scholarly journals Cloning and Expression of kiss2 in the Zebrafish and Medaka

Endocrinology ◽  
2009 ◽  
Vol 150 (2) ◽  
pp. 821-831 ◽  
Author(s):  
Takashi Kitahashi ◽  
Satoshi Ogawa ◽  
Ishwar S. Parhar
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Mature Female ◽  
Cloning And Expression ◽  
Hypothalamic Nucleus ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
The Novel ◽  
Female Zebrafish

Newly discovered kisspeptin (metastin), encoded by the Kiss1/KISS1 gene, is considered as a major gatekeeper of puberty through the regulation of GnRH. In the present study, we cloned a novel kisspeptin gene (kiss2) in the zebrafish Danio rerio and the medaka Oryzias latipes, which encodes a sequence of 125 and 115 amino acids, respectively, and its core sequence (FNLNPFGLRF, F-F form) is different from the previously characterized kiss1 (YNLNSFGLRY, Y-Y form). Our in silico data mining shows kiss1 and kiss2 are highly conserved across nonmammalian vertebrate species, and we have identified two putative kisspeptins in the platypus and three forms in Xenopus. In the brain of zebrafish and medaka, in situ hybridization and laser capture microdissection coupled with real-time PCR showed kiss1 mRNA expression in the ventromedial habenula and the periventricular hypothalamic nucleus. The kiss2 mRNA expression was observed in the posterior tuberal nucleus and the periventricular hypothalamic nucleus. Quantitative real-time PCR analysis during zebrafish development showed a significant increase in zebrafish kiss1, kiss2 (P < 0.002), gnrh2, and gnrh3 (P < 0.001) mRNA levels at the start of the pubertal phase and remained high in adulthood. In sexually mature female zebrafish, Kiss2 but not Kiss1 administration significantly increased FSH-β (2.7-fold, P < 0.05) and LH-β (8-fold, P < 0.01) mRNA levels in the pituitary. These results suggest that the habenular Kiss1 and the hypothalamic Kiss2 are potential regulators of reproduction including puberty and that Kiss2 is the predominant regulator of gonadotropin synthesis in fish. Habenular kisspeptin-1 (Kiss1) and the novel hypothalamic Kiss2 are potential regulators of puberty. Kiss2 is the predominant regulator of gonadotropin synthesis in fish.

scholarly journals Effect of dietary α-lipoic acid on the mRNA expression of genes involved in drug metabolism and antioxidation system in rat liver

2014 ◽  
Vol 112 (3) ◽  
pp. 295-308 ◽  
Author(s):  
Takashi Ide
Keyword(s):  
Drug Metabolism ◽  
Mrna Expression ◽  
Real Time ◽  
Dna Microarray ◽  
Lipoic Acid ◽  
Real Time Pcr ◽  
Glutathione Transferase ◽  
Redox System ◽  
Pcr Analysis ◽  
Mrna Levels

In the present study, the mRNA levels of hepatic proteins involved in the drug metabolism of rats fed α-lipoic acid were evaluated by DNA microarray and real-time PCR analyses. Experimental diets containing 0, 0·1, 0·25 and 0·5 % (w/w) α-lipoic acid were fed to four groups of rats consisting of seven animals each for 21 d. DNA microarray analysis revealed that the diet containing 0·5 % α-lipoic acid significantly (P< 0·05) increased the mRNA levels of various phase I drug-metabolising enzymes up to 15-fold and phase II enzymes up to 52-fold in an isoenzyme-specific manner. α-Lipoic acid also up-regulated the mRNA levels of some members of the ATP-binding cassette transporter superfamily, presumed to be involved in the exportation of xenobiotics, up to 6·6-fold. In addition, we observed that α-lipoic acid increased the mRNA levels of many proteins involved in antioxidation, such as members of the thiol redox system (up to 5·5-fold), metallothioneins (up to 12-fold) and haeme oxygenase 1 (1·5-fold). These results were confirmed using real-time PCR analysis, and α-lipoic acid dose dependently increased the mRNA levels of various proteins involved in drug metabolism and antioxidation. Consistent with these observations, α-lipoic acid dose dependently increased the hepatic concentration of glutathione and the activities of glutathione reductase and glutathione transferase measured using 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates, but decreased the hepatic and serum concentrations of malondialdehyde. In conclusion, the present study unequivocally demonstrated that α-lipoic acid increases the mRNA expression of proteins involved in drug metabolism and antioxidation in the liver.

Investigation of the effect of Prunus Amygdalus Amara on the expression of some genes of apoptosis and immortality in breast cancer cells (MCF-7)

2021 ◽  
Vol 13 ◽  
Author(s):  
Maryam Abdolahi-Majd ◽  
Gholamhossein Hassanshahi ◽  
Mahboubeh Vatanparast ◽  
Mojgan Noroozi Karimabad
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Prunus Amygdalus ◽  
Mcf7 Cells ◽  
Significant Difference ◽  
Anti Cancer ◽  
Bitter Almond ◽  
Mcf 7

Background: Anti-cancer effects of almond nuts or oil have been approved, but there are a few pieces of research that have evaluated, in detail, almond and other seeds' effects on cancer. Therefore, in the present project, the aim was to explore the regulatory effect of the bitter almond extract (Prunus amygdalus Batsch) on the apoptotic and anti-cancer potency of MCF-7 cells. Objectives: In the current experimental research, the Almond effect on MCF7 cells was evaluated by investigating the expression and the balance between Bcl-2, Bax genes to unmark the potential molecular mechanism. Methods: For 24 and 48h, the MCF7 cells were treated with the bitter almond extract (187.5-3000 µg/mL). MTT assay was used to assess the viability, and Real-time-PCR was applied to determine the expression of Bax and Bcl-2, facing β-actin. Results: Our results revealed a significant difference between different extract concentrations on the viability of MCF7 cell lines in 24 and 48 h; cell viability decreased time-dependently (P < 0.05). After 24 and 48h of extract facing MCF7 cells, the evaluated IC50 value was 3000 and 1500 µg/mL, respectively. Based on Real Time-PCR analysis, after 24 and 48 h, the mRNA levels of BCL-2 decreased by the extract, whereas BAX was in the MCF-7 cell line. Conclusion: From the results, it can be concluded that bitter almond extract has anti-cancer properties that may influence the apoptotic pathways by regulating relative gene expression.

p29ING4 Regulates the Production of Pro-Angiogenic Molecules by Human Myeloma Cells in Normoxic and Hypoxic Conditions Being Involved in Myeloma-Induced Angiogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 515-515
Author(s):  
Sara Tagliaferri ◽  
Francesca Morandi ◽  
Paolo Lunghi ◽  
Simona Colla ◽  
Mirca Lazzaretti ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Mrna Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Interleukin 8 ◽  
Mrna Levels ◽  
Quantitative Real Time Pcr ◽  
Angiogenic Switch ◽  
Hypoxic Conditions

Abstract Multiple myeloma (MM) cells produce several angiogenic molecules as VEGF, Angiopoietin-1 (Ang-1), interleukin-8 (IL-8) and osteopontin (OPN), however the molecular mechanisms underlying the angiogenic switch are not completely elucidated. The candidate tumor suppressor gene inhibitor of growth family member 4 (p29ING4) has been recently implicated in solid tumors as a repressor of angiogenesis and tumor growth through the suppression of angiogenic related molecules including interleukin-8 (IL-8) and the hypoxia inducible factor (HIF)-1 alpha. In this study we investigate the potential involvement of p29ING4 in the angiogenic switch in MM. First using quantitative real time PCR we compared p29ING4 with VEGF, Ang-1, IL-8 and OPN mRNA levels in eight human myeloma cell lines (HMCLs). A significantly negative correlation was observed between ING4 and IL-8 and a trend of correlation with OPN. Following we transfected HMCLs JJN3, OPM-2 and RPMI-8226 with specific siRNA to completely block the expression of p29ING4 checking the effect on the expression and production of the myeloma-related angiogenic molecules VEGF, Ang-1, IL-8 and OPN by quantitative real time PCR and ELISA assay. p29ING4 suppression in HMCLs did not affect VEGF and Ang-1 production but induced a strong up-regulation of IL-8 mRNA and IL-8 protein secretion. Similarly an induction of OPN mRNA expression as well as OPN secretion was induced by siRNA anti-p29ING4. Moreover conditioned media of HMCLs transfected with siRNA anti p29ING4 significantly increased vessel formation in an experimental in vitro model of angiogenesis (ANGIO kit) as compared to controls. Further we investigate the role of p29ING4 in the production of angiogenic molecule by MM cells in hypoxic condition compared to normoxic one as well as its potential relationship with HIF-1alpha system. Hypoxia induced HIF-1alpha expression at nuclear level and its activity in HMCLs and p29ING4 suppression by siRNA further induced HIF-1alpha transcriptional activity with an increase of its target gene Nip-3 in HMCLs. In turn the block of HIF1-alpha by specific siRNA up-regulated p29ING4 and suppressed IL-8 and OPN mRNA expression by HMCLs suggesting a relationship between p29ING4 and HIF-1alpha activity. These in vitro observations have been extended in vivo by the finding of a significant correlation between bone marrow (BM) plasma IL-8 levels and p29ING4 mRNA expression in purified MM cells obtained from 40 newly diagnosed MM patients (R=−0.58 Spearman’s 2-tailed test: p=0.04), consistently MM patients with higher BM IL-8 levels have a significantly lower p29ING4 mRNA levels. Similarly MM patients positive for OPN expression with high OPN BM levels had a significant lower p29ING4 levels (p=0.02). Finally we found that MM patients with high microvalscular density (MVD&gt;30) have significant lower p29ING4 levels as compared to those with low MVD (&lt;30) (p=0.04) and that MM patients with histological high grade had significant lower p29ING4 mRNA levels (Mann-Whitney 2-tailed: p=0.05). In conclusion, our data indicate that the tumor suppressor p29ING4 regulate the production of angiogenic molecules by MM cells both in normoxic and hypoxic conditions being involved in MM-induced angiogenesis and potentially in tumor progression.

Elevated mRNA Expression of FGF2 and TGFB1 Genes in Myelofibrosis and Essential Thrombocythemia Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5179-5179
Author(s):  
Daniela Prudente Teixeira Nunes ◽  
Luciene Terezina Lima ◽  
Guilherme Wataru Gomes ◽  
Maria de Lourdes L. F. Chauffaille ◽  
Maria Regina Regis Silva ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Essential Thrombocythemia ◽  
Real Time Pcr ◽  
Sao Paulo ◽  
World Health ◽  
Mrna Levels ◽  
São Paulo ◽  
Mutation Status ◽  
Allele Burden

Abstract Background: Myelofibrosis (MF) and essential thrombocythemia (ET) are clonal hematopoietic stem cell malignancies classified as myeloproliferative neoplasms (MPNs). FGF2 and TGF-β were associated with pathophysiology and progression of these diseases. Although FGF2 and TGF-β roles in angiogenesis and fibrosis are known, their mRNA expression in leukocytes of MPNs patients was not yet evaluated. Furthermore, the detailed mechanism of TGF-β signaling in the pathophysiology of these diseases remains unclear. In regular signaling process, Smads are a group of proteins critical for transmitting signals from TGF-β superfamily of the cell surface to the nucleus. Thus, Smads signaling plays an important role in the regulation of hematopoiesis and it could modulate TGF-β action in MPNs. Aims: The aim of this study was to investigate if there are differences in mRNA expression of FGF2, TGFB1 and SMADS 1 to 7 in MPNs patients and healthy controls and if it is related to JAK2 mutation status and allele burden. Methods: Fifteen primary myelofibrosis (PMF), 15 myelofibrosis post essential thrombocytemia (MPET), and 22 essential trombocythemia (ET) patients diagnosed according to the World Health Organization criteria (2008) were selected from the Hematology Department of the Federal University of de Sao Paulo and from the Hematology Division of the Catholic University of Sao Paulo. Other group with healthy subjects was also included. The control group was matched by age and gender with MPNs patients: 30 for PMF, 17 for MPET, and 34 for ET. Expressions of FGF2, TGFB1 and SMADs 1 to 7 mRNA were evaluated in total leukocytes by Real Time PCR, using Taqman assays. JAK2V617F mutation status and allele burden and MPL W515K/L analysis were performed in total leukocytes DNA by Real Time PCR, using Taqman MGB probes. Results: No difference was found between frequencies of JAK2V617F mutated subjects (P=0.520) and in allele burden percentage (P=0.415) in MPNs groups. Allele burden was also not associated with mRNA levels of studied genes in each group of MPNs, and none of the patients presented MPL W515K/L mutations. MF and ET patients had higher TGFB1 and FGF2 mRNA levels than its controls (P<0.05), while MPET patients had higher FGF2 levels than controls (P< 0.001). Both MPET and ET patients had higher SMAD6 expression than its controls (P<0.05), whereas ET patients also presented higher SMAD1 levels than controls (P=0.01). The SMADs expressions were similar in MF and their controls. Conclusions: Our findings suggest that FGF mRNA is overexpressed in leukocytes of MF, MPET and ET patients. It seems that higher TGFB1 mRNA levels do not affect the molecular signaling by SMADs in MF patients, although SMAD6 and SMAD1 mRNA levels in ET and MPET patients might be upregulated. Financing: FAPESP 2012/ 12957-5 Disclosures No relevant conflicts of interest to declare.

scholarly journals Real-Time PCR and Immunocytochemical Study of Chondroitin Sulfate Proteoglycans after Scratch Wounding in Cultured Astrocytes / PCR I IMUNOCITOHEMIJSKA STUDIJA EKSPRESIJE HONDROITIN-SULFATNIH PROTEOGLIKANA NAKON POVREDE ASTROCITA U KULTURI

2013 ◽  
Vol 32 (4) ◽  
pp. 398-405
Author(s):  
Ana Parabucki ◽  
Anja Santrač ◽  
Danijela Savić ◽  
Sanja Dacić ◽  
Ivana Bjelobaba ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Complex Disease ◽  
In Vitro Models ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Cultured Astrocytes ◽  
Scratch Wound

Summary Background: Various in vivo and in vitro models have been described in order to elucidate the pathobiology underlying the traumatic brain injury (TBI) and test potentially suitable treatments. Since TBI is a complex disease, models differ in regard to the aspect of TBI that is being investigated. One of the used in vitro models is the scratch wound assay, first established as a reproducible, low-cost assay for the analysis of cell migration in vitro. The aim of the present study was to further investigate the relevancy of this model as a counter- part of in vivo TBI models. Methods: We have examined the astrocytic response to a mechanical injury in terms of expression of chondroitin sul- fate proteoglycans (CSPGs) - phosphacan, neurocan and brevican, using real-time PCR and immunocytochemistry. Results: Our results indicate that in vitro scratch wounding alters the expression profile of examined CSPGs. Four hours after the scratch injury of the astrocytic monolayer, real-time PCR analysis revealed upregulation of mRNA levels for phos- phacan (3-fold) and neurocan (2-fold), whereas brevican mRNA was downregulated (2-fold). Immunofluorescent sig- nal for phosphacan and neurocan was more intense in astro- cytes close to the injury site, while brevican was scarcely present in cultured astrocytes. Conclusions: Obtained results indicate that CSPGs are differ- entially expressed by astrocytes after scratch wounding, demonstrating that the scratch wound model might be suit- able for investigation of astrocyte-derived response to injury.

scholarly journals The Role of Endocannabinoid System Based on mRNA Expression During the Late Luteal Phase and Estrus in the Bovine Endometrium

2019 ◽  
Vol 19 (4) ◽  
pp. 979-989
Author(s):  
Essa Dirandeh ◽  
Zarbakht Ansari-Pirsaraei ◽  
Hamid Deldar
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Estrous Cycle ◽  
Real Time Pcr ◽  
Luteal Phase ◽  
Fatty Acid Amide Hydrolase ◽  
Acid Amide ◽  
Endocannabinoid System ◽  
Pcr Analysis ◽  
Late Luteal Phase

AbstractThere are several findings indicating that the endocannabinoid system (ECS) is an important factor, acting in multiple ways in regulating reproductive function but changes of this system in the bovine endometrium have rarely been investigated; therefore, this study was designed to consider an association between endometrial ECS expression and different stages of the estrous cycles. MRNA expressions of the ECS were investigated during the late luteal phase and estrus using real-time PCR. Following estrous synchronization of sixteen Holstein dairy cows (34±1.3 kg/day of milk production), using two PGF2α injections given 14 days apart, at 30 and 44 days in milk (DIM), blood samples and ultrasonography (US) were performed every other day from the day of second PGF2α injection (44 DIM) until the start of the next estrous cycle (67±2 DIM) to verify CL development and ovulation. Based on blood and US results endometrial tissue was collected on days 16 (late luteal phase) and 21 (estrus) of the synchronized estrous cycle (ovulation = d 0). Real-time PCR analysis of ECS mRNA expression revealed endocannabinoid receptor (CNR2), diacylglycerol lipase (DAGL), cyclooxygenase-2 (COX-2), fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGLL) had significant fold differences when comparing two different stages of the estrous cycle (late luteal phase vs. estrus). CNR2 and DAGL showed 2.01 and 2.57 fold increase, respectively (P=0.04 and P=0.02), in estrous cows. Among the analyzed genes FAAH (P=0.01) and MGLL (P=0.02) were significantly down-regulated in estrous cows, with a 5.01- and 2.44-fold difference in mRNA expression, respectively. Overall, this study highlights an association between the expression of the ECS in the bovine endometrium and stage of the estrous cycle.

scholarly journals Simultaneous Quantitative Detection of Relevant Biomarkers in Breast Cancer by Quantitative Real-Time PCR

2006 ◽  
Vol 21 (1) ◽  
pp. 30-39 ◽  
Author(s):  
M. Labuhn ◽  
V. Vuaroqueaux ◽  
F. Fina ◽  
A. Schaller ◽  
I. Nanni-Metellus ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Mrna Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Mrna Levels ◽  
Quantitative Real Time Pcr ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Mrna Expression Levels

The assessment of ERα, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERα, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERα, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Real-time PCR Analysis of Phosphodiesterase (PDE) 4D Splice-variant mRNA Expression and Selective PDE Inhibitor Action in Highly Purified Human Granulosa/luteal Cell Cultures.

2008 ◽  
Vol 78 (Suppl_1) ◽  
pp. 150-151
Author(s):  
David W. Schomberg ◽  
Ioanna Konidari ◽  
Jon A. Proctor ◽  
Justin E. Eller ◽  
Grace M. Couchman ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Cell Cultures ◽  
Real Time Pcr ◽  
Splice Variant ◽  
Luteal Cell ◽  
Pcr Analysis

Quantitative real time-PCR of CD133 mRNA: A potential surrogate angiogenic marker of response for patients with metastatic sarcoma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 20034-20034
Author(s):  
E. H. Lin ◽  
Y. Li ◽  
J. Trent ◽  
S. M. Patel ◽  
M. A. Burgess ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Progressive Disease ◽  
Tumor Response ◽  
Mrna Levels ◽  
Paired Samples ◽  
Before And After ◽  
Metastatic Sarcoma ◽  
Mrna Expression Levels

20034 Background: CD133 antigen is a specific surface marker for circulating endothelial progenitor cells (CEPs), pivotal in post-natal angiogenesis. We hypothesize that changes in levels of the CD133 mRNA expression and vascular endothelial growth factor (VEGF) may correlate with tumor response. Methods: After informed consent, we obtained 48 peripheral blood samples from patients with metastatic sarcoma including gastrointestinal stroma tumors (GIST). There were 16-paired samples before and after treatment including chemotherapy, or surgery, or imatinib. Of 24 patients with metastatic GIST enrolled, seven were paired samples. We measured CD133 mRNA levels by method (Lin E et al AACR 2003# 5392) and VEGF levels by ELISA (Genzyme, MA). The measurements were done in duplicates in two experiments. Results: The mean CD133 levels and VEGF levels before and after treatment was in Table 1 . The treatment resulted in significant reduction of CD133 mRNA expression (p = 0.035) as well as the level of VEGF (p = 0.014). Three patients experienced increase in CD133 expression had progressive disease. Among the paired GIST patients, there was a trend of increased CD133 mRNA expression levels in patients with progressive disease, death, or tumor recurrence. CD133 mRNA levels appeared elevated among the imatinib naïve patients and imatinib appeared to reset CD133 mRNA levels among the responding GIST patients to that of healthy volunteers. Conclusion: CD133 mRNA expression levels in sarcoma patients measured by real time-PCR assay appeared to correlate with tumor response. Unpublished independent work with NASBA assay platform in other solid tumors supported our findings ( www.primagen.com ). Further work is needed to reduce assay variability, and to determine the sensitivity and specificity of assay comparing to flow cytometry and to test assay’s applicability in the antiangiogenic therapy of cancer and the angiogenic therapy of cardiovascular diseases. [Table: see text] No significant financial relationships to disclose.

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