Orexin-A exerts equivocal role in atherosclerosis process depending on the duration of exposure : in vitro study

Orexin-A is a peptide hormone that plays a crucial role in feeding regulation and energy homeostasis. Diurnal intermittent fasting (DIF) has been found to increase orexin-A plasma levels during fasting hours, while Ramadan fasting which resembles DIF, has led to beneficial effects on endothelial function. Herein, we aimed to investigate the effects of orexin-A on the expression of molecules involved in the atherogenesis process: Monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase-1 and 2 (TIMP-1 and TIMP-2), in human aortic endothelial cells (HAECs). HAECs were incubated with orexin-A at concentrations of 40 ng/mL, 200 ng/mL and 400 ng/mL for 6, 12 and 24 h. The mRNA levels of MCP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 and orexin-1 receptor were measured by real-time qPCR. We also evaluated the MMP-2, p38, phospho-p38, NF-κΒ/p65 as well as TIMP-1 protein levels by Western blot and ELISA, respectively. MMP-2 activity was measured by gelatin zymography. Short-term 6-h incubation of HAECs with orexin-A at a high concentration (400 ng/mL) decreased MCP-1, MMP-2 expression, MMP-2/TIMP-1 ratio (p < 0.05), and MMP-2 activity, while incubation for 24 h increased MCP-1, MMP-2 expression (p < 0.05), MMP-2/TIMP-1 and MMP-2/TIMP-2 ratio (p < 0.01 and p < 0.05, respectively) as well as MMP-2 activity. The dual effects of orexin-A are mediated, at least in part, via regulation of p38 and NF-κΒ pathway. Orexin-A may have an equivocal role in atherosclerosis process with its effects depending on the duration of exposure

Systemic hypertension augments, whereas insulin-dependent diabetes down-regulates, endothelin A receptor expression in the mammary artery in coronary artery disease patients

Background: Endothelin (ET) A receptor antagonism causes decreased vasodilation in hypertensive coronary arteries and decreased effects on coronary artery compliance in diabetic patients. Methods: We investigate the mRNA expression of ET-1, ETA and ETB receptors, using real time RT-PCR, in biopsies from the internal mammary artery obtained from 49 patients, 18 diabetics and 34 hypertensives, all undergoing coronary artery bypass grafting. Results: Hypertensive patients had higher ET-1 mRNA expression (16438 [8417, 23917]), than normotensive patients (2974 [2283, 18055], p=0.008). Diabetic patients had significantly lower ETA receptor levels than non-diabetic patients (455 [167, 1496] vs. 1660 [700, 3190], respectively, p = 0.003). Conclusions: Multivariate analysis demonstrated that the presence of systemic hypertension was the only independent predictor of log ETA receptor expression and log ET-1 expression, while insulin-dependent diabetes was negatively correlated with ETA receptor expression. ETB receptor expression was not correlated with any predictor. Systemic hypertension is associated with increased ET-1 and ETA receptor mRNA expression, whereas insulin-dependent diabetes down-regulates ETA receptor mRNA expression in the internal mammary artery in patients with coronary artery disease undergoing bypass grafting

Quantitative RT-PCR luminometric hybridization assay with an RNA-internal standard for cytokeratin-19 mRNA in peripheral blood of patients with breast cancer. Clin Biochem

6 normal PBMC and is highly specific as none of the 26 healthy controls tested had detectable CK-19 mRNA levels, while 10 out of 14 (71.4%) and 9 out of 37 (24.3%) patients with stage IV and stage I/II breast cancer, respectively, were tested positive. Conclusion: The developed quantitative RT-PCR hybridization assay for CK-19 is reproducible, highly sensitive and specific, and can be used for a large-scale prospective evaluation of clinical samples

Effect of antineoplastic agents on the expression of human telomerase reverse transcriptase b plus transcript in MCF-7 cells

Abstract Objectives: To evaluate the effect of antineoplastic agents on the expression of human telomerase reverse transcriptase (hTERT) splice variants in MCF-7 cells. Design and methods: We have developed a luminometric hybridization assay for hTERT beta plus transcript. MCF-7 cells were isolated before and after treatment with antineoplastic agents. A combination of nested RT-PCR and the developed luminometric hybridization assay was used for the specific detection of hTERT beta plus transcript in treated and untreated MCF-7 cells. Amplification of all hTERT splicing variants by nested PCR in the same samples was also performed. Results: MCF-7 cells treated with taxol and etoposide were found positive for all hTERT splicing variants, while the expression of hTERT beta plus transcript did not differ significantly before and after exposure. MCF-7 cells treated with doxorubicin and 5-fluorouracil did not express any of hTERT splicing variants. In the presence of cisplatin, three splicing variants of hTERT were detected. Conclusions: The developed hybridization assay is highly sensitive and specific for the detection of hTERT beta plus transcript in clinical samples

A Sandwich Electrochemical Immunosensor Using Magnetic DNA Nanoprobes for Carcinoembryonic Antigen

A novel magnetic nanoparticle-based electrochemical immunoassay of carcinoembryonic antigen (CEA) was designed as a model using CEA antibody-functionalized magnetic beads [DNA/Fe3O4/ZrO2; Fe3O4 (core)/ZrO2 (shell) nano particles (ZMPs)] as immunosensing probes. To design the immunoassay, the CEA antibody and O-phenylenediamine (OPD) were initially immobilized on a chitosan/nano gold composite membrane on a glassy carbon electrode (GCE/CS-nano Au), which was used for CEA recognition. Then, horseradish peroxidase (HRP)-labeled anti-CEA antibodies (HRP-CEA Ab2) were bound to the surface of the synthesized magnetic ZMP nanoparticles as signal tag. Thus, the sandwich-type immune complex could be formed between secondary antibody (Ab2) modified DNA/ZMPs nanochains tagged by HRP and GCE/CS-nano Au. Unlike conventional nanoparticle-based electrochemical immunoassays, the recognition elements of this immunoassay included both electron mediators and enzyme labels, which obviously simplifies the electrochemical measurement process. The sandwich-type immunoassay format was used for online formation of the immunocomplex of CEA captured in the detection cell with an external magnet. The electrochemical signals derived from HRP during the reduction of H2O2 with OPD as electron mediator were measured. The method displayed a high sensitivity for CEA detection in the range of 0.008–200 ng/mL, with a detection limit of 5 pg/mL (estimated at a signal-to-noise ratio of 3). The precision, reproducibility, and stability of the immunoassay were good. The use of the assay was evaluated with clinical serum samples, and the results were in excellent accordance with those obtained using the standard enzyme-linked immunosorbent assay (ELISA) method. Thus, the magnetic nanoparticle-based assay format is a promising approach for clinical applications, and it could be further developed for the detection of other biomarkers in cancer diagnosis

Markers for the identification of late breast cancer recurrence

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