Induction of Winter Flounder Antifreeze Protein Messenger RNA at 4 C in                vivo and in vitro

AbstractMicroRNAs (miRNAs) are short (18–24 nucleotides) non-coding RNA sequences that regulate gene expression via binding of messenger RNA. It is estimated that miRNAs co-regulate the expression of more than 70% of all human genes, many of which fulfil important roles in bone metabolism and muscle function. In-vitro and in-vivo experiments have shown that the targeted loss of miRNAs in distinct bone cell types (osteoblasts and osteoclasts) results in altered bone mass and bone architecture. These results emphasize the biological relevance of miRNAs for bone health.MiRNAs are not only considered as novel bone biomarkers because of their biological importance to bone metabolism, but also on the basis of other favorable properties: 1) Secretion of miRNAs from cells enables “minimally invasive” detection in biological fluids such as serum. 2) High stability of miRNAs in serum enables the retrospective analysis of frozen blood specimens. 3) Quantification of miRNAs in the serum is based on the RT-PCR - a robust method that is considered as the gold standard for the analysis of nucleic acids in clinical diagnostics.With regard to osteoporosis, it has been shown that many of the known risk factors are characterized by distinct miRNA profiles in the affected tissues: i) age-related loss of bone mass, ii) sarcopenia, iii) changes in estrogen metabolism and related changes Loss of bone mass, and iv) diabetes. Therefore, numerous studies in recent years have dealt with the characterization of miRNAs in the serum of osteoporosis patients and healthy controls, and were able to identify recurring miRNA patterns that are characteristic of osteoporosis. These novel biomarkers have great potential for the diagnosis and prognosis of osteoporosis and its clinical outcomes.The aim of this article is to give a summary of the current state of knowledge on the research and application of miRNA biomarkers in osteoporosis.

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Differential Effects of Halothane and Thiopental on Surfactant Protein C Messenger RNA In Vivo and In Vitro in Rats

Anesthesiology ◽

10.1097/00000542-200009000-00030 ◽

2000 ◽

Vol 93 (3) ◽

pp. 805-810 ◽

Cited By ~ 17

Author(s):

Catherine Paugam-Burtz ◽

Serge Molliex ◽

Bernard Lardeux ◽

Corinne Rolland ◽

Michel Aubier ◽

...

Keyword(s):

Messenger Rna ◽

Protein C ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Alveolar Type Ii Cells ◽

Mechanically Ventilated ◽

Mrna Content

Background Pulmonary surfactant is a complex mixture of proteins and phospholipids synthetized by alveolar type II cells. Volatile anesthetics have been shown to reduce surfactant phospholipid biosynthesis by rat alveolar type II cells. Surfactant-associated protein C (SP-C) is critical for the alveolar surfactant functions. Our goal was to evaluate the effects of halothane and thiopental on SP-C messenger RNA (mRNA) expression in vitro in rat alveolar type II cells and in vivo in mechanically ventilated rats. Methods In vitro, freshly isolated alveolar type II cells were exposed to halothane during 4 h (1, 2, 4%) and 8 h (1%), and to thiopental during 4 h (10, 100 micrometer) and 8 h (100 micrometer). In vivo, rats were anesthetized with intraperitoneal thiopental or inhaled 1% halothane and mechanically ventilated for 4 or 8 h. SP-C mRNA expression was evaluated by ribonuclease protection assay. Results In vitro, 4-h exposure of alveolar type II cells to thiopental 10 and 100 micrometer increased their SP-C mRNA content to 145 and 197%, respectively, of the control values. In alveolar type II cells exposed for 4 h to halothane 1, 2, and 4%, the SP-C mRNA content increased dose-dependently to 160, 235, and 275%, respectively, of the control values. In vivo, in mechanically ventilated rats, 4 h of halothane anesthesia decreased the lung SP-C mRNA content to 53% of the value obtained in control (nonanesthetized, nonventilated) animals; thiopental anesthesia increased to 150% the lung SP-C mRNA content. Conclusions These findings indicate that halothane and thiopental used at clinically relevant concentrations modulate the pulmonary SP-C mRNA content in rats. In vivo, the additive role of mechanical ventilation is suggested.

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Relative messenger RNA expression profiling of collagenases and aggrecanases in human articular chondrocytes in vivo and in vitro

Arthritis & Rheumatism ◽

10.1002/art.10531 ◽

2002 ◽

Vol 46 (10) ◽

pp. 2648-2657 ◽

Cited By ~ 262

Author(s):

Brigitte Bau ◽

Pia M. Gebhard ◽

Jochen Haag ◽

Thomas Knorr ◽

Eckart Bartnik ◽

...

Keyword(s):

Expression Profiling ◽

Messenger Rna ◽

Articular Chondrocytes ◽

Human Articular Chondrocytes ◽

Rna Expression ◽

Rna Expression Profiling

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An Electron-Microscope Study of the in vitro Transformation of Human Leucocytes

Journal of Cell Science ◽

10.1242/jcs.2.3.359 ◽

1967 ◽

Vol 2 (3) ◽

pp. 359-370

Author(s):

J. A. CHAPMAN ◽

M. W. ELVES ◽

J. GOUGH

Keyword(s):

Endoplasmic Reticulum ◽

Electron Microscope ◽

Messenger Rna ◽

Human Peripheral Blood ◽

Limited Extent ◽

Single Particles ◽

Blast Cells ◽

Protein Secreting

Electron-microscope studies of cultured small lymphocytes from human peripheral blood transforming into larger blastoid cells in the presence of phytohaemagglutinin (PHA) show that the transformed cell possesses the preliminary stages of development of a protein-synthesizing system. The transformed blastoid cell has abundant ribosomes, although, in contrast with in vivo protein-secreting cells, many of these occur as single particles with only a small proportion Linked in polysomal clusters. Endoplasmic reticulum membranes occur to a very limited extent and with a marked paucity of attached ribosomal particles; the few attached particles are usually located in groups. Some endoplasmic reticulum membranes revealed degenerative changes in otherwise normal cells. A moderately well-developed Golgi apparatus was a characteristic feature of the cells. Apart from the relatively low proportion of polysomes, in vitro PHA-transformed blastoid cells are identical in fine structure to in vivo blast cells (otherwise known as immunoblasts, haemocytoblasts, etc.) occurring in the immune response. It is suggested that messenger-RNA production in PHA-stimulated transformed cells may be reduced and that this could explain the limited number of polysomes and the restricted development of the endoplasmic reticulum.

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LncRNA HCG18 contributes to the progression of hepatocellular carcinoma via miR-214-3p/CENPM axis

The Journal of Biochemistry ◽

10.1093/jb/mvaa073 ◽

2020 ◽

Vol 168 (5) ◽

pp. 535-546 ◽

Cited By ~ 1

Author(s):

Yuepei Zou ◽

Zhonghua Sun ◽

Shuangming Sun

Keyword(s):

Hepatocellular Carcinoma ◽

Messenger Rna ◽

Animal Experiments ◽

Non Coding Rna ◽

Diphenyltetrazolium Bromide ◽

And Migration ◽

Hcc Cells ◽

Proliferation And Migration

Abstract Long non-coding RNA (lnc) HCG18 has been reported to contribute progression of a variety of tumours. However, its roles in hepatocellular carcinoma (HCC) remains unknown. In the current study, we intended to uncover the biological functions of HCG18 in HCC. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to detect the expression of HCG18, microRNA-214-3p (miR-214-3p) and centromere protein M (CENPM) messenger RNA (mRNA). The role of HCG18 in the growth and migration were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, wound healing assay and flow cytometry in vitro and animal experiments in vivo. The results showed that HCG18 was highly expressed in HCC tissues. HCG18 silencing inhibited the proliferation and migration while induced the apoptosis of HCC cells. Besides, miR-214-3p was down-regulated in HCC cells. Further experiments revealed that miR-214-3p could directly bind to HCG18 and exerted an anti-tumour role to counteracted siHCG18-1-mediated influence in HCC cells. Moreover, miR-214-3p could directly interact with CENPM mRNA and down-regulating the expression of CENPM. While HCG18 could up-regulate the expression of CENPM through acting as a sponge of miR-214-3p. Therefore, those results suggested HCG18 functioned as an oncogene to promote the proliferation and migration of HCC cells via miR-214-3p/CENPM axis.

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Evaluation of biomarkers for in vitro prediction of drug-induced nephrotoxicity in RPTEC/TERT1 cells

Toxicology Research ◽

10.1093/toxres/tfaa005 ◽

2020 ◽

Vol 9 (2) ◽

pp. 91-100 ◽

Cited By ~ 1

Author(s):

Xuan Qiu ◽

Yufa Miao ◽

Xingchao Geng ◽

Xiaobing Zhou ◽

Bo Li

Keyword(s):

Transcriptional Activation ◽

Messenger Rna ◽

Aristolochic Acid ◽

Kidney Injury ◽

Drug Induced ◽

Vitro Assay ◽

Protein Levels ◽

Aristolochic Acid I

Abstract There have been intensive efforts to identify in vivo biomarkers that can be used to monitor drug-induced kidney damage before significant impairment occurs. Kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, clusterin, β2-microglobulin and cystatin C (CysC) have been validated as clinical or preclinical biomarkers in urinary and plasma predictive of acute and chronic kidney injuries and diseases. A high-throughput in vitro assay predictive of nephrotoxicity could potentially be implemented in early drug discovery stage to reduce attrition at later stages of drug development. To assess the potential of these known in vivo biomarkers for in vitro evaluation of drug-induced nephrotoxicity, we selected four nephrotoxic agents (cisplatin, cyclosporin, aristolochic acid I and gentamicin) and detected their effects on the protein levels of nephrotoxic biomarkers in RPTEC/TERT1 cells. The protein levels of clusterin, CysC, GSTπ and TIMP-1 significantly increased in the conditioned media of RPTEC/TERT1 cells treated with cisplatin, cyclosporin, aristolochic acid I and gentamicin. The messenger RNA levels of clusterin, CysC, GSTπ and TIMP-1 also increased in RPTEC/TERT1 cells treated with cisplatin, cyclosporin, aristolochic acid I and gentamicin, indicating that drug-induced upregulation involves transcriptional activation. Taken together, the results clearly demonstrate that among the known in vivo nephrotoxic biomarkers, clusterin, CysC, GSTπ and TIMP-1 can be effectively used as in vitro biomarkers for drug-induced nephrotoxicity in RPTEC/TERT1 cells.

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Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

The Journal of Cell Biology ◽

10.1083/jcb.74.2.414 ◽

1977 ◽

Vol 74 (2) ◽

pp. 414-427 ◽

Cited By ~ 52

Author(s):

J Kruppa ◽

DD Sabatini

Keyword(s):

Ionic Strength ◽

Rat Liver ◽

Messenger Rna ◽

High Salt ◽

Salt Medium ◽

Ribosomal Subunits ◽

Microsomal Membranes ◽

Low Ionic Strength

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

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Controversial Regulation of Gene Expression and Protein Transduction of Aquaporins under Drought and Salinity Stress

Plants ◽

10.3390/plants9121662 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1662

Author(s):

Lucía Yepes-Molina ◽

Gloria Bárzana ◽

Micaela Carvajal

Keyword(s):

Plasma Membrane ◽

Messenger Rna ◽

Regulation Of Gene Expression ◽

Protein Transduction ◽

Mrna Synthesis ◽

Plasma Membrane Intrinsic Protein ◽

Entire Plant ◽

The One

Enhancement of the passage of water through membranes is one of the main mechanisms via which cells can maintain their homeostasis under stress conditions, and aquaporins are the main participants in this process. However, in the last few years, a number of studies have reported discrepancies between aquaporin messenger RNA (mRNA) expression and the number of aquaporin proteins synthesised in response to abiotic stress. These observations suggest the existence of post-transcriptional mechanisms which regulate plasma membrane intrinsic protein (PIP) trafficking to the plasma membrane. This indicates that the mRNA synthesis of some aquaporins could be modulated by the accumulation of the corresponding encoded protein, in relation to the turnover of the membranes. This aspect is discussed in terms of the results obtained: on the one hand, with isolated vesicles, in which the level of proteins present provides the membranes with important characteristics such as resistance and stability and, on the other, with isolated proteins reconstituted in artificial liposomes as an in vitro method to address the in vivo physiology of the entire plant.

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Renal sugar transport in the winter flounder: V. secretion of 2-deoxy-D-galactose

AJP Renal Physiology ◽

10.1152/ajprenal.1978.234.5.f424 ◽

1978 ◽

Vol 234 (5) ◽

pp. F424-F431

Author(s):

J. B. Pritchard ◽

G. Booz ◽

A. Kleinzeller

Keyword(s):

Sugar Transport ◽

Winter Flounder ◽

Free Sugar ◽

Renal Tubules ◽

Pseudopleuronectes Americanus ◽

Clearance Ratio ◽

Renal Handling ◽

Cell Clearance

Isolated renal tubules and renal clearance techniques were used to characterize the renal handling of 2-deoxy-D-galactose (2-d-Gal) by the winter flounder (Pseudopleuronectes americanus). In vitro, energy-dependent, pH-sensitive uptake of 2-d-Gal (2–100 micron) was seen at the antiluminal face of the cell. Clearance measurements showed net secretion of 2-d-Gal in vivo. The mean clearance of 2-d-Gal in 18 fish was 0.98 +/- 0.16 ml/h while the glomerular filtration rate (GFR) was only 0.37 +/- 0.10 ml/h. Secretion was associated with marked renal accumulation of both 2-d-Gal and phosphorylated derivatives (2-d-Gal-1-phosphate). Tissue-to-plasma ratios (T/P) averaged 19 for free sugar and 59 for total sugar. Both clearance ratio and T/P were reduced to approximately 1 by injection of galactose (2.5 mmol/kg) simultaneously with 2-d-Gal (25 mumol/kg). Phlorizin (2.5 mumol/kg) increased net 2-d-Gal secretion, whereas glucose (2.5 mmol/kg) produced no change in secretion. Both compounds depressed 2-d-Gal T/P. This result suggests the presence of readsorptive transport at the brush border, sensitive to glucose and phlorizin.

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Growth- and differentiation-associated expression of bcl-2 in B-chronic lymphocytic leukemia cells

Blood ◽

10.1182/blood.v79.11.2981.2981 ◽

1992 ◽

Vol 79 (11) ◽

pp. 2981-2989 ◽

Cited By ~ 150

Author(s):

M Schena ◽

LG Larsson ◽

D Gottardi ◽

G Gaidano ◽

M Carlsson ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Messenger Rna ◽

Phorbol Esters ◽

Lymphocytic Leukemia ◽

Mantle Zone ◽

Cell Stage ◽

High Level

Abstract The bcl-2 gene is translocated into the Ig loci in about 80% of human follicular lymphomas and in 10% of B-type chronic lymphocytic leukemias (B-CLL), resulting in a high level of expression. We have compared the expression of bcl-2 transcripts and protein in B-CLL cells in their normal equivalent CD5+ B cells and in normal B-cell populations representative of different in vivo and in vitro stages of activation and proliferation. We report here that bcl-2 was expressed in 11 of 11 cases of CD5+ B-CLL clones, contrasting with the absent expression in normal CD5+ B cells. Activation of 173 and 183 B-CLL cells by phorbol esters (12-O-tetradecanoylphorbol-13-acetate [TPA]) to IgM secretion without concomitant DNA synthesis resulted in a rapid but transient downregulation of bcl-2 expression. In contrast, the reduction of bcl-2 at both the messenger RNA and protein levels was sustained after mitogenic stimulation, suggesting that bcl-2 expression and proliferation are inversely related in these cells. This notion was further supported by immunocytochemical analysis showing that bcl-2 was primarily expressed in small resting lymphocytes and in cells differentiating to the plasma cell stage, but less expressed in Ki67- positive proliferating B blasts. Moreover, it was also supported by the low level of bcl-2 in exponentially growing Epstein-Barr virus-carrying lymphoblastoid and B-CLL cell lines. The regulation of bcl-2 expression in B-CLL resembled that of normal tonsillar follicular B cells, in which a high level of expression was found in resting mantle zone B cells but not in the proliferating germinal center B cells. Based on these findings and the role of bcl-2 in maintaining B-cell memory, we propose that the phenotype of B-CLL cells corresponds to a mantle zone memory-type B cell.

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