237 POTASSIUM CONCENTRATION AND mRNA LEVELS OF POTASSIUM CHANNELS DECREASED IN CYSTIC OVARIAN FOLLICLE FLUID

2007 ◽  
Vol 19 (1) ◽  
pp. 234
Author(s):  
D. Kang ◽  
C. Choe ◽  
E. S. Kim ◽  
H. Y. Yang ◽  
C. G. Hur ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Ovarian Follicle ◽  
Ion Concentration ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Significant Difference ◽  
Task Channels ◽  
Cell Layers ◽  
K Concentration ◽  
Theca Interna

Cystic ovarian follicle (COF) is one of the most frequently diagnosed ovarian diseases and a major cause of reproductive failure in cattle. Despite an abundance of reports on this subject, the exact pathogenesis of COF still remains unclear. It is generally accepted that disruption of the hypothalmo-pituitary-gonadal axis, by endogenous and/or exogenous factors, causes cystic formation. We here examined whether ion concentration and expression of ion channels are altered in the follicle fluid derived from a Korean native cow with COF. In an ovary with a cystic follicle, granulosa cell layers were exfoliated; the theca interna was thinner than that in an ovary without cystic follicle, based on histological examination. Concentrations of K+, Na+, and Cl- in COF fluid (COFF) were 10.4 � 3.5 mM, 138 � 12 mM, and 104.9 � 7.0 mM, respectively. In COFF, K+ concentration showed a significant difference from the value observed in normal follicle fluid (NFF) (P < 0.05; NFF: 10.4 � 3.5 mM vs. COFF: 6.2 � 0.8 mM). The total numbers of follicles observed (normal, 3–5 mm in diameter vs. COF, 20–30 mm in diameter) were 200 and 20 in normal and COF, respectively. To compare mRNA expression of K+ channels, we performed semiquantitative RT-PCR using follicle fluid and ovaries with or without cystic follicles. RT-PCR showed that mRNA levels of TASK channels (TASK-1, TASK-3, and TASK-5) decreased by 50% in COFF and an ovary with cystic follicles compared to NFF and a normal ovary. TASK channels are involved in apoptosis of mammalian cells. Our results suggest that potassium may play an important role in the pathogenesis of COF.

NDRG1 Expression Is Inhibited in AML and Its Knockdown Attenuates Neutrophil Differentiation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 927-927
Author(s):  
Mario P. Tschan ◽  
Deborah Shan ◽  
Judith Laedrach ◽  
Marianne Eyholzer ◽  
Elisabeth Oppliger Leibundgut ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Line ◽  
Cell Phenotype ◽  
Mrna Levels ◽  
U937 Cells ◽  
Rt Pcr ◽  
Atra Treatment ◽  
Significant Difference ◽  
Blast Cells ◽  
Neutrophil Differentiation

Abstract The N-myc down-regulated gene 1 (NDRG1) is a stressed induced protein whose expression is associated with growth arrest and differentiation of tumor cells. Although the exact function of NDRG1 protein remains unknown, various studies support its role as a suppressor of tumor metastasis. In prostate, colon and breast cancer its expression is associated with a better disease prognosis and patient survival. In hematopoietic cells, NDRG1 was identified in a differential display screen for differentiation-related genes in human myelomonocytic U937 cells. In the present study, we sought to investigate the role of NDRG1 in myeloid differentiation. To this end we first evaluated NDRG1 mRNA expression in acute myeloid leukemia (AML; n=82) patient samples as well as in CD34+ progenitor cells (n=5) and neutrophils (n=6) of healthy donors using quantitative real-time RT-PCR. We found significantly higher NDRG1 mRNA levels in granulocytes as compared to CD34+ (p=0.0043) or AML blast cells (p<0.0001), whereas no significant difference between CD34+ progenitor and AML blast cells was seen (Figure A). Moreover, we found that NDRG1 mRNA levels were increased in 4/5 APL patients upon ATRA therapy. In contrast, the closest relative of NDRG1, NDRG2, did not show significantly different expression in these primary cells, thus indicating a unique role for NDRG1 in granulocyte differentiation. Next we examined NDRG1 expression using quantitative RT-PCR and Western blotting in two different cell line models for ATRA-induced neutrophil differentiation. ATRA treatment of NB4 and HT93 acute promyelocytic leukemia (APL) cells induced NDRG1 mRNA 2.3- and 14.3- fold, respectively. Increased NDRG1 mRNA expression was paralleled by an increase of NDRG1 protein as well as a decrease in c-myc protein. Earlier reports described that NDRG1 is also suppressed by c-myc suggesting that down-regulation of c-myc in our cell line models allowed for an increase of NDRG1. In line with these observations, lentivirus-driven short hairpin (sh)RNA-mediated silencing of NDRG1 diminished ATRA-induced neutrophil differentiation of NB4 and U937 cells as measured by CD11b, CD11c and CD18 surface expression. In NB4 NDRG1 knockdown versus non-targeting shRNA expressing cells mean fluorescent intensities (MFI) for CD11b, CD11c and CD18 upon six days of ATRA-treatment were 99±17 vs 146±7, 20±2 vs 32±10 and 19±2 vs 45±6, respectively (Figure B). Similarly, U937 NDRG1 knockdown versus control cells displayed the following MFIs for CD11b and CD18 upon neutrophil differentiation: 61±1 vs 102±2 and 11±4 vs 33±13, respectively. In conclusion, we report here for the first time an association of low NDRG1 levels with an immature hematopoietic cell phenotype. Using RNAi technology we further provide evidence that NDRG1 is functionally involved in neutrophil maturation. Figure Figure

scholarly journals Expression profiles of myostatin and calpastatin genes and analysis of shear force and intramuscular fat content of yak longissimus muscle

2011 ◽  
Vol 56 (No. 12) ◽  
pp. 545-550 ◽  
Author(s):  
Y.C. Zheng ◽  
Y.Q. Lin ◽  
Y. Yue ◽  
Y.O. Xu ◽  
S.Y. Jin
Keyword(s):  
Expression Profiles ◽  
Muscle Growth ◽  
Intramuscular Fat ◽  
Mrna Levels ◽  
Longissimus Muscle ◽  
Rt Pcr ◽  
Yellow Cattle ◽  
Significant Difference ◽  
Different Ages ◽  
Imf Content

The main objective of this study was to reveal the expression profiles of two negative regulators, myostatin (MSTN) and calpastatin (CAST)genes, of skeletal muscle growth in highland yaks (Bos grunniens). mRNA levels of both genes were quantified in different yak tissues by semi-quantitative RT-PCR to reveal the tissue expression pattern, and real-time quantitative RT-PCR was employed to compare the mRNA levels of MSTN and CAST in longissimus muscles of yaks at different ages and adult Yellow cattle. Intramuscular fat (IMF) content, tenderness and pH of longissimus muscle of yaks at different ages and of adult Yellow cattle were also measured. The results showed that MSTN and CAST expressions have tissue specificity and both exhibited a high level in longissimus muscle and a low level in adipose tissue. Yak calves had lower mRNA levels of both MSTN and CAST in longissimus muscle compared with adult yaks. The analysis of meat quality traits of longissimus muscle showed that the shear forces of raw longissimus muscle of yak calves were significantly lower than those of adult yaks and Yellow cattle, no significant difference was found between adult yaks and Yellow cattle of similar age. IMF content in longissimus muscle was lower in yaks than in Yellow cattle. Although yaks were smaller in body size than Yellow cattle, adult yaks showed lower levels of MSTN and similar level of CAST mRNA in longissimus muscle compared to Yellow cattle. These data indicate that the expression of both MSTN and CAST in longissimus muscle differs between adult yaks and yak calves, and the yak longissimus muscle shows a lower IMF content compared to cattle. &nbsp;

scholarly journals COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS

2014 ◽  
Vol 15 (1) ◽  
Author(s):  
Vasila Packeer Mohamed ◽  
Yumi Z. H-Y. Hashim ◽  
A. Amid ◽  
M. Mel
Keyword(s):  
Mammalian Cells ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Primary Concern ◽  
Rt Pcr ◽  
Total Rna ◽  
Rna Purification ◽  
Dnase Treatment ◽  
Study Gene Expression ◽  
Significant Difference

ABSTRACT: Various methods have been described to extract RNA from adherent mammalian cells. RNA isolation in conjunction with reverse transcription polymerase chain reaction (RT-PCR) is a valuable tool used to study gene expression profiling. This approach is now being used in mammalian cell bioprocessing to help understand and improve the system. The objective of this study was to compare and determine the most suitable RNA extraction method for CHO-K1 cells in a setting where a relatively large amount of samples was involved. Total RNA was extracted using Total RNA purification kit (without DNase treatment; Norgen, Canada) and RNeasy mini kit (with DNase treatment; Qiagen, USA) respectively. The extracted RNA was then reverse transcribed, and the cDNA was subjected to PCR-amplifying 18S. Yield from RNeasy kit was significantly higher (0.316 ± 0.033 µg/µl; p=0.004) than Total RNA purification kit (0.177 ± 0.0243 µg/µl). However, RNA purity for both methods was close to 2.0 and there was no significant difference between the methods. Total RNA purification kit is less expensive than RNeasy kit. Since there is no DNase treatment step in the former, extraction time for RNA is shorter. When the extracted RNA was subjected to RT-PCR, both methods were able to show detection of 18S at 219 bp.   Therefore, this study demonstrates that both protocols are suitable for RNA extraction for CHO-K1 cells. RNeasy mini kit (Qiagen) is recommended if higher yields is the primary concern and Total RNA Purification kit (Norgen) is recommended if time and cost are concerned. ABSTRAK: Pelbagai kaedah telah digunakan untuk mengekstrak RNA daripada sel mamalia lekat.  Pemencilan RNA dengan menggunakan reaksi rantai polimerase transkripsi berbalik (RT-PCR) merupakan kaedah penting yang digunakan dalam mengkaji pernyataan gen berprofil.  Pendekatan ini kini digunakan dalam pemprosesan bio sel mamalia untuk memahami dan menambah baik sistem.  Tujuan kajian dijalankan adalah untuk menentukan dan membandingkan kaedah ekstraksi RNA yang paling sesuai bagi sel CHO-K1 di persekitaran di mana kadar sampel yang agak besar terlibat. Jumlah RNA  diekstrak menggunakan kit penulenan Jumlah RNA (tanpa rawatan DNase; Norgen, Canada) dan kit mini RNeasy (dengan rawatan DNase; Qiagen, USA).  RNA yang diekstrak kemudiannya diterbalikkan transkripsi, dan cDNA menjalani penguat PCR 18S. Hasil daripada kit RNeasy adalah lebih tinggi (0.316 ± 0.033 µg/µl; p=0.004) berbanding dengan kit penulenan Jumlah RNA (0.177 ± 0.0243 µg/µl). Walaupun begitu, kaedah penulenan RNA untuk kedua-duanya hampir 2.0 dan tidak terdapat perbezaan yang ketara antara keduanya. Kit penulenan Jumlah RNA adalah lebih murah berbanding dengan kit RNeasy. Memandangkan tidak ada langkah rawatan DNase dengan penggunaan kit Jumlah RNA, tempoh ekstrak RNA nya lebih pendek. Apabila RNA yang telah diekstrak menjalani RT-PCR, kedua-dua kaedah berjaya mengesan 18S pada 219 bp.   Kesimpulannya, kajian ini menunjukkan kedua-dua kaedah sesuai untuk mengekstrak RNA bagi sel CHO-K1. Kit mini RNeasy (Qiagen) lebih sesuai jika hasil yang tinggi diinginkan dan kit penulenan Jumlah RNA (Norgen) pula ideal, jika kos dan masa berkepentingan.

Negative Correlation Between Serum Levels of Homocysteine and Apolipoprotein M

2019 ◽  
Vol 19 (2) ◽  
pp. 120-126
Author(s):  
J. Wei ◽  
Y. Yu ◽  
Y. Feng ◽  
J. Zhang ◽  
Q. Jiang ◽  
...  
Keyword(s):  
Negative Correlation ◽  
Hepg2 Cells ◽  
Serum Levels ◽  
Significant Negative Correlation ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Apolipoprotein M ◽  
Protein Mass ◽  
Protein Levels ◽  
Phosphoinositide 3 Kinase

Background: Homocysteine (Hcy) has been suggested as an independent risk factor for atherosclerosis. Apolipoprotein M (apoM) is a constituent of the HDL particles. The goal of this study was to examine the serum levels of homocysteine and apoM and to determine whether homocysteine influences apoM synthesis. Methods: Serum levels of apoM and Hcy in 17 hyperhomocysteinemia (HHcy) patients and 19 controls were measured and their correlations were analyzed. Different concentrations of homocysteine (Hcy) and LY294002, a specific phosphoinositide 3- kinase (PI3K) inhibitor, were used to treat HepG2 cells. The mRNA levels were determined by RT-PCR and the apoM protein mass was measured by western blot. Results: We found that decreased serum apoM levels corresponded with serum HDL levels in HHcy patients, while the serum apoM levels showed a statistically significant negative correlation with the serum Hcy levels. Moreover, apoM mRNA and protein levels were significantly decreased after the administration of Hcy in HepG2 cells, and this effect could be abolished by addition of LY294002. Conclusions: resent study demonstrates that Hcy downregulates the expression of apoM by mechanisms involving the PI3K signal pathway.

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

scholarly journals DNA Aptamer Functionalized Hydrogels for Interferometric Fiber-Optic Based Continuous Monitoring of Potassium Ions

Biosensors ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 266
Author(s):  
Nataša Žuržul ◽  
Bjørn Torger Stokke
Keyword(s):  
Conformational Transition ◽  
Polymer Network ◽  
Potassium Ion ◽  
Ion Concentration ◽  
Dna Aptamer ◽  
Blood Levels ◽  
Label Free ◽  
Practical Applications ◽  
Hydrogel Matrix ◽  
K Concentration

In the present paper, we describe a potassium sensor based on DNA-aptamer functionalized hydrogel, that is capable of continuous label-free potassium ion (K+) monitoring with potential for in situ application. A hydrogel attached to the end of an optical fiber is designed with di-oligonucleotides grafted to the polymer network that may serve as network junctions in addition to the covalent crosslinks. Specific affinity toward K+ is based on exploiting a particular aptamer that exhibits conformational transition from single-stranded DNA to G-quadruplex formed by the di-oligonucleotide in the presence of K+. Integration of this aptamer into the hydrogel transforms the K+ specific conformational transition to a K+ concentration dependent deswelling of the hydrogel. High-resolution interferometry monitors changes in extent of swelling at 1 Hz and 2 nm resolution for the hydrogel matrix of 50 µm. The developed hydrogel-based biosensor displayed high selectivity for K+ ions in the concentration range up to 10 mM, in the presence of physiological concentrations of Na+. Additionally, the concentration dependent and selective K+ detection demonstrated in the artificial blood buffer environment, both at room and physiological temperatures, suggests substantial potential for practical applications such as monitoring of potassium ion concentration in blood levels in intensive care medicine.

scholarly journals Polypurine Reverse-Hoogsteen Hairpins as a Tool for Exon Skipping at the Genomic Level in Mammalian Cells

2021 ◽  
Vol 22 (7) ◽  
pp. 3784
Author(s):  
Véronique Noé ◽  
Carlos J. Ciudad
Keyword(s):  
Dna Sequencing ◽  
Rare Diseases ◽  
Mammalian Cells ◽  
Mrna Level ◽  
Exon Skipping ◽  
Selective Medium ◽  
Rt Pcr ◽  
Exon 2 ◽  
Reading Frame ◽  
Additional Exon

Therapeutic strategies for rare diseases based on exon skipping are aimed at mediating the elimination of mutated exons and restoring the reading frame of the affected protein. We explored the capability of polypurine reverse-Hoogsteen hairpins (PPRHs) to cause exon skipping in NB6 cells carrying a duplication of exon 2 of the DHFR gene that causes a frameshift abolishing DHFR activity. Methods: Different editing PPRHs were designed and transfected in NB6 cells followed by incubation in a DHFR-selective medium lacking hypoxanthine and thymidine. Surviving colonies were analyzed by DNA sequencing, RT-PCR, Western blotting and DHFR enzymatic activity. Results: Transfection of editing PPRHs originated colonies in the DHFR-selective medium. DNA sequencing results proved that the DHFR sequence in all these colonies corresponded to the wildtype sequence with just one copy of exon 2. In the edited colonies, the skipping of the additional exon was confirmed at the mRNA level, the DHFR protein was restored, and it showed high levels of DHFR activity. Conclusions: Editing-PPRHs are able to cause exon skipping at the DNA level and could be applied as a possible therapeutic tool for rare diseases.

scholarly journals RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor)

2005 ◽  
Vol 86 (12) ◽  
pp. 3419-3424 ◽  
Author(s):  
Constanze Yue ◽  
Elke Genersch
Keyword(s):  
Varroa Destructor ◽  
Pcr Analysis ◽  
Body Parts ◽  
Rt Pcr ◽  
Larval Food ◽  
Viral Pathogen ◽  
Deformed Wing Virus ◽  
Significant Difference ◽  
Almost All ◽  
Viral Sequences

Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding.

Mechanical activity in heart regulates translation of α-myosin heavy chain mRNA but not its localization

1999 ◽  
Vol 276 (6) ◽  
pp. H2013-H2019 ◽  
Author(s):  
Gordana Nikcevic ◽  
Maria C. Heidkamp ◽  
Merja Perhonen ◽  
Brenda Russell
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Mrna Translation ◽  
Neonatal Rat ◽  
Mechanical Activity ◽  
Mrna Levels ◽  
Rat Cardiomyocytes ◽  
Significant Difference ◽  
Polysomal Fraction ◽  
Mechanical Transduction

Mechanical inactivity depresses protein expression in cardiac muscle tissue and results in atrophy. We explore the mechanical transduction mechanism in spontaneously beating neonatal rat cardiomyocytes expressing the α-myosin heavy chain (α-MyHC) isoform by interfering with cross-bridge function [2,3-butanedione monoxime (BDM), 7.5 mM] without affecting cell calcium. The polysome content and α-MyHC mRNA levels in fractions from a sucrose gradient were analyzed. BDM treatment blocked translation at initiation (162 ± 12% in the nonpolysomal RNA fraction and 43 ± 6% in the polysomal fraction, relative to control as 100%; P < 0.05). There was an increase in α-MyHC mRNA from the nonpolysomal fraction (120.5 ± 7.7%; P < 0.05 compared with control) with no significant change in the heavy polysomes. In situ hybridization of α-MyHC mRNA was used to estimate message abundance as a function of the distance from the nucleus. The mRNA was dispersed through the cytoplasm in spontaneously beating cells as well as in BDM-treated cells (no significant difference). We conclude that direct inhibition of contractile machinery, but not calcium, regulates initiation of α-MyHC mRNA translation. However, calcium, not pure mechanical signals, appears to be important for message localization.

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