Sequential transformation of 4-androstenedione into dihydrotestosterone in prostate carcinoma (DU-145) cells indicates that 4-androstenedione and not testosterone is the substrate of 5α-reductase

2010 ◽  
Vol 1 (2) ◽  
Author(s):  
Melanie Samson ◽  
Fernand Labrie ◽  
Van Luu-The
Keyword(s):  
Real Time ◽  
Prostate Carcinoma ◽  
Real Time Pcr ◽  
Profile Analysis ◽  
Reductase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Pcr Analysis ◽  
No Production ◽  
Reduction Step ◽  
Type 3

Abstract: Although it is well recognized that 5α-reductases possess higher affinity for 4-androstenedione than testosterone, and the affinity of 4-androstenedione is higher for 5α-reductases than 17β-hydroxysteroid dehydrogenases, it is generally believed that dihydrotestosterone is necessarily produced by the transformation of testosterone into dihydrotestosterone, suggesting that the step catalyzed by 17β-hydroxysteroid dehydrogenase precedes the step catalyzed by 5α-reductase. This interpretation is in contradiction with the enzymatic kinetic law that suggests that the 5α-reduction step that catalyzes the transformation of 4-dione into 5α-androstane-3,17-dione precedes the 17keto-reduction step.: To verify which of these two pathways is operative, we quantified mRNA expression levels of steroidogenic enzymes in prostate carcinoma DU-145 cells by real-time PCR and determined the metabolites produced after incubation with [: Real-time PCR analysis strongly suggests that the new type 3 5α-reductase is responsible for 5α-reductase activity in DU-145 cells. Steroid profile analysis shows that in the absence of inhibitor 5α-androstanedione is first produced, followed by the production of androsterone and dihydrotestosterone. The concentration of testosterone was not detectable. In the presence of Finasteride, an inhibitor of 5α-reductase, there was no transformation of 4-androstenedione and also there was no production of testosterone. The present data clearly indicate that the biosynthesis of dihydrotestosterone in DU-145 cells does not require testosterone as intermediate, and the step catalyzed by 5α-reductase precedes the step catalyzed by 17β-hydroxysteroid dehydrogenase.

Autophagy modulation altered differentiation capacity of CD146+ cells toward endothelial cells, pericytes, and cardiomyocytes

2020 ◽  
Author(s):  
Mehdi Hassanpour ◽  
Jafar Rezaie ◽  
Masoud Darabi ◽  
Amirataollah Hiradfar ◽  
Reza Rahbarghazi ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Real Time ◽  
Western Blotting ◽  
Real Time Pcr ◽  
Unsaturated Fatty Acids ◽  
Profile Analysis ◽  
Cardiac Regeneration ◽  
Pcr Analysis ◽  
Pcr Assay ◽  
Protein Levels

Abstract Background: To date, many attempts are employed to increase the regenerative potential of stem cells. In this study, we evaluated the hypothesis whether an autophagy modulation could induce/reduce CD146+ cells differentiation into mature pericyte, endothelial and cardiomyocyte lineage. Methods In this study, CD146+ cells were enriched from human bone marrow aspirates and trans-differentiated into mature EC, PC and CM after exposure to autophagy stimulator (50 µM Met)/inhibitor (15 µM HCQ). The protein levels of autophagy proteins were monitored by western blotting. NO content was measured using the Griess assay. Using real-time PCR assay and western blotting, we monitored the lineage protein and gene levels. The fatty acid change was determined by gas chromatography. Pro-inflammatory cytokine and angiocrine factors were measured by ELISA. The exosome secretion capacity was assessed by AChE activity and real-time PCR assay. Result Data revealed the modulation of autophagy factors, Beclin-1, P62 and LC3 II/I ratio in differentiating CD146+ cells after exposure to Met and HCQ (p<0.05). The inhibition of autophagy increased released NO content and decreased intracellular NO content compared to the Met-treated cells (p<0.05). Real-time PCR analysis showed that the treatment of CD146+ cells with autophagy modulators altered the expression of VE-cadherin, cTnI and α-SMA. Our data demonstrated that the stimulation of autophagy signaling in CD146+ cells with Met increased the expression of VE-cadherin, α-SMA, and cTnI compared to HCQ-treated cells (p<0.05) while western blotting revealed the protein synthesis of all lineage-specific proteins in under the stimulation and inhibition of autophagy. Fatty acid profile analysis revealed the increase of unsaturated fatty acids after exposure to HCQ (p<0.05). None statistically significant differences were found in the levels of Tie-1, Tie-2, VEGFR-1 and VEGFR-2 after autophagy modulation. The treatment of cells with HCQ increased the levels of TNF-α and IL-6 compared to the Met-treated cells. Data revealed the increase of exosome biogenesis and secretion to the supernatant in cells treated with HCQ compared to the Met groups (p<0.05).ConclusionsIn summary, autophagy modulation could be altered differentiation potency of CD146+ cells and could be novel and applicable cardiac cell therapy in the cardiac regeneration field.

ID: 316 Real-time PCR analysis of BCL2L12, a novel member of BCL2 family of the apoptosis-related genes, in human leukemia.

2006 ◽  
Vol 4 (s1) ◽  
pp. 82-82
Author(s):  
K. Floros ◽  
H. Thomadaki ◽  
S. Pavlovic ◽  
M. Talieri ◽  
M. Colovic ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Analysis ◽  
Human Leukemia ◽  
Bcl2 Family

Improved diagnosis of gastrointestinal infections using a semi-automated multiplex real-time PCR for detection of enteropathogens

2021 ◽  
Vol 70 (9) ◽  
Author(s):  
Berta Fidalgo ◽  
Elisa Rubio ◽  
Victor Pastor ◽  
Marta Parera ◽  
Clara Ballesté-Delpierre ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Enteric Pathogens ◽  
Pcr Analysis ◽  
Culture Methods ◽  
Gastrointestinal Infections ◽  
Content Type ◽  
Link Type ◽  
Microbiological Culture ◽  
Micro Organisms

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis. Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination. Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures. Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013–2018) included stool culture, microscopy and immunochromatography. Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013–2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis. Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.

scholarly journals Real-time PCR analysis of the apoptosis related genes in ATRA treated APL t(15;17) patients

2003 ◽  
Vol 35 (5) ◽  
pp. 454-459 ◽  
Author(s):  
Hakan Savli ◽  
Sema Sirma ◽  
Balint Nagy ◽  
Melih Aktan ◽  
Guncag Dincol ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Analysis

scholarly journals Touchable 3D hierarchically structured polyaniline nanoweb for capture and detection of pathogenic bacteria

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Kyung Hoon Kim ◽  
MinHo Yang ◽  
Younseong Song ◽  
Chi Hyun Kim ◽  
Young Mee Jung ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Bacterial Pathogens ◽  
Physical Contact ◽  
Structural Deformation ◽  
Pcr Analysis ◽  
Mechanical Resistance ◽  
Fluorescence Signal ◽  
The Real

AbstractA bacteria-capturing platform is a critical function of accurate, quantitative, and sensitive identification of bacterial pathogens for potential usage in the detection of foodborne diseases. Despite the development of various nanostructures and their surface chemical modification strategies, relative to the principal physical contact propagation of bacterial infections, mechanically robust and nanostructured platforms that are available to capture bacteria remain a significant problem. Here, a three-dimensional (3D) hierarchically structured polyaniline nanoweb film is developed for the efficient capture of bacterial pathogens by hand-touching. This unique nanostructure ensures sufficient mechanical resistance when exposed to compression and shear forces and facilitates the 3D interfacial interactions between bacterial extracellular organelles and polyaniline surfaces. The bacterial pathogens (Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus) are efficiently captured through finger-touching, as verified by the polymerase chain reaction (PCR) analysis. Moreover, the real-time PCR results of finger-touched cells on a 3D nanoweb film show a highly sensitive detection of bacteria, which is similar to those of the real-time PCR using cultured cells without the capturing step without any interfering of fluorescence signal and structural deformation during thermal cycling. Graphic Abstract

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