Transcriptional and translational expression of PPARY in the human colorectal cancer dell llne, colo 205

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors of the nuclear hormone receptor superfamily that includes the receptor for steroid, retinoid and thyroid hormone. These nuclear receptors are characterised by their ability to bind to specific DNA sequences and, when activated by a ligand, PPARs regulate the expression of various genes and their functions (Auwerx 1999; Vamecq eta/., 1999). PPAR was first cloned from the mouse liver by lsseman and Green in 1990. The PPAR family consists of three distinct isoforms, namely PPARa, PPARB (or PPARf3) and PPARy. Each isoform is encoded by a separate gene (Tontonoz eta/. 1994). PPARy is further divided into the PPARy1, PPARy2, PPARy3 and PPARy4 isoforms (Sundvold and Lien, 2001) whereby PPARy2 has an additional30 amino acids at its N-terminus but is believed to have similar functions as those of PPARy1 (Sundvold eta/. 1995). PPARy3 is only found in humans and the expressed protein is identical to that of PPARy1 (Fajas eta/., 1998). PPARy is found mainly in adipocytes and cells of the immune system (Braissant et a/1996; Lemberger et a/ 1996) and is involved in the regulation of adipogenesis, glucose metabolism and macrophage development and function (reviewed in Kersten eta/., 2000). PPARr and carcinogenesis Although it has long been known that PPARa ligands such as hypolipidaemic drugs cause hepatocarcinogenesis in rodents (Reddy and Chu 1996), the possible involvement of PPAR in human neoplasms has only recently emerged. PPARy has been shown to be expressed in human colon cancer (Sarraf eta/., 1998), prostate cancer (Mueller eta/., 2000) and breast cancer (Clay eta/., 1999) cell lines. However the involvement of PPARy activity in inducinggrowth inhibition in such tumours have been contradictory. For example, it has previously been shown that PPARy can inhibit the proliferation of human colorectal carcinomas (Brockman eta/., 1998). In sharp contrast with this, however, was the report that activation of PPARy promotes the development of colon tumors in C57BU6J-APCMin/+mice (Lefebvre et at., 1998), a clinically relevant model for both human familial adenomatous polyposis and sporadic colon canc~r (Suet~/., 1992). Recent evidence suggests that PPARy ligands could have an anti-tumor effect in human$ as these compounds decrease cell growth and induce apoptosis in several malignant human cell types, including colorectal carcinomas (S~rraf eta/., 1998). Specific ligands of PPARy such as the antidiabetic thiazolidinediones (TZDs), natural fatty acid derivatives, non-steroidal anti-inflammatory drugs (NSAIDs) and certain polyunsaturated fatty acids have been identified (Palmer et a/., 1998). In agreement with the potential role of PPARy ligands for the treatment of cancer, our present study was aimed at observing the growth inhibition of the colorectal cancer cell line, COL0205 by the PPARy ligand, ciglitazone. This aim was slightly different from the original one proposed under this grant whereby the determination of both PPARa and PPARy expression by two colorectal cell lines, HT-29 and COL0205 was proposed. The change was necessary because the amount of funds approved was about 45% less than that requested and also based on current developments in this research area including the use of a more advanced technique of gene quantification, namely, using real time PCR. In addition, we propose to correlate these findings with the expression of the corresponding proteins by the cell line

The expression of ppars gamma in the cervical cancer line, bela and the effects of specific PPAR ligand on cell growth

To quantify the mRNA expression levels of PPARy in the cervical cancer cell line, HeLa, using real-time PCR. To correlate the findings in (1) with the corresponding protein expression levels by Western blotting. To correlate the transcriptional and translational levels of PPARy with markers of apoptosis in these cells. To determine whether addition of specific PPARy ligands to the tumour cell line would modulate the above parameters

Gene quantitation by a competitive quantitative PCR technique using a homologous standard

The project aimed to develop a quantitative PCR technique to accurately quantity gene expression. We tested the accuracy of the method by determining the expression levels of the transcription factor, PPARy. This was done firstly in a mock system (utilizing cloned PPARy in a plasmid with known concentrations) and later on activated monocytes. The method utilized a homologous internal standard in a competitive PCR. This method has the advantage over using housekeeping genes as control since the latter requires different primers and amplification conditions. The current method also has advantage over using PCR MIMIC which may have different amplification efficiency due to the different gene fragment used. The homologous standard was constructed from the same target gene with a slightly shorter length to allow for visualization and analysis following competition with the target gene. The mock experiment showed that the method worked successfully. Using cDNA prepared from activated monocytes we were also able to show that it is useful for determining changes in the levels of PPARy expression in cells expressing the transcription factor

The Modulation of PPARγ1 and PPARγ2 mRNA Expression by Ciglitazone in CD3/CD28-Activated Naïve and Memory CD4+ T Cells

Given their roles in immune regulation, the expression of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) 1 and 2 isoforms was investigated in human naïve (CD45RA+) and memory (CD45RO+) CD4+ T cells. Stimulation of both types of cells via the CD3/CD28 pathway resulted in high expression of both PPARγ receptors as measured by real-time PCR. Treatment with the PPARγ agonist, ciglitazone, increased PPARγ1 expression but decreased PPARγ2 expression in stimulated naïve and memory cells. Furthermore, when present, the magnitude of both PPARγ receptors expression was lower in naïve cells, perhaps suggesting a lower regulatory control of these cells. Similar profiles of selected proinflammatory cytokines were expressed by the two cell types following stimulation. The induction of PPARγ1 and suppression of PPARγ2 expressions in naïve and memory CD4+ T cells in the presence of ciglitazone suggest that the PPARγ subtypes may have different roles in the regulation of T-cell function

Ciglitazone inhibits growth and induces apoptosis in human colorectal cancer cell lines independently of PPARγ

The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) γ is a ligand-activated transcription factor that regulates several biological processes such as adipogenesis, glucose homeostasis and cell growth

Compatibility of Porous Chitosan Scaffold with the Attachment and Proliferation of human Adipose-Derived Stem Cells In Vitro

Adipose-derived stem cells (ASCs) have potential applications in the repair and regeneration of various tissues and organs. The use of various scaffold materials as an excellent template for mimicking the extracellular matrix to induce the attachment and proliferation of different cell types has always been of interest in the field of tissue engineering because ideal biomaterials are in great demand. Chitosan, a marine polysaccharide, have wide clinical applications and it acts as a promising scaffold for cell migration and proliferation. ASCs, with their multi-differentiation potential, and chitosan, with its great biocompatibility with ASCs, were investigated in the present study. ASCs were isolated and were characterized by two different methods: immunocytochemistry and flow cytometry, using the mesenchymal stem cell markers CD90, CD105, CD73 and CD29. The ASCs were then induced to differentiate into adipogenic, osteogenic and chondrogenic lineages. These ASCs were incorporated into a porous chitosan scaffold (PCS), and their structural morphology was studied using a scanning electron microscope and hematoxylin and eosin staining. The proliferation rate of the ASCs on the PCS was assessed using a PrestoBlue viability assay. The results indicated that the PCS provides an excellent template for the adhesion and proliferation of ASCs. Thus, this study revealed that PCS is a promising biomaterial for inducing the proliferation of ASCs, which could lead to successful tissue reconstruction in the field of tissue engineering

The role of REST and HDAC2 in epigenetic dysregulation of Nav1.5 and nNav1.5 expression in breast cancer

Background: Increased expression of voltage-gated sodium channels (VGSCs) have been implicated with strong metastatic potential of human breast cancer in vitro and in vivo where the main culprits are cardiac isoform Nav1.5 and its ‘neonatal’ splice variant, nNav1.5. Several factors have been associated with Nav1.5 and nNav1.5 gain of expression in breast cancer mainly hormones, and growth factors. Aim: This study aimed to investigate the role of epigenetics via transcription repressor, repressor element silencing transcription factor (REST) and histone deacetylases (HDACs) in enhancing Nav1.5 and nNav1.5 expression in human breast cancer by assessing the effect of HDAC inhibitor, trichostatin A (TSA). Methods: The less aggressive human breast cancer cell line, MCF-7 cells which lack Nav1.5 and nNav1.5 expression was treated with TSA at a concentration range 10–10,000 ng/ml for 24 h whilst the aggressive MDA-MB-231 cells was used as control. The effect of TSA on Nav1.5, nNav1.5, REST, HDAC1, HDAC2, HDAC3, MMP2 and N-cadherin gene expression level was analysed by real-time PCR. Cell growth (MTT assay) and metastatic behaviors (lateral motility and migration assays) were also measured. Results: mRNA expression level of Nav1.5 and nNav1.5 were initially very low in MCF-7 compared to MDA-MB-231 cells. Inversely, mRNA expression level of REST, HDAC1, HDAC2, and HDAC3 were all greater in MCF-7 compared to MDA-MB-231 cells. Treatment with TSA significantly increased the mRNA expression level of Nav1.5 and nNav1.5 in MCF-7 cells. On the contrary, TSA significantly reduced the mRNA expression level of REST and HDAC2 in this cell line. Remarkably, despite cell growth inhibition by TSA, motility and migration of MCF-7 cells were enhanced after TSA treatment, confirmed with the up-regulation of metastatic markers, MMP2 and N-cadherin. Conclusions: This study identified epigenetics as another factor that regulate the expression level of Nav1.5 and nNav1.5 in breast cancer where REST and HDAC2 play important role as epigenetic regulators that when lacking enhances the expression of Nav1.5 and nNav1.5 thus promotes motility and migration of breast cancer. Elucidation of the regulatory mechanisms for gain of Nav1.5 and nNav1.5 expression may be helpful for seeking effective strategies for the management of metastatic diseases

Expression of P-selectin, TXA2, TGF-β1 and PDGF-AB in the Presence of Bioadhesive Chitosan Derivatives

Chitosan is a natural macromolecular polysaccharide, derived from chitin extracted from the exoskeleton of some marine invertebrates. This study was designed to evaluate the expression of P-selectin, thromboxane A2 (TXA2), transforming growth factor-β1 (TGF- β1) and platelet derived growth factor-AB (PDGF-AB) using enzyme-linked immunosorbent assays (ELISAs). The chitosan derivatives utilized were 7% N,Ocarboxymethylchitosan (NO-CMC) (with 0.45 mL collagen), 8% NO-CMC, oligo - chitosan (O-C) and oligo -chitosan 53 (O-C 53). We found that O-C showed a significantly superior effect compared to NO-CMC in achieving hemostasis. Post-hoc multiple comparisons using Scheffe test indicated that p-values between the mean differences of all the assayed chitosan derivatives were not significant. However, our analyses successfully identified distinctions between the O-C and NO-CMC test groups and blood alone. These results may expand the understanding of the ability of chitosan derivatives to promote inflammatory responses and angiogenesis depending on their physical and chemical structure

Eksperimental Variasi Kecepatan Putar Screw Feeding dengan Kecepatan Putar PIsau Pengupas terhadap Kualitas Hasil Pengupasan pada Mesin Pengupas Kulit Pinang

The processing of areca nut at the present time is still done manually and requires a long working time. To facilitate the skinner process and optimize the results that required a technology of machine which paring the areca nut skin. The Skinner machine of areca nut at this point still has deficiency. For resolving the problems that exist on the areca nut skinner machine which has type of screw then troubleshoot by varying the rotational speed of the screw feeding toward skinner tool of areca nut. After tested with variations of the rotational speed of the screw feeding to ward skinner tool of areca nut, then obtained the optimal results as much as 6 pieces and other 4 pieces of areca nut already broken on screw feeding speed at 37 rpm and 800 rpm on a skinner tool. The time that required to perform the paring process on this rotational speed is 21.7 seconds. The optimal skinner Results of areca nut increases to 9 pieces after made the casing modification which the efficiency of time is 73.5

Report on von Willebrand Disease in Malaysia

BACKGROUND: Von Willebrand disease (vWD) is an inherited hemostatic disorder that affects the hemostasis pathway. The worldwide prevalence of vWD is estimated to be 1% of the general population but only 0.002% in Malaysia.AIM: Our present paper has been written to disclose the statistical counts on the number of vWD cases reported from 2011 to 2013.MATERIAL AND METHODS: This article is based on sociodemographic data, diagnoses and laboratory findings of vWD in Malaysia. A total of 92 patients were reported to have vWD in Malaysia from 2011 to 2013.RESULTS: Sociodemographic-analysis revealed that 60% were females, 63% were of the Malay ethnicity, 41.3% were in the 19-44 year old age group and 15.2% were from Sabah, with the East region having the highest registered number of vWD cases. In Malaysia, most patients are predominately affected by vWD type 1 (77.2%). Factor 8, von Willebrand factor: Antigen and vWF: Collagen-Binding was the strongest determinants in the laboratory profiles of vWD.CONCLUSION: This report has been done with great interest to provide an immense contribution from Malaysia, by revealing the statistical counts on vWD from 2011-2013