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Heat Shock Proteins (HSPs) are a class of ubiquitously expressed and functionally related proteins found in all living organisms from humans to bacteria. Their expression is increased in response to various cellular stressors in what is referred to as the heat shock response. The induction of one particular HSP, HSP60, has been found to be correlated with mitochondria specific cell stress. Recently HSP60 has been found to be secreted and expressed extracellularly, after first being thought to be strictly intracellular. However, the mechanisms of these mechanisms of translocation and secretion have not been clearly identified. The aim of this study was to develop a model in vitro system for HSP60 expression in human cells so that treatments which resulted in mitochondrial impairment could be investigated. This study looked at several ...
Heat shock protein 60 (Hsp60) is a mitochondrial stress protein that is elevated in response to mitochondrial impairment. It is involved in protein folding and, more recently, it has been identified as a signalling molecule. It has been found elevated in patients suffering from diabetes, atherosclerosis and cardiomyopathy. In order to investigate the translocation of Hsp60 from mitochondria we employed RG224428, a plasmid housing genes for a human Hsp60 connected to a green fluorescent protein (GFP) bound at the C-terminus. The plasmid was cloned inside DH5α E. coli (DH5α) and purified using an endotoxin-free plasmid extraction kit. Using endotoxin-free DNA we transfected the human cervical cancer cell line HeLa with the Hsp60-GFP DNA. It has been found that endotoxin (or otherwise known as lipopolysaccharide) can affect Hsp60 expressi...
Milk from dairy cows is an important human food. It contains the protein beta-lactoglobulin (BLG) which is not present in human milk and thought to be a major allergen in the milk of cows. RNA interference (RNAi), mediated by sequence-specific micro RNAs (miRNAs), provides a new molecular tool to reduce the levels of undesirable proteins. In this study, I have screened ten miRNAs targeting the BLG mRNA for their potential to knockdown BLG expression using a cell-based in vitro assay. I identified four miRNAs which showed substantial bovine BLG (bBLG) (78-97%) and ovine BLG (oBLG) (33-94%) knockdown activity in vitro. Tandem miRNA constructs with combinations of these four single miRNAs did not result in significantly increased knockdown efficiency compared to the respective single miRNA constructs in the in vitro assay (P>0.05). Beca...
Myostatin is a secreted protein which is known to be a negative regulator of skeletal muscle growth. Numerous studies have demonstrated that down-regulating the expression of myostatin results in an increase in muscle mass. This increase in skeletal muscle is accompanied by a marked change in the muscle fibre composition from one reliant on mitochondrial oxidative metabolism to one more focused on glycolytic metabolism. A comparative proteomics study was undertaken to investigate the effect of this altered metabolism has on the mitochondria from the gastrocnemius muscle of myostatin null mice, compared with those from wild type mice. The majority of the proteins identified showed no significant modulation between the two phenotypes, but gives an interesting insight into observations made in previous studies. Several proteins were shown...
Heat Shock Proteins 60 & 70 (HSP60 & HSP70) are intracellular protein that has been shown to be present at elevated levels in systemic circulation in Type 2 Diabetes mellitus (T2DM) patients. Conditions that lead to its secretion, and the mechanism of its translocation from cells, have not yet been defined. The aim of this study was to determine if specific cell stressors associated with T2DM, namely hyperglycaemia and oxidative stress, result in the upregulation of HSP60 in human cells in vitro. Human HeLa cells were grown in media supplemented with 100 mM glucose, 200 μM hydrogen peroxide (H₂O₂), and 50 μM sodium azide. Initially, the effect of these treatments on cell growth rate was examined, with each treatment significantly inhibiting growth rate. LDH and MTT assays were also used to successfully demonstrate that these treatments...
C5a is thought to play a role during complement-activated neuronal apoptotic cell death in the central nervous system. The mechanisms responsible are however not well-understood. As mitochondria play a key role during apoptosis, we investigated mitochondria as a potential target for C5a. Using PC12 cells, we demonstrated that exposure to C5a led to inhibition of mitochondrial respiration, dehydrogenase and cytochrome c oxidase activities. Interestingly, an increase in expression of the mitochondrial stress protein chaperonin 60 was also observed, confirming a marked effect of C5a on mitochondrial functions. These observations are the first documented intracellular effects noted for the complement molecule C5a in in-vitro cultured cells.
There is increasing evidence that mitochondrial dysfunction and oxidative stress may be integral to the pathogenesis of type 2 diabetes mellitus. Heat shock protein (Hsp60) is a mitochondrial stress protein known to be induced under conditions of mitochondrial impairment. Although this intracellular protein is normally found in the mitochondrion, several studies have shown that this protein is also present in systemic circulation. In this study, we report the presence of elevated levels of Hsp60 in both saliva and serum of type 2 diabetic patients compared to non-diabetic controls. Hsp60 was detectable in the saliva of 10% of control and 93% of type 2 diabetic patients. Levels detected were in the range of 3–7 ng/ml in control and 3–75 ng/ml in type 2 diabetic patients. Serum Hsp60 levels in the range of 3–88 ng/ml were detected in 33%...
Metformin is in widespread clinical use for the treatment of diabetes mellitus in patients. It has been shown to inhibit mitochondrial bioenergetic functions by inhibiting complex I of the electron transport chain. The expression of mitochondrial-specific molecular stress protein Hsp60 is a key consequence of mitochondrial impairment. Since this protein has important immune-modulatory properties, we have investigated the expression of Hsp60 in human THP-1 monocyte cells exposed to metformin. In this study, we demonstrate significant up-regulation of Hsp60 at both mRNA and protein levels when these cells were exposed to metformin at therapeutic dosage levels. Interestingly, there was also an increase in expression of CD14 mRNA in these cells. This suggested a possible modulation of the differentiation rates of the THP-1 cells during exp...
Angiopoietin-like protein 2 (Angptl2) levels are increased by obesity and obesity-related pathological conditions, and it is considered to be an important adipocyte-derived inflammatory mediator. In contrast, the multifunctional cytokine TGF-β1 has been reported to be augmented in obesity of rodents and humans, but inhibits adipocyte differentiation in vitro. Here we demonstrate that TGF-β1 induces expression of the Angptl2 gene through a Smad3-dependent pathway in RAW264.7 macrophage cells, primary peritoneal macrophages, and differentiated 3T3-L1 adipocytes. Transcriptional induction of the Angptl2 gene by TGF-β1 was dependent on the Smad3 protein which binds to the Smad Binding Element (SBE) region located on the Angptl2 promoter. Macrophages with Smad3 knocked down by small interfering RNA showed reduction of TGF-β1-induced Angptl2...
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