Project description:Although ADAMTS-4 and ADAMTS-5 are the principal aggrecanases in mice and humans, mice lacking both these enzymes (ADAMTS-4/-5Dcat) are healthy, viable, with no overt skeletal phenotype, suggesting the presence of an alternative aggrecanase in these mice. We previously identified a novel aggrecanase activity that was regulated by retinoic acid, but not IL-1a. The upregulated activity cleaved aggrecan at E↓A bonds. This study aimed to identify the alternative aggrecanase. Femoral head cartilage from TS-4/5Dcat mice was stimulated with IL-1a or retinoic acid and total RNA was analysed by microarray. Candidate, upregulated genes identified by Quantile Normalisation included ADAMTS, MMPm cathepsin and calpain families as putative aggrecanases. Three criteria, including quantitative RT-PCR analyses, sensitivity to retinoic acid treatment and sensitivity to TIMP-3 inhibition, were then used as readouts to identify candidate extracellular proteinases whose mRNA expression levels were increased by retinoic acid, but not by IL-1a treatment. Thereafter, gene silencing was used to identify the most likely aggrecanase in chondrocytes from TS-4/5Dcat mice. Aggrecanase activity was monitored by Western blotting and immunohistochemistry with neoepitope antibodies. The microarray analyses identified ADAMTS-9, MMP-11 and calpain-5 as candidate aggrecanases that were upregulated by retinoic acid. While qPCR confirmed this finding, calpain 5 was excluded from the candidate list because it was not inhibited by TIMP-3. MMP-11 was also excluded because it was upregulated by IL-1a. ADAMTS-9 therefore emerged as a candidate for the novel aggrecanase. Gene silencing confirmed ADAMTS-9 as the putative aggrecanase. Immunohistochemistry revealed that ADAMTS-9 in mouse knee joints was expressed in the proliferative zone of both wildtype and TS-4/5Dcat growth plates, and also in the calcified cartilage of the wildtype hypertrophic zone. In conclusion, ADAMTS-9 is an aggrecanase that cleaves at FREEE1467↓1468GLGS in the chondroitin sulphate-rich region of aggrecan.
Project description:Insulin degrading enzyme (IDE) is a major enzyme responsible for insulin degradation in the liver. The modulation of insulin degrading enzyme activity is hypothesized to be a link between T2DM and liver cancer. Results provide insight into role of IDE in proliferation and other cell functions.
Project description:Insulin degrading enzyme (IDE) is a major enzyme responsible for insulin degradation in the liver. The modulation of insulin degrading enzyme activity is hypothesized to be a link between T2DM and liver cancer. Results provide insight into role of IDE in proliferation and other cell functions. HepG2 cells were transfected with 96nM siRNA for IDE or AllStars Negative Control siRNA (Qiagen) using Lipofectamine 2000 (Invitrogen). 16 h after transfection, cells were treated with 10 nM insulin (Sigma Aldrich) or vehicle for 24 h in serum starvation condition. Total RNA was extracted. For each of the 4 conditions, 3 biological replicates were included.
Project description:Insulin-degrading enzyme (IDE) is a protein with proteolytic and non-proteolytic functions that regulates glucose homeostasis.We hypothesize that IDE deficiency alters glucagon signaling and thereby gluconeogenesis. To test this hypothesis we performed microarray studies in HepG2 cells with a complete deficiency of IDE gene. Results show that IDE deficiency led to upregulation of genes involved in cellular functions related to membranes, organelles and signaling receptors. We used microarrays to find genes whose expression is affected by the absence of IDE gene expression in hetapome human cells
Project description:RATIONALE: Shark cartilage extract may help shrink or slow the growth of colorectal cancer or breast cancer cells.
PURPOSE: Randomized phase III trial to determine the effectiveness of shark cartilage in treating patients who have advanced colorectal cancer or advanced breast cancer.
Project description:Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD) model mice showed decreased insulin-degrading enzyme (IDE) levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa-/-) mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa-/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3); Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa-/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD.
Project description:The plant cell wall degrading enzyme LipA (Lipase/Esterase A) is a Type II secretion system secreted protein of Xanthomonas oryzae pv. oryzae (Xoo; the casual of bacterial leaf blight of rice). LipA is an Xoo virulence factor. However, LipA is a double edged sword for Xoo as it induces rice defense responses such as programmed cell death/hypersensitive response like reaction (HR) and callose deposition. Prior treatment with LipA enhances resistance against subseqent Xoo infection. In order to understand the molecular events associated with Esterase (LipA) induced innate immune responsein rice , whole genome transcriptional profiling was performed using Affymetrix Rice GeneChips