol. The transcriptional activity of STAT3 was assessed by m67 luciferase reporter assay.</p> <p>Results</p> <p>In human papillary thyroid cancer, the expression levels of PLD2 protein were higher than those in the corresponding paired normal tissues. PLD and RET/PTC could be co-immunoprecipitated from cells where each protein was over-expressed. In addition, the activation of PLD by pervanadate triggered phosphorylation of tyrosine 705 residue on STAT-3, and its phosphorylation was dramatically higher in TPC-1 cells (from papillary carcinoma) that have an endogenous RET/PTC1 than in ARO cells (from anaplastic carcinoma) without alteration of total STAT-3 expression. Moreover, the RET/PTC-mediated transcriptional activation of STAT-3 was synergistically increased by over-expression of PLD, whereas the PLD activity as a lipid hydrolyzing enzyme was not affected by RET/PTC.</p> <p>Conclusion</p> <p>These findings led us to suggest that the PLD synergistically functions to activate the STAT3 signaling by interacting directly with the thyroid oncogenic kinase RET/PTC.</p

DJ-1 Null Dopaminergic Neuronal Cells Exhibit Defects in Mitochondrial Function and Structure: Involvement of Mitochondrial Complex I Assembly

DJ-1 is a Parkinson's disease-associated gene whose protein product has a protective role in cellular homeostasis by removing cytosolic reactive oxygen species and maintaining mitochondrial function. However, it is not clear how DJ-1 regulates mitochondrial function and why mitochondrial dysfunction is induced by DJ-1 deficiency. In a previous study we showed that DJ-1 null dopaminergic neuronal cells exhibit defective mitochondrial respiratory chain complex I activity. In the present article we investigated the role of DJ-1 in complex I formation by using blue native-polyacrylamide gel electrophoresis and 2-dimensional gel analysis to assess native complex status. On the basis of these experiments, we concluded that DJ-1 null cells have a defect in the assembly of complex I. Concomitant with abnormal complex I formation, DJ-1 null cells show defective supercomplex formation. It is known that aberrant formation of the supercomplex impairs the flow of electrons through the channels between respiratory chain complexes, resulting in mitochondrial dysfunction. We took two approaches to study these mitochondrial defects. The first approach assessed the structural defect by using both confocal microscopy with MitoTracker staining and electron microscopy. The second approach assessed the functional defect by measuring ATP production, O2 consumption, and mitochondrial membrane potential. Finally, we showed that the assembly defect as well as the structural and functional abnormalities in DJ-1 null cells could be reversed by adenovirus-mediated overexpression of DJ-1, demonstrating the specificity of DJ-1 on these mitochondrial properties. These mitochondrial defects induced by DJ-1mutation may be a pathological mechanism for the degeneration of dopaminergic neurons in Parkinson's disease

The Effect of Rural-to-Urban Migration on Obesity and Diabetes in India: A Cross-Sectional Study

Shah Ebrahim and colleagues examine the distribution of obesity, diabetes, and other cardiovascular risk factors among urban migrant factory workers in India, together with their rural siblings. The investigators identify patterns of change of cardiovascular risk factors associated with urban migration

Principles of genetic circuit design

Cells navigate environments, communicate and build complex patterns by initiating gene expression in response to specific signals. Engineers seek to harness this capability to program cells to perform tasks or create chemicals and materials that match the complexity seen in nature. This Review describes new tools that aid the construction of genetic circuits. Circuit dynamics can be influenced by the choice of regulators and changed with expression 'tuning knobs'. We collate the failure modes encountered when assembling circuits, quantify their impact on performance and review mitigation efforts. Finally, we discuss the constraints that arise from circuits having to operate within a living cell. Collectively, better tools, well-characterized parts and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.National Institute of General Medical Sciences (U.S.) (Grant P50 GM098792)National Institute of General Medical Sciences (U.S.) (Grant R01 GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (EEC0540879)Life Technologies, Inc. (A114510)National Science Foundation (U.S.). Graduate Research FellowshipUnited States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant 4500000552

Association of LETM1 and mrpl36 contributes to the regulation of mitochondrial ATP production and necrotic cell death

Leucine zipper/EF hand-containing transmembrane-1 (LETM1) is a mitochondrial inner membrane protein that was first identified in Wolf-Hirschhorn syndrome, and was deleted in nearly all patients with the syndrome. LETM1 encodes for the human homologue of yeast Mdm38p, which is a mitochondria-shaping protein of unclear function. Here, we describe LETM1-mediated regulation of mitochondrial ATP production and biogenesis. We show that LETM1 overexpression can induce necrotic cell death in HeLa cells, in which LETM1 reduces mitochondrial biogenesis and ATP production. LETM1 acts as an anchor protein and associates with mitochondrial ribosome protein L36. Adenovirus-mediated overexpression of LETM1 reduced mitochondrial mass and expression of many mitochondrial proteins. LETM1-mediated inhibition of mitochondrial biogenesis enhanced glycolytic ATP supply and activated protein kinase B activity and cell survival signaling. The expression levels of LETM1 were significantly increased in multiple human cancer tissues compared with normals. These data suggest that LETM1 serves as an anchor protein for complex formation with the mitochondrial ribosome and regulates mitochondrial biogenesis. The increased expression of LETM1 in human cancer suggests that dysregulation of LETM1 is a key feature of tumorigenesis

Modulatory role of phospholipase D in the activation of signal transducer and activator of transcription (STAT)-3 by thyroid oncogenic kinase RET/PTC-5

Ds. The tissue extracts and transfectant controls of PLD1/PLD2 were applied to SDS-PAGE for Western blotting with antibodies directed against PLD and β-actin. (B) Immunohistochemistry of PLD in normal and cancerous tissue. PLD2 was highly expressed in papillary thyroid cancer tissue () compared to faintly stained normal thyroid tissue (). () Negative isotype control with preimmune mouse IgG1. Original magnification, 100×. (C) Expression of RET/PTC-1 in malignant papillary thyroid cancer tissue. After the normal and cancer tissues were lysed and the total RNA was isolated, RT-PCR was performed with the primers specific to the human RET/PTC1 and GAPDH. After the PCR amplification, the products were separated by agarose gel electrophoresis and visualized using ethidium bromide staining. The results are representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Modulatory role of phospholipase D in the activation of signal transducer and activator of transcription (STAT)-3 by thyroid oncogenic kinase RET/PTC"</p><p>http://www.biomedcentral.com/1471-2407/8/144</p><p>BMC Cancer 2008;8():144-144.</p><p>Published online 23 May 2008</p><p>PMCID:PMC2412888.</p><p></p

Modulatory role of phospholipase D in the activation of signal transducer and activator of transcription (STAT)-3 by thyroid oncogenic kinase RET/PTC-0

Ds. The tissue extracts and transfectant controls of PLD1/PLD2 were applied to SDS-PAGE for Western blotting with antibodies directed against PLD and β-actin. (B) Immunohistochemistry of PLD in normal and cancerous tissue. PLD2 was highly expressed in papillary thyroid cancer tissue () compared to faintly stained normal thyroid tissue (). () Negative isotype control with preimmune mouse IgG1. Original magnification, 100×. (C) Expression of RET/PTC-1 in malignant papillary thyroid cancer tissue. After the normal and cancer tissues were lysed and the total RNA was isolated, RT-PCR was performed with the primers specific to the human RET/PTC1 and GAPDH. After the PCR amplification, the products were separated by agarose gel electrophoresis and visualized using ethidium bromide staining. The results are representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Modulatory role of phospholipase D in the activation of signal transducer and activator of transcription (STAT)-3 by thyroid oncogenic kinase RET/PTC"</p><p>http://www.biomedcentral.com/1471-2407/8/144</p><p>BMC Cancer 2008;8():144-144.</p><p>Published online 23 May 2008</p><p>PMCID:PMC2412888.</p><p></p