Current Issue : January - March Volume : 2013 Issue Number : 1 Articles : 6 Articles
Background: The p38a mitogen-activated protein kinase (MAPK) is a critical mediator of myoblast differentiation,\r\nand does so in part through the phosphorylation and regulation of several transcription factors and chromatin\r\nremodelling proteins. However, whether p38a is involved in processes other than gene regulation during\r\nmyogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear.\r\nMethods: To further characterise the involvement of p38a during myoblast differentiation, we developed and\r\napplied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In\r\naddition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in\r\nthe same experiment, and we made use of this to study the substrate specificities of the p38a and b isoforms.\r\nResults: Applying the technique to p38a resulted in the identification of seven in vivo phosphorylation sites on six\r\nproteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison\r\nwith p38b revealed that substrate specificity does not discriminate these two isoforms, but rather that their\r\ndistinguishing characteristic appears to be cellular localisation.\r\nConclusion: Our results suggest p38a has a novel cytoplasmic role during myogenesis and that its unique cellular\r\nlocalisation may be why p38b and other isoforms cannot compensate for its absence. The substrate-finding\r\napproach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and\r\nfor uncovering the deeper mechanisms of phosphorylation-dependent cell signalling....
Animal model have been used extensively to study of experimental ulcerative colitis syndromes. Some compounds and chemical agents like acetic acid (AA), trinitrobenzene sulfonic acid (TNBS), iodoacetamide, indomethacin, oxazolone, dexran sodium sulphate (DSS) and peptidoglycan polysaccharide used for the induction of colitis. Induction of colitis by chemical/compounds is an easily inducible model of inflammatory bowel disease and the inflammatory phase bears some resemblance to acute human intestinal inflammation. Recent studies suggest that the search for the new agent (compounds/medicinal plants/herbs) and its mechanism for colitis continue in animal models. Despite the difference in anatomy, physiology, and nutritional factors between animal model systems and the human body, the fact remains that one or more of these model systems may reflect the same mechanism or etiologic agent which occurs in the human ulcerative colitis....
For studying proliferation and determination of survival of cancer cells after irradiation, the multiple MTT assay,\r\nbased on the reduction of a yellow water soluble tetrazolium salt to a purple water insoluble formazan dye by\r\nliving cells was modified from a single-point towards a proliferation assay. This assay can be performed with a\r\nlarge number of samples in short time using multi-well-plates, assays can be performed semi-automatically with a\r\nmicroplate reader. Survival, the calculated parameter in this assay, is determined mathematically. Exponential\r\ngrowth in both control and irradiated groups was proven as the underlying basis of the applicability of the\r\nmultiple MTT assay. The equivalence to a clonogenic survival assay with its disadvantages such as time\r\nconsumption was proven in two setups including plating of cells before and after irradiation. Three cell lines (A\r\n549, LN 229 and F 98) were included in the experiment to study its principal and general applicability....
Background: Antigen-specific CD8+ cytotoxic T lymphocytes represent potent effector cells of the adaptive\r\nimmune response against viruses as well as tumours. Therefore assays capable at exploring the generation and\r\nfunction of cytotoxic T lymphocytes represent an important objective for both clinical and experimental settings.\r\nMethods: Here we show a simple and reproducible assay for the evaluation of antigen-specific CD8+ cytotoxic T\r\nlymphocytes based on a LysiSpot technique for the simultaneous determination of antigen-specific IFN-g\r\nproduction and assessment of tumor cytolysis. The assay was developed within an experimental model of\r\ncolorectal carcinoma, induced by the colorectal tumor cell line DHD-K12 that induces tumors in BDIX rats and, in\r\nturn, elicits a tumor- specific immune response.\r\nResults: Using DHD-K12 cells transfected to express Escherichia coli b-galactosidase as target cells, and by the fine\r\nsetting of spot colours detection, we have developed an in vitro assay that allows the recognition of cytotoxic T\r\nlymphocytes induced in BDIX rats as well as the assessment of anti-tumour cytotoxicity. The method highlighted\r\nthat in the present experimental model the tumour antigen-specific immune response was bound to killing target\r\ncells in the proportion of 55%, while 45% of activated cells were not cytotoxic but released IFN-g. Moreover in this\r\nmodel by an ELISPOT assay we demonstrated the specific recognition of a nonapeptide epitope called CSH-275\r\nconstitutionally express in DHD-K12 cells.\r\nConclusions: The assay proved to be highly sensitive and specific, detecting even low frequencies of cytotoxic/\r\nactivated cells and providing the evaluation of cytokine-expressing T cells as well as the extent of cytotoxicity\r\nagainst the target cells as independent functions. This assay may represent an important tool to be adopted in\r\nexperimental settings including the development of vaccines or immune therapeutic strategies....
Background: Nanogold has been investigated in a wide variety of biomedical applications because of the antiinflammatory\r\nproperties. The purpose of this study was to evaluate the effects of TPU (Therapeutic Pulsed\r\nUltrasound) with gold nanoparticles (GNP) on oxidative stress parameters and the expression of pro-inflammatory\r\nmolecules after traumatic muscle injury.\r\nMaterials and methods: Animals were divided in nine groups: sham (uninjured muscle); muscle injury without\r\ntreatment; muscle injury + DMSO; muscle injury + GNP; muscle injury + DMSO + GNP; muscle injury + TPU; muscle\r\ninjury + TPU + DMSO; muscle injury + TPU + GNP; muscle injury + TPU + DMSO + GNP. The ROS production was\r\ndetermined by concentration of superoxide anion, modulation of antioxidant defenses was determined by the\r\nactivity of superoxide dismutase, catalase and glutathione peroxidase enzymes, oxidative damage determined by\r\nformation of thiobarbituric acid-reactive substance and protein carbonyls. The levels of interleukin-1b (IL-1b) and\r\ntumor necrosis factor-a (TNF-a) were measured as inflammatory parameters.\r\nResults: Compared to muscle injury without treatment group, the muscle injury + TPU + DMSO + GNP gel group\r\npromoted a significant decrease in superoxide anion production and lipid peroxidation levels (p < 0.050). It also\r\nshowed a significant decrease in TNF-a and IL-1b levels (p < 0.050) when compared to muscle injury without\r\ntreatment group.\r\nConclusions: Our results suggest that TPU + DMSO + GNP gel presents beneficial effects on the muscular healing\r\nprocess, inducing a reduction in the production of ROS and also the expression of pro-inflammatory molecules....
Introduction: Endothelial progenitor cells (EPC) capable of initiating or augmenting vascular growth were recently\r\nidentified within the small population of CD34-expressing cells that circulate in human peripheral blood and which\r\nare considered hematopoietic progenitor cells (HPC). Soon thereafter human HPC began to be used in clinical trials\r\nas putative sources of EPC for therapeutic vascular regeneration, especially in myocardial and critical limb\r\nischemias. However, unlike HPC where hematopoietic efficacy is related quantitatively to CD34+ cell numbers\r\nimplanted, there has been no consensus on how to measure EPC or how to assess cellular graft potency for\r\nvascular regeneration. We employed an animal model of spontaneous neovascularization to simultaneously\r\ndetermine whether human cells incorporate into new vessels and to quantify the effect of different putative\r\nangiogenic cells on vascularization in terms of number of vessels generated. We systematically compared\r\ncompetence for therapeutic angiogenesis in different sources of human cells with putative angiogenic potential, to\r\nbegin to provide some rationale for optimising cell procurement for this therapy.\r\nMethods: Human cells employed were mononuclear cells from normal peripheral blood and HPC-rich cell sources\r\n(umbilical cord blood, mobilized peripheral blood, bone marrow), CD34+ enriched or depleted subsets of these,\r\nand outgrowth cell populations from these. An established sponge implant angiogenesis model was adapted to\r\ndetermine the effects of different human cells on vascularization of implants in immunodeficient mice.\r\nAngiogenesis was quantified by vessel density and species of origin by immunohistochemistry.\r\nResults: CD34+ cells from mobilized peripheral blood or umbilical cord blood HPC were the only cells to promote\r\nnew vessel growth, but did not incorporate into vessels. Only endothelial outgrowth cells (EOC) incorporated into\r\nvessels, but these did not promote vessel growth.\r\nConclusions: These studies indicate that, since EPC are very rare, any benefit seen in clinical trials of HPC in\r\ntherapeutic vascular regeneration is predominantly mediated by indirect proangiogenic effects rather than through\r\ndirect incorporation of any rare EPC contained within these sources. It should be possible to produce autologous\r\nEOC for therapeutic use, and evaluate the effect of EPC distinct from, or in synergy with, the proangiogenic effects\r\nof HPC therapies....
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