Relevant bibliographies by topics / Protein and mRNA levels

Academic literature on the topic 'Protein and mRNA levels'

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Published: 6 September 2023

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Journal articles on the topic "Protein and mRNA levels"

Berger, Lloyd C., Jnanankur Bag, and Bruce H. Sells. "Translation of poly(A)-binding protein mRNA is regulated by growth conditions." Biochemistry and Cell Biology 70, no. 9 (September 1, 1992): 770–78. http://dx.doi.org/10.1139/o92-117.

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Translational efficiency of a minor group of mRNAs is regulated by serum levels in 3T6 fibroblasts. Included within this group is the poly(A)-binding protein (PABP) mRNA. We analyzed the distribution of PABP mRNA in polysome profiles and found a large percentage of this mRNA to be translationally repressed in both actively growing (~ 60%) and resting cells (~ 70%). Elevated serum levels induced a distinct bimodal distribution of this mRNA between actively translated and repressed fractions. Similarly, treatment of cells with low doses of cycloheximide also generated a partial shift of repressed PABP mRNA into the actively translated fraction. In an attempt to characterize the factors which regulate PABP mRNA translation we have identified the proteins which bind to this mRNA in vitro. Sequences within the 5′ untranslated region were found to be sufficient for binding of all proteins to this mRNA. We suggest that this region and the proteins associated with it may be essential for translation control of PABP mRNA.Key words: translation, mRNP, poly(A) binding protein.

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Gerstenfeld, L. C., M. H. Finer, and H. Boedtker. "Altered beta-actin gene expression in phorbol myristate acetate-treated chondrocytes and fibroblasts." Molecular and Cellular Biology 5, no. 6 (June 1985): 1425–33. http://dx.doi.org/10.1128/mcb.5.6.1425-1433.1985.

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Phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, was shown to have opposite effects on the cellular morphology and steady-state levels of beta-actin mRNA in embryonic chicken muscle fibroblasts and sternal chondrocytes. When fibroblasts were treated with PMA, they formed foci of densely packed cells, ceased to adhere to culture plates, and had significantly reduced levels of beta-actin mRNA and protein. Conversely, when treated with PMA, floating chondrocytes attached to culture dishes, spread out, and began to accumulate high levels of beta-actin mRNA and proteins. In the sternal chondrocytes the stimulation of the beta-actin mRNA production was accompanied by increased steady-state levels of fibronectin mRNAs and protein. These alterations were concomitant with a fivefold reduction in type II collagen mRNA and a cessation in its protein production. After fibronectin and actin mRNAs and proteins reached their maximal levels, type I collagen mRNA and protein synthesis were turned on. Removal of PMA resulted in reduced beta-actin mRNA levels in chondrocytes and in a further alteration in the cell morphology. These observed correlations between changes in cell adhesion and morphology and beta-actin expression suggest that the effect of PMA on cell shape and adhesion may result in changes in the microfilament organization of the cytoskeleton which ultimately lead to changes in the extracellular matrix produced by the cells.

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Willis, Dianna E., Erna A. van Niekerk, Yukio Sasaki, Mariano Mesngon, Tanuja T. Merianda, Gervan G. Williams, Marvin Kendall, Deanna S. Smith, Gary J. Bassell, and Jeffery L. Twiss. "Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs." Journal of Cell Biology 178, no. 6 (September 4, 2007): 965–80. http://dx.doi.org/10.1083/jcb.200703209.

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Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein–β-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous β-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.

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Veletza, S. V., K. V. Nichols, I. Gross, H. Lu, D. W. Dynia, and J. Floros. "Surfactant protein C: hormonal control of SP-C mRNA levels in vitro." American Journal of Physiology-Lung Cellular and Molecular Physiology 262, no. 6 (June 1, 1992): L684—L687. http://dx.doi.org/10.1152/ajplung.1992.262.6.l684.

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We have studied hormonal regulation of the surfactant protein C (SP-C) in fetal 18-dah rat lung explants. SP-C mRNA was detected in Northern blots with a specific rat SP-C cDNA probe and quantified by densitometry. Treatment of the explants with dexamethasone resulted in a dose-dependent increase of the SP-C mRNA level. Transcriptional assays have shown that the regulation of SP-C mRNA by dexamethasone involves a transcriptional step. Administration of the cAMP analogues, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), produced a dose-dependent increase of SP-C mRNA levels, with maximum stimulation observed at 200 microM. The thyroid hormone T3 had no effect on SP-C mRNA levels, whether administered alone or in combination with dexamethasone. Variation in the effects of the above hormones on three surfactant protein mRNAs, SP-A, SP-B and SP-C, indicates that the hormonal regulation of the surfactant proteins is a complex process and that each gene is, in part, differentially regulated.

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Maicas, E., F. G. Pluthero, and J. D. Friesen. "The accumulation of three yeast ribosomal proteins under conditions of excess mRNA is determined primarily by fast protein decay." Molecular and Cellular Biology 8, no. 1 (January 1988): 169–75. http://dx.doi.org/10.1128/mcb.8.1.169-175.1988.

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The suggestion that compensation for overabundant mRNA of the genes for Saccharomyces cerevisiae ribosomal protein (r-protein) L3, L29, or rp59 occurs by translation repression has been reinvestigated. First, analysis of the distribution of these three mRNAs in polysome profiles revealed no differences between normal and mRNA-overproducing strains, indicating that initiation of r-protein translation is not repressed under conditions of mRNA overaccumulation. Second, experiments involving radioactive pulse-labeling of proteins were done by using a modified method of data collection and analysis that allows quantitation and correction for fast decay during the pulse. These measurements revealed that the synthesis rate of the three r-proteins is increased when their mRNA levels are elevated and that their decay rate is also high, with half-lives ranging from a fraction of a minute to more than 10 min. We conclude that accumulation of excess r-protein mRNA has no effect on translation rate; rapid decay of protein during the course of the labeling period can account for the apparent discrepancy between mRNA levels and protein synthesis rates. Yeast r-proteins, when produced in excess, are among the most rapidly degraded proteins so far described.

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Chen, Xu-Qiao, Carlos A. Barrero, Rodrigo Vasquez-Del Carpio, E. Premkumar Reddy, Chiara Fecchio, Salim Merali, Alessia Deglincerti, Cheng Fang, Jack Rogers, and Maria L. Maccecchini. "Posiphen Reduces the Levels of Huntingtin Protein through Translation Suppression." Pharmaceutics 13, no. 12 (December 7, 2021): 2109. http://dx.doi.org/10.3390/pharmaceutics13122109.

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Posiphen tartrate (Posiphen) is an orally available small molecule that targets a conserved regulatory element in the mRNAs of amyloid precursor protein (APP) and α-synuclein (αSYN) and inhibits their translation. APP and αSYN can cause neurodegeneration when their aggregates induce neurotoxicity. Therefore, Posiphen is a promising drug candidate for neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Posiphen’s safety has been demonstrated in three independent phase I clinical trials. Moreover, in a proof of concept study, Posiphen lowered neurotoxic proteins and inflammatory markers in cerebrospinal fluid of mild cognitive impaired patients. Herein we investigated whether Posiphen reduced the expression of other proteins, as assessed by stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry (MS)-based proteomics. Neuroblastoma SH-SY5Y cells, an in vitro model of neuronal function, were used for the SILAC protein profiling response. Proteins whose expression was altered by Posiphen treatment were characterized for biological functions, pathways and networks analysis. The most significantly affected pathway was the Huntington’s disease signaling pathway, which, along with huntingtin (HTT) protein, was down-regulated by Posiphen in the SH-SY5Y cells. The downregulation of HTT protein by Posiphen was confirmed by quantitative Western blotting and immunofluorescence. Unchanged mRNA levels of HTT and a comparable decay rate of HTT proteins after Posiphen treatment supported the coclusion that Posiphen reduced HTT via downregulation of the translation of HTT mRNA. Meanwhile, the downregulation of APP and αSYN proteins by Posiphen was also confirmed. The mRNAs encoding HTT, APP and αSYN contain an atypical iron response element (IRE) in their 5′-untranslated regions (5′-UTRs) that bind iron regulatory protein 1 (IRP1), and Posiphen specifically bound this complex. Conversely, Posiphen did not bind the IRP1/IRE complex of mRNAs with canonical IREs, and the translation of these mRNAs was not affected by Posiphen. Taken together, Posiphen shows high affinity binding to the IRE/IRP1 complex of mRNAs with an atypical IRE stem loop, inducing their translation suppression, including the mRNAs of neurotoxic proteins APP, αSYN and HTT.

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Fortelny, Nikolaus, Christopher M. Overall, Paul Pavlidis, and Gabriela V. Cohen Freue. "Can we predict protein from mRNA levels?" Nature 547, no. 7664 (July 2017): E19—E20. http://dx.doi.org/10.1038/nature22293.

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Watkins, David C., Peter J. Rapiejko, Manuel Ros, Hsien-yu Wang, and Craig C. Malbon. "G-Protein mRNA levels during adipocyte differentiation." Biochemical and Biophysical Research Communications 165, no. 3 (December 1989): 929–34. http://dx.doi.org/10.1016/0006-291x(89)92692-2.

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Dissertations / Theses on the topic "Protein and mRNA levels"

Simion, Oana-Maria. "Uncoupling proteins mRNA levels in mice lacking acylation-stimulating protein." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=33018.

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The etiology of obesity involves imbalanced energy intake and utilization. ASP is an adipose tissue hormone that facilitates adipocyte uptake of serum fatty acids and their storage. Mice lacking ASP have less adipose tissue mass, despite increased food intake, than wild-type littermates. We hypothesize that the unstored fuels are oxidized through UCP (thermogenic mitochondrial carriers).
In male ASP-deficient mice mRNA levels were measured by semi-quantitative RT-PCR and the following changes were observed: UCP-1 decreased in all tested tissues, UCP-2 increased by 15% and 6 fold in muscle and white adipose tissue and UCP-3 increased 2.5 and 10 fold in muscle and epididymal adipose tissue, respectively. In female ASP-deficient mice UCP-1 decreased in all tissues, UCP-2 increased by 10% and 40% in inguinal and brown adipose tissue, respectively, and UCP-3 remained stable in all tissues. High fat diet nullified these differences, and decreased all wild-type UCP levels.
We propose that UCP-2 and 3 assume the role of UCP-1 in fuel utilization, thus helping mice face an increased energy load in the absence of ASP.

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Byström, Jonas. "Eosinophil Cationic Protein : Expression Levels and Polymorphisms." Doctoral thesis, Uppsala University, Department of Medical Sciences, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-2059.

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The eosinophil cationic protein (ECP) is usually associated with the eosinophil granulocyte. In this thesis the presence and production of this protein has been studied in two other cells. The circulating monocyte was found to contain ECP mRNA and small amounts of ECP, one thousand times less than that found in the eosinophil. The production decreased by differentiation of the myelomonoblastic cell line U937 into a macrophage phenotype. Submucosal lung macrophages did not stain for ECP and alveolar macrophages did not contain ECP mRNA. The circulating neutrophil contains ECP at a level hundred fold less than the eosinophil. We found that the protein is located to the primary granules of the neutrophil but could detect no ECP mRNA in the cell. It was shown in vitro that the protein was taken up by the cell and partly transported to the primary granules. The uptake did not seem to be receptor mediated. Upon stimulation of the neutrophils, ECP previously taken up, was re-secreted.

The ECP protein is heterogeneous both to molecular characteristics and to function. To evaluate if a genetic component is involved, the ECP gene was analysed in 70 individuals. Three single nucleotide polymorphisms (SNP´s) were found, denoted 277(C>T), 434(G>C) and 562(G>C). The two first were located to the mature peptide-coding region and would change the amino acids, arg45cys and arg97thr. The prevalence of the most common SNP, 434, was evaluated in two eosinophil-related diseases, allergy/asthma and Hodgkin Lymphoma (HL). Forty-three HL patients were evaluated and it was found that the 434GG was significantly more prevalent in patients having nodular sclerosis (NS) as compared to other histologies (p=0.03). Erythrocyte sedimentation rate was also related to the 434GG genotype (p=0.009). In 209 medical students 434GG was more common (p=0.002) in those who indicated allergy. The genotype was unrelated to the production of IgE antibodies to allergens. In analysis of 76 subjects with asthma it was found that the 434GG genotype was significantly more common among allergic asthmatics (p=0.04). Asthma and HL-NS are characterized by fibrosis and eosinophils and ECP has been suggested in fibrosis development.

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Kachra, Zarin. "Regulation of insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) mRNA levels in cultured rat hepatocytes." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41300.

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The liver is a major site of production of circulating levels of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs). We have used primary cultured rat hepatocytes maintained under serum free conditions to explore the regulatory role of various hormones on hepatic IGF-I and IGFBP-1 mRNA levels.
IGF-I mRNA levels were stimulated 2.0 to 2.5 fold by bovine growth hormone (bGH) and 1.8 to 2.0 fold by glucagon but on combining bGH and glucagon, a synergistic effect was observed and IGF-I mRNA level was augmented 10 to 12 fold. Octreotide blocked the hGH induced stimulation of IGF-I production in serum and hepatic IGF-I mRNA levels in hypophysectomized rats. This effect could have been partly due to the low levels of glucagon in serum when hypophysectomized rats were treated with hGH and octreotide. Octreotide was also found to inhibit GH stimulated IGF-I mRNA levels in rat hepatocytes.
The unique synergy observed with glucagon and bGH on IGF-I mRNA levels in hepatocytes was not reproduced by T$ sb3$, oPRL, dexamethasone, EGF or insulin when each was added in combination with bGH or glucagon. Like glucagon, the addition of IBMX or (Bu)$ sb2$cAMP stimulated IGF-I mRNA levels 1.8 to 2.0 fold, but in the presence of bGH, IGF-I mRNA levels were stimulated 10 to 12 fold. PMA stimulated IGF-I mRNA levels 1.2 to 1.4 fold but displayed no synergism when added with bGH. The stimulatory effect of bGH plus glucagon on IGF-I mRNA levels was inhibited in PKC depleted cells, in the presence of inhibitors of PKC and in the presence of cycloheximide. bGH had no posttranscriptional effect on IGF-I mRNA stability whereas glucagon or (Bu)$ sb2$cAMP stabilized IGF-I mRNA at a posttranscriptional level.
In summary, the major hormonal regulators of hepatic IGF-I mRNA levels appear to be GH and glucagon. Hepatic IGF-I mRNA levels are regulated by pathways involving protein kinase C and, protein kinase A as well as by synthesis of one or more protein(s).
Glucagon and dexamethasone each stimulated IGFBP-1 mRNA levels 3 to 4 fold whereas bGH and T$ sb3$ each inhibited IGFBP-1 mRNA levels 45 to 70%. Insulin, which inhibited IGFBP-1 mRNA levels 95%, was the most powerful inhibitor and was also found to inhibit IGFBP-1 mRNA levels in the presence of dexamethasone. IBMX and (Bu)$ sb2$cAMP stimulated IGFBP-1 mRNA levels 6 to 8 fold whereas PMA inhibited IGFBP-1 mRNA levels 40 to 50%. The inhibitory effect of bGH on IGFBP-1 mRNA levels was abolished in PKC depleted cells and also in the presence of inhibitors of PKC. In the presence of cycloheximide, IGFBP-1 mRNA was superinduced by bGH. bGH had no posttranscriptional effect on IGFBP-1 mRNA whereas glucagon and (Bu)$ sb2$cAMP stabilized IGFBP-1 mRNA at a postranscriptional level.
In summary, bGH, T$ sb3$ and insulin inhibited whereas dexamethasone and glucagon stimulated IGFBP-1 mRNA levels in hepatocytes. Effect of glucagon may be via elevation of cAMP levels, whereas the effect of bGH may be via activation of PKC levels. The inhibitory effect of bGH appears to require synthesis of one or more protein(s) besides stimulation of PKC levels.

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Schwanhäußer, Björn. "Global analysis of cellular protein dynamics by pulse-labeling and quanti tati ve mass spectrometry." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16305.

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Der erste Teil der Arbeit beschreibt die Etablierung einer modifizierten Form des klassichen SILAC-Verfahrens, das in der quantitativen Massenspektrometrie zur Bestimmung von relativen Änderungen in Proteinmengen benutzt wird. Im sog. „pulsed SILAC (pSILAC)“ Verfahren werden Zellen im Zuge einer differentiellen Behandlung in Kulturmedien transferiert, die unterschiedlich Isotop-markierte Aminosäuren enthalten. Da hier die Quantifizierung auf dem Verhältnis der neusynthetisierten Proteinmengen beruht, können gezielt Unterschiede in der Proteinproduktion bestimmt werden. Mit Hilfe von pSILAC konnte im zweiten Teil der Arbeit erstmals quantitativ erfasst werden, welchen Einfluss microRNAs auf die Proteinsynthese ausüben. So konnte gezeigt werden, dass sowohl die Überexpression als auch die Repression einzelner microRNAs die Produktion hunderter Proteine beeinflussen kann. Außerdem konnten Genprodukte identifiziert werden, die ausschließlich translational reguliert werden. Die Messung von Proteinneusynthese ermöglichte auch die Bestimmung von Proteinumsatzraten, dargestellt im dritten Teil der Arbeit. Zusammen mit mRNA-Umsatzraten sowie Protein- und mRNA-Mengen bilden sie die Grundlage für eine dynamische Beschreibung zelluärer Genexpression. Durch den gleichzeitigen Einsatz des Nukleosidanalogons 4-Thiouridin (4sU) und von schweren Aminosäuren (SILAC) konnte eine metabolische Markierung neusynthetiserter mRNAs und Proteine in murinen Fibroblasten erreicht und damit eine Berechnung von Protein- und mRNA-Halbwertszeiten und absoluten Mengen für ca. 5,000 Gene ermöglicht werden. Während mRNA- und Proteinenmengen deutlich korrelierten, war zwischen mRNA- und Proteinhalbwertszeiten nur eine äußerste schwache Korrelation zu erkennen. Dennoch stehen mRNA- und Proteinumsatzraten nicht einem willkürlichen Zusammhang zu einander, da bestimmte Kombinationen von mRNA- und Proteinhalbwertszeiten eine Optimierung von Genen hinsichtlich ihrer biologischen Funktionen erkennen ließen.
The first part of the thesis describes the establishment of a modified version of the classic SILAC approach routinely used in quantitative mass spectrometry (MS) to assay relative changes in protein levels. In the newly-devised approach termed pulsed SILAC (pSILAC) differentially treated cells are transferred to culture medium supplemented with different versions of stable-isotope labeled heavy amino acids. As MS-based relative quantification is exclusively based on the newly-synthesized heavy protein amounts the method enables the detection of differences in protein production resulting from the treatment. The second part of the thesis shows the use of pSILAC to globally quantify the impact of microRNAs onto the proteome. Ectopic over-expression or knock-down of a single microRNA both affected protein production of hundreds of proteins. pSILAC identified several target genes as exclusively translationally regulated as changes in corresponding transcript levels were virtually absent. Measuring newly-synthesized protein amounts with heavy amino acids in a pulsed-labeling fashion has also been used to determine turnover rates of individual proteins, described in the third part of the present work. Along with transcript turnover as well as mRNA and protein levels they are essential for a dynamic description of gene expression. Simultaneous application of the nucleoside analogue 4-thiouridine (4sU) and heavy amino acids (SILAC) to metabolically label newly-produced mRNAs and proteins in mouse fibroblasts resulted in the calculation of mRNA and protein lifetimes and absolute levels for approximately 5,000 genes. While mRNA and protein levels were overall well correlated, a correlation between mRNA and protein half-lives was virtually absent. Yet this seemingly chaotic distribution of mRNA and protein half-lives was highly instructive since specific gene subsets have obviously evolved distinct combinations of half-lives that relate to their biological functions.

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Van, Rooyen Marina. "The modulating effect of myo-inositol and other antidepressants on the mRNA levels and protein expression of selected subcellular enzymes / Marina van Rooyen." Thesis, North-West University, 2005. http://hdl.handle.net/10394/504.

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myo-lnositol (mIns), a natural component of the human diet and essential precursor of several signalling pathways, including that of G protein-coupled receptors, has also been shown to be effective in the treatment of psychiatric disorders such as depression, obsessive compulsive disorder and panic disorder. Most likely since mlns is a simple isomer of glucose, no serious side effects have been reported with its use, even at high oral doses of mlns. Previous studies suggest that the therapeutic action of mlns may include reduced serotonin 5HTzA and muscarinic acetylcholine receptor function. An important signal transduction system that may possibly be involved in the mechanism of action of antidepressants is phosphoinositide (PI) turnover. In this signalling system PI-phospholipase C (PLCpl), that is implicated in the in the mechanism of action of antidepressants and anxiolytics, is activated. The mechanism of action of mlns, however, still remains elusive and needs further investigation. In this study a possible modulatory role of 24-hour pre-treatment of human neuroblastoma cell line (SH-SY5Y) with mlns on mRNA levels and protein expression of phospholipase C-p1 (PLCP1) and glycogen synthase kinase 3P (GSK3p) was investigated. The effects of mlns were also compared to that of other prototype antidepressants, such as fluoxetine (a selective serotonin reuptake inhibitor), imipramine (a tricyclic antidepressant), lithium and another drug with potential antidepressant effects, sildenafil (phosphodiesterase 5-type (PDE5) inhibitor). Real-time reverse transcription Polymerase Chain Reaction (RTPCR) was performed in order to investigate the mRNA levels, while protein expression in membranes and the cytosol fraction of cells were quantified with Western blots. The expression of PLCPl was decreased after pre-treatments with imipramine or myoinositol in combination with fluoxetine. In addition, sildenafil alone or in combination with myo-inositol, also decreased the expression of membrane-bound PLCp1. However, a 24- hour pre-treatment with lithium did not alter PLCPl expression significantly. Determined mRNA levels for the expression of PLCPl were consistent in these findings, except for the inhibition of the mRNA for the expression of PLCPl also after lithium treatment. The reduced PLCpl mRNA levels after lithium pre-treatment may suggest the involvement of posttranscriptional modification (or delayed translational effects) of PLCpl after lithium treatment. The data from the current study suggest that antidepressant action may include downregulation of PLCPl expression and that modulators of the nitric oxidecGMP pathway (e.g. sildenafil as a PDE5 inhibitor) may exhibit similar properties.
Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

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Peter, Martina Andrea. "Growth hormone-dependent expression and regulation of insulin-like growth factor (IGF) I and IGF binding protein mRNA levels in rat tissues in vivo /." [S.l.] : [s.n.], 1993. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10291.

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Kogan, Cary. "The expression of neurofilament protein and mRNA levels in the lateral geniculate nucleus and area V1 of the developing and adult vervet monkey (Ceorcopithicus aethiops) /." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0028/MQ50807.pdf.

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Beyerle, Jolantha [Verfasser], and Heinz [Akademischer Betreuer] Schmeiser. "Assessment of mRNA, protein levels and activities of xenobiotic metabolizing enzymes in colon and rectal mucosa of colorectal cancer patients / Jolantha Beyerle ; Betreuer: Heinz Schmeiser." Heidelberg : Universitätsbibliothek Heidelberg, 2016. http://d-nb.info/1180736281/34.

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Arslan, Sevki. "Effects Of Benzene On Liver, Kidney And Lung Cyp1a, Cyp2b4, Cyp2e1 And Cyp3a6 Mrna, Protein Level, And Drug Metabolizing Enzyme Activities And Toxicity In Diabetic Rabbits." Phd thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/3/12609446/index.pdf.

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The effects of diabetes on cytochrome P450 dependent drug metabolizing enzymes have not to be clarified yet. The most widely used animals in these studies have been rats, and information regarding the effects of diabetes on cytochrome P450 dependent procarcinogen/carcinogen metabolism in rabbits is limited. In the present study, we investigated, for the first time, the influence of benzene on liver, kidney and lung microsomal cytochrome P450 dependent drug metabolizing enzyme activities, protein and mRNA levels in diabetic and non-diabetic rabbits. Male New Zealand rabbits were made diabetic by a single dose of alloxan treatment in this study. AST, ALT and LDH enzyme activities in the blood serum and lipid peroxidation in liver microsomes were found to increase in diabetic, benzene treated and benzene treated diabetic rabbits. Besides these, CYP2E1 dependent NDMA N-demethylase and p-nitrophenol hydroxylase activities and CYP2E1 protein level were found to increase in liver and kidney of diabetic and benzene-treated rabbits. The combined effects of benzene and diabetes on these activities and protein level were found to be additive. Although diabetes caused induction of pulmonary CYP2E1 protein level and associated enzyme activities, benzene treatment of rabbits resulted in no change in enzyme activities and protein level in lung. The level of mRNA was investigated by Real-Time PCR. Accordingly, hepatic CYP2E1 mRNA level was increased 6.71-, 10.53- and 12.93-fold in diabetic, benzene treated and benzene treated diabetic rabbits with respect to the control animals. Similarly, renal CYP2E1 mRNA level was found in increase in these rabbits. In addition to CYP2E1, CYP3A6 associated enzyme activity, erythromycin N-demethylase, CYP3A6 protein and mRNA level were found to increase in diabetic rabbit liver and lung. Unlike diabetes, benzene treatment caused suppression of CYP3A6 protein and inhibition of associated enzyme activity in liver. There was no significant change in the erythromycin N-demethylase activity and CYP3A6 level of liver and lung as a result of benzene treatment of diabetic rabbits. Moreover, diabetes induced CYP1A2 protein and mRNA level and CYP1A associated enzyme activities in the rabbit liver. On the other hand, benzene caused statistically insignificant decreases in CYP1A dependent enzyme activities and CYP1A2 protein level in liver. CYP1A associated enzyme activities, CYP1A2 protein and mRNA levels were not changed in the liver of benzene treated diabetics. The results of the present work indicate that both diabetes and benzene stimulate metabolic activation toxic chemicals metabolized by CYP2E1 such as NDMA and benzene by inducing CYP2E1 which results in the formation of increased amounts of reactive metabolites. Application of benzene to diabetic rabbits further elevates expression and activities of the CYP2E1. As a result of additive induction of the CYP2E1 in benzene treated diabetics, further increase the risk of hepatotoxicity produced by toxins may be observed when compared to the separate treatments. This may in turn further potentiate the risk of organ toxicity and mutagenesis in liver and kidney of these subjects. As in the case of CYP2E1, the risk of carcinogenesis due to induction of CYP1A may be increased in diabetic subjects. Moreover, in diabetic and benzene exposed subjects, alteration of drug clearance and clinical drug toxicity may be observed due to induction or suppression of CYP3A.

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Beresford, Guy William. "Control of glucokinase and mRNA levels in hepatocytes." Thesis, University of Newcastle Upon Tyne, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386292.

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Books on the topic "Protein and mRNA levels"

Lynne, Maquat, ed. Nonsense-mediated mRNA decay. Georgetown, Tex: Landes Bioscience, 2006.

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Berry, Fred Brandon. Calmodulin protein and mRNA expression during postmatal development of the rat brain. Ottawa: National Library of Canada, 1995.

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J, Kay, Ballard F. J, and Mayer R. J, eds. Gene expression: Regulation at the RNA and protein levels. London: Biochemical Society, 1989.

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Kovalchuke, Lyudmila. Regulation of Parkin Protein Levels by L-3,4-dihydroxyphenylalanine. [New York, N.Y.?]: [publisher not identified], 2018.

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D, Takezawa, and United States. National Aeronautics and Space Administration., eds. Calmodulin gene family in potato: Developmental and touch-induced expression of the mRNA encoding a novel isoform. [Washington, D.C: National Aeronautics and Space Administration, 1997.

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Nargolwalla, Cyra. Modulation of mRNA levels for tPA, PAI-1 & TGF-gbs in rat testicular somatic cells. Ottawa: National Library of Canada = Bibliothèque nationale du Canada, 1991.

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Manzerra, Pasquale. Expression of constitutive hsc70 and stress-inducible hsp70 mRNA and protein in the rabbit central nervous system. Ottawa: National Library of Canada = Bibliothèque nationale du Canada, 1997.

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Stevens, Miata Yvette. Corticotropin-releasing hormone receptor subtype 1 and subtype mRNA expression and protein localization in the myometruim in pregnanct. Ottawa: National Library of Canada, 1998.

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Myllynen, Jenni Eileen. The effect of nerve-mediated activity on the expression of Muscle Regulatory Factor and Myosin Heavy Chain Protein mRNA transcripts. Sudbury, Ont: Laurentian University, Behavioural Neuroscience Program, 1997.

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Owh, Phillip. Myelin basic protein up-regulates CD44 levels in an astrocytoma cell line in vitro. Ottawa: National Library of Canada, 1996.

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Book chapters on the topic "Protein and mRNA levels"

Matsumoto, Ayaka, Nariaki Matsuura, and Miki Hieda. "Detection of SUN1 Splicing Variants at the mRNA and Protein Levels in Cancer." In The LINC Complex, 307–19. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-8691-0_21.

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Arnoldi, Michele, Giulia Zarantonello, Stefano Espinoza, Stefano Gustincich, Francesca Di Leva, and Marta Biagioli. "Design and Delivery of SINEUP: A New Modular Tool to Increase Protein Translation." In Methods in Molecular Biology, 63–87. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2010-6_4.

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AbstractSINEUP is a new class of long non-coding RNAs (lncRNAs) which contain an inverted Short Interspersed Nuclear Element (SINE) B2 element (invSINEB2) necessary to specifically upregulate target gene translation. Originally identified in the mouseAS-Uchl1 (antisense Ubiquitin carboxyl-terminal esterase L1) locus, natural SINEUP molecules are oriented head to head to their sense protein coding, target gene (Uchl1, in this example). Peculiarly, SINEUP is able to augment, in a specific and controlled way, the expression of the target protein, with no alteration of target mRNA levels. SINEUP is characterized by a modular structure with the Binding Domain (BD) providing specificity to the target transcript and an effector domain (ED)—containing the invSINEB2 element—able to promote the loading to the heavy polysomes of the target mRNA. Since the understanding of its modular structure in the endogenous AS-Uchl1 ncRNA, synthetic SINEUP molecules have been developed by creating a specific BD for the gene of interest and placing it upstream the invSINEB2 ED. Synthetic SINEUP is thus a novel molecular tool that potentially may be used for any industrial or biomedical application to enhance protein production, also as possible therapeutic strategy in haploinsufficiency-driven disorders.Here, we describe a detailed protocol to (1) design a specific BD directed to a gene of interest and (2) assemble and clone it with the ED to obtain a functional SINEUP molecule. Then, we provide guidelines to efficiently deliver SINEUP into mammalian cells and evaluate its ability to effectively upregulate target protein translation.

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Zhou, Haiyan. "Design of Bifunctional Antisense Oligonucleotides for Exon Inclusion." In Methods in Molecular Biology, 53–62. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2010-6_3.

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AbstractBifunctional antisense oligonucleotide (AON) is a specially designed AON to regulate pre-messenger RNA (pre-mRNA) splicing of a target gene. It is composed of two domains. The antisense domain contains sequences complementary to the target gene. The tail domain includes RNA sequences that recruit RNA binding proteins which may act positively or negatively in pre-mRNA splicing. This approach can be designed as targeted oligonucleotide enhancers of splicing, named TOES, for exon inclusion; or as targeted oligonucleotide silencers of splicing, named TOSS, for exon skipping. Here, we provide detailed methods for the design of TOES for exon inclusion, using SMN2 exon 7 splicing as an example. A number of annealing sites and the tail sequences previously published are listed. We also present methodology of assessing the effects of TOES on exon inclusion in fibroblasts cultured from a SMA patient. The effects of TOES on SMN2 exon 7 splicing were validated at RNA level by PCR and quantitative real-time PCR, and at protein level by western blotting.

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Smeland, Erlend B., Tore Godal, Klaus Beiske, Rosemary Watt, Susan Pfeifer-Ohlsson, and Rolf Ohlsson. "Regulation of c-myc mRNA and Protein Levels During Activation of Normal Human B Cells." In Current Topics in Microbiology and Immunology, 290–96. Berlin, Heidelberg: Springer Berlin Heidelberg, 1986. http://dx.doi.org/10.1007/978-3-642-71562-4_43.

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Bradbury, Joshua J., Holly E. Lovegrove, Marta Giralt-Pujol, and Shane P. Herbert. "Analysis of mRNA Subcellular Distribution in Collective Cell Migration." In Cell Migration in Three Dimensions, 389–407. New York, NY: Springer US, 2023. http://dx.doi.org/10.1007/978-1-0716-2887-4_22.

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AbstractThe movement of groups of cells by collective cell migration requires division of labor between group members. Therefore, distinct cell identities, unique cell behaviors, and specific cellular roles are acquired by cells undergoing collective movement. A key driving force behind the acquisition of discrete cell states is the precise control of where, when, and how genes are expressed, both at the subcellular and supracellular level. Unraveling the mechanisms underpinning the spatiotemporal control of gene expression in collective cell migration requires not only suitable experimental models but also high-resolution imaging of messenger RNA and protein localization during this process. In recent times, the highly stereotyped growth of new blood vessels by sprouting angiogenesis has become a paradigm for understanding collective cell migration, and consequently this has led to the development of numerous user-friendly in vitro models of angiogenesis. In parallel, single-molecule fluorescent in situ hybridization (smFISH) has come to the fore as a powerful technique that allows quantification of both RNA number and RNA spatial distribution in cells and tissues. Moreover, smFISH can be combined with immunofluorescence to understand the precise interrelationship between RNA and protein distribution. Here, we describe methods for use of smFISH and immunofluorescence microscopy in in vitro angiogenesis models to enable the investigation of RNA and protein expression and localization during endothelial collective cell migration.

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Mirlekar, Bhalchandra, Daniel Michaud, and Yuliya Pylayeva-Gupta. "IL-35 Detection in B Cells at the mRNA and Protein Level." In Methods in Molecular Biology, 125–47. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1237-8_8.

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Fawcett, Tony, William J. Simon, John Shanklin, and Antoni R. Slabas. "Expression of MRNA and Steady-State Levels of Protein Isoforms of Enoyl-ACP Reductase From Brassica napus." In Plant Lipid Metabolism, 90–92. Dordrecht: Springer Netherlands, 1995. http://dx.doi.org/10.1007/978-94-015-8394-7_25.

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Boudreau-Larivière, Céline, Roxanne Y. Y. Chan, and Bernard J. Jasmin. "Activity-Linked Regulation of Acetylcholinesterase mRNA Levels Involves Distinct Molecular Mechanisms in Developing Versus Adult Skeletal Muscles." In Structure and Function of Cholinesterases and Related Proteins, 128–29. Boston, MA: Springer US, 1998. http://dx.doi.org/10.1007/978-1-4899-1540-5_26.

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Lackner, K. J., W. März, G. Wolter, H. Sartor, and W. Gross. "Anabolic Steroids do not Change mRNA Levels and Protein Secretion of Apolipoprotein A-I and B-100 in HepG2 Cells." In Recent Developments in Lipid and Lipoprotein Research, 151–56. Berlin, Heidelberg: Springer Berlin Heidelberg, 1993. http://dx.doi.org/10.1007/978-3-642-84855-1_18.

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Matsuno, Akira, Yudo Ishii, Mineko Murakami, Katsumi Hoya, Shoko M. Yamada, Shinya Miyamoto, Jae-Hyun Son, et al. "Quantum Dot-Based In Situ Hybridization and Immunohistochemistry to Detect mRNA and Protein at Subcellular Levels, Comparison with Studies Using Electron Microscopy." In In Situ Hybridization Methods, 417–25. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-2303-8_22.

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Conference papers on the topic "Protein and mRNA levels"

Ohlsson, M., A. J. W. Hsueh, and T. Ny. "HORMONE REGULATION OF THE FIBRINOLYTIC SYSTEM IN THE OVARY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644389.

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In the ovary, the release of oocytes from graafian follicles during hormone-induced ovulation has been found to be associated with substantial increases in follicular plasminogen activator (PA) activity. Most of the PA activity comes from the granulosa cells that have been shown to produce tPA, uPA as well as the type-1 PA-inhibitor,(PAI-1).We have studied the molecular mechanism of follicle stimulating hormone (FSH) and gonadotropin releasing hormone (GnRH) on the synthesis of tPA in primary cultures of rat granulosa cells. FSH and GnRH were both found to induce tPA in granulosa cells in a time and dose dependent manner. The effect of FSH and GnRH on the levels of tPA mRNA was also studied by northern and slot blot hybridizations. FSH and GnRH were both found to increase the level of tPA mRNA. The stimulation was up to 18 -fold compared to untreated cells.The induction of tPA mRNA by FSH and GnRH was additive and the time courses of the stimulation by the hormones differed, suggesting that different cellular mechanisms are involved. Consistent with the ability of FSH to activate the cAMP dependent protein kinase A pathway, the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine further enhanced the FSH induction of tPA mRNA.GnRH is known to activate the phospholipid-dependent protein kinase C pathway. Likewise the effect of GnRH can be mimicked by the kinase C activator, phorbol myristate acetate.It is concluded that FSH and GnRH regulates tPA production by differnt molecular mechanisms, and that the increase in tPA activity is mediated via an increase in the levels tPA mRNA. Since both gonadotropins and GnRH cause ovulation in hyposectomized animals, similar stimulatory actions of these hormones on the tPA activity suggest a correlative relationship between this enzyme and the ovulatory process.

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Msdcalf, R. L., E. K. O. Kruithof, and W.-D. Schleuning. "TRANSIENT INDUCTION OF PLASMINOGEN ACTIVATOR INHIBITOR 2 (PAI-2) GENE TRANSCRIPTION BY THE TUMOUR PROMOTING PHORBOL ESTER PMA IN THE HUMAN MACROPHAGE-LIKE CELT, LINE U-937." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642857.

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The human hematopoietic cell line U-937 differentiates to a macrophage/monocyte phenotype after exposure to the tumour promoter PMA. We have recently shown that PMA concomitantly induces PAI-2 biosynthesis in these cells. Now, we have employed an 1880 base pair cDNA to study PAI-2 biosynthesis on the level of transcription and mRNA stability. By in vitro elongation of initiated PAI-2 transcripts in isolated nuclei in the presence of 32p-labelled UTP followed by hybridization to cloned DNA (“run-on” transcription assay), we have demonstrated that PAI-2 gene transcription rates are induced 50-fold after exposure of the cells to PMA for 4 h in the presence of 5% fetal bovine serum. Transcription rates subsequently declined to base-line levels within 48 h. This transient increase of PAI-2 transcription rate was reflected by a transient induction of PAI-2 mRNA with maximal levels (60-fold) occuring after 16 h and subsiding to near base-levels after 48 h. Similar experiments performed in the absence of fetal bovine serum revealed that maximal levels of PAI-2 mRNA occured after only 4 hours exposure to PMA and were virtually absent after 16 h. Our results indicate that PMA induction of PAI-2 biosynthesis occurs at the level of gene transcription and is influenced by the presence of serum. Since PAI-2 is a major protein of U-937 cells, these date also suggest a role for hormonally modulated PAI-2 biosynthesis in macrophage physiology.

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Apostolatos, Christopher, Tracess Smalley, and Mildred A. Duncan. "Abstract 3934: Analysis of PKC-ζ protein and mRNA levels in normal and malignant breast tissue." In Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA. American Association for Cancer Research, 2016. http://dx.doi.org/10.1158/1538-7445.am2016-3934.

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Schützle, H., M. Weis-Klemm, RE Gay, S. Gay, and WK Aicher. "THU0049 Protein kinase inhibition enhances steady state mrna levels encoding il -16 but reduces il-18 encoding message levels in fibroblasts." In Annual European Congress of Rheumatology, Annals of the rheumatic diseases ARD July 2001. BMJ Publishing Group Ltd and European League Against Rheumatism, 2001. http://dx.doi.org/10.1136/annrheumdis-2001.846.

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Andreasen, P. A., A. Riccio, L. R. Lund, K. G. Welinder, F. Blasi, and K. Danø. "PLASMINOGEN ACTIVATOR INHIBITOR TYPE 1: STUDIES ON STRUCTURE AND REGULATION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642810.

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Human plasminogen activator inhibitor type-1 is an Mr∼54,000 protein which specifically inhibits urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators. During inhibition, u-PA and t-PA convert PAI-1 to an inactive form with Mr∼50,000. We have determined the amino-terminal amino acid sequence of native and converted PAI-1, and isolated and partly sequenced PAI-1 cDNA. The data show that the conversion of PAI-1 consists of cleavage of an Arg-Met bond 33 residues from the carboxy-terminus, thus localizing the reactive center of the inhibitor to that position, and identifying PAI-1 as an "arg-serpin". PAI-1 activity is known to be influenced by a number of agents; we have studied the mechanisms of the stimulation of PAI-1 activity by transforming growth factor-β (TGF-β) and the synthetic glucocorticoid dexame-thasone in human WI-38 lung fibroblasts and HT-1080 fibrosarcoma cells. Bytheuse of PAI-1 cDNA, TGF-β was found to course a rapid increase in PAI-1 mRNA level in WI-38 cells, reaching a maximal 50-fold enhancement after 8 hours. Dexamethasone caused a 10-fold increase in PAI-1 mRNA in HT-1080 cells, which was detectable after 4 hours and became maximal after 16 hours. In both cases, the 3.4 as well as the 2.4 Kb-PAI-1-mRNA species were increased. Quantitative studies on the effect of these agents on PAI-1 protein levels in cell extracts and culture media by ELISA gave results consistent with the effects on PAI-1 mRNA. These studies suggest that TGF-β and glucocorticoids may exert important controls over plasminogen activation-mediated extracellular proteolysis through an enhancement of PAI-1 gene transcription.

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Antonacopoulou, A., A. Kottorou, V. Tzelepi, F. Dimitrakopoulos, T. Makatsoris, A. Koutras, and H. Kalofonos. "PO-513 The alternative NF-κB pathway in colorectal cancer. from genetic polymorphisms through mRNA to protein levels." In Abstracts of the 25th Biennial Congress of the European Association for Cancer Research, Amsterdam, The Netherlands, 30 June – 3 July 2018. BMJ Publishing Group Ltd, 2018. http://dx.doi.org/10.1136/esmoopen-2018-eacr25.1014.

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Medcalf, R. L., E. van den Berg, and W.-D. Schleuning. "THE INFLUENCE OF GLUCOCORTICOID HORMONES ON THE GENE TRANSCRIPTION OF POUR COMPONENTS OF THE FIBRINOLYTIC SYSTEM." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644611.

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The hormonal regulation of plasminogen activator (urokinase type (u-PA) and tissue-type (t-PA)) biosynthesis plays an important role in fibrinolysis and extracellular matrix turnover during invasive growth and cell migration. Recently, two genetically distinct inhibitors of both PA's (PA inhibitor 1 (PAI-1) and PA inhibitor 2 (PAI-2)) have been described which may contribute to the modulation of matrix stability. We have employed cloned cDNA probes to study the regulation of biosynthesis of these proteins in the human fibrosarcoma line HT1080. These cells constitutively express high levels of pro-u-PA. PAI-1, PAI-2 and t-PA are also present at relatively low levels. Treatment of the cells with the glucocorticoid dexamethasone (Dex; 10−7 M), almost completely suppresses u-PA gene transcription, as determined by measurement of in vitro elongation of initiated u-PA transcripts in isolated nuclei ("run-on" transcription assay). Concomitantly, Dex also induces PAI-1 and t-PA gene transcription, whereas PAI-2 gene transcription appeared to remain ' unaffected. These changes in transcription rates are also reflected at the level of mRNA: u-PA mRNA is decreased, whereas PAI-1 and t-PA mRNA are simultaneously induced. PAI-2 mRNA is apparently unchanged. These results demonstrate that glucocorticoid hormones reprogramne the expression of components of the fibrinolytic system, and are of possible relevance in the context of inflammatory disease and malignancy.

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You, Jun, Clare E. Yellowley, Henry J. Donahue, and Christopher R. Jacobs. "Physiological Levels of Substrate Deformation Are Less Stimulatory to Bone Cells Compared to Fluid Flow." In ASME 1999 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 1999. http://dx.doi.org/10.1115/imece1999-0427.

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Abstract It is believed that bone cells can sense mechanical loading and alter bone external shape and internal structure to efficiently support the load bearing demands placed upon it. However, the mechanism by which bone cells sense and respond to their mechanical environment is still poorly understood. In particular, the load-induced signals to which bone cells respond, e.g. fluid flow, substrate deformation, electrokinetic effects etc., are unclear. Furthermore, there are few studies focused on the effects of physiological strain (strain < 0.5%, Burr, 1996; Owan, 1997) on bone cells. The goal of this study was to investigate cytosolic Ca2+ mobilization (a very early signaling event) in response to different substrate strains (physiological or supra-physiological strains), and to distinguish the effects of substrate strain from those of fluid flow by applying precisely controlled strain without induced fluid flow. In addition, we quantified the effect of physiologically relevant fluid flow (Cowin, 1995) and substrate stretch on the expression of mRNA for the bone matrix protein osteopontin (OPN). A computer controlled stretch device was employed to apply different substrate strains, 0.1%, 1%, 5% and 10%. A parallel plate flow chamber was used to test cell responses to steady and oscillating flows (20dyn/cm2, 1Hz). Our data demonstrate that physiological strain (< 0.5%) does not induce [Ca2+]i responses in primary rat osteoblastic cells (ROB) in vitro. However, there was a significant (p < 0.05) increase in the number of responding cells at supra-physiological strains of 1, 5, and 10% suggesting that the cells were capable of a biological response. Similar results for human fetal osteoblastic cells (hFOB 1.19) and osteocyte-like cells (ML0-Y4) were obtained. Furthermore, compared to physiological substrate deformation, physiological fluid flow induced greater [Ca2+]i responses for hFOB cells, and these [Ca2+]i responses were quantitatively similar to those obtained for 10% substrate strain. Moreover we found no change in osteopontin mRNA expression after 0.5% strain stretch. Conversely, physiological oscillating flow (20dyn/cm2, 1Hz) caused a significant increase in osteopontin mRNA. These data suggest that, relative to fluid flow, substrate deformation may play less of a role in bone cell mechanotransduction.

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Killian, Megan L., and Tammy L. Haut Donahue. "Effect of Pathological and Physiological Loads on Interleukin-1α Protein Production in Porcine Menisci." In ASME 2008 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2008. http://dx.doi.org/10.1115/sbc2008-192145.

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The meniscus performs several functions for the maintenance of knee joint health, such as load transmission and joint stability. Meniscal lesions have been suggested as a precursor to the onset of osteoarthritis (OA)[1]. Such lesions often lead to surgical removal of the torn portion of the meniscus, increasing cartilage to cartilage contact area. Partial meniscectomies have been shown using finite element analysis and histology to lead to altered and increased mechanical loading on the remaining meniscus and underlying articular cartilage[2,3]. Consequently, pathological compressive strains of more than 15% have been shown to increase proteoglycan breakdown and meniscal matrix degradation[4]. Preliminary investigations in our laboratory have demonstrated an increase in interleukin-1α (IL-1α) gene expression of meniscal explants subjected to pathological levels of dynamic compressive strain [6,7]. This inflammatory cytokine has been attributed to apoptosis and matrix degradation[5]. However, gene expression measurements merely suggest possible matrix remodeling mechanisms and do not necessarily result in protein syntheses from which matrix changes occur. Therefore, the purpose of this study was to quantify protein synthesis of IL-1α in porcine meniscal implants after compressive strain exercises. It was hypothesized that, similar to mRNA expression, protein synthesis for pathologically loaded (0 or 20% dynamic strain) samples would be greater than samples loaded to physiological levels (10% strain).

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Wheeler, Sarah, Raja Seethala, Doris Siwak, Kathleen Cieply, Carol Sherer, Gordon Mills, Jennifer R. Grandis, and Ann Marie Egloff. "Abstract 4646: Evaluation of EGFR gene amplification status, mRNA, protein, and phosphoprotein levels expression in head and neck cancer patient tissues." In Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC. American Association for Cancer Research, 2010. http://dx.doi.org/10.1158/1538-7445.am10-4646.

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Reports on the topic "Protein and mRNA levels"

Barash, Itamar, and Robert E. Rhoads. Translational Mechanisms that Govern Milk Protein Levels and Composition. United States Department of Agriculture, November 2004. http://dx.doi.org/10.32747/2004.7586474.bard.

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Original objectives: The long term objective of the project is to achieve higher content of protein in the milk of ruminants by modulating the translational machinery in the mammary gland. The first specific aim of the BARD proposal was to characterize responsiveness of various experimental systems to combination of lactogenic hormones and amino acids with particular emphasis on discrimination between the control of total protein synthesis and milk protein synthesis. Based on the results, we planned to proceed by characterizing the stage of protein synthesis in which the stimulation by lactogenic hormones and amino acid occur and finally we proposed to identify which components of the translation machinery are modified. Background to the topic: Milk protein is the most valuable component in milk, both for direct human consumption and for manufacturing cheese and other protein-based products. Attempts to augment protein content by the traditional methods of genetic selection and improved nutritional regimes have failed. The proposal was based on recent results suggesting that the limiting factor for augmenting protein synthesis in the bovine mammary gland is the efficiency of converting amino acids to milk proteins. Major conclusions, solutions, achievements: Insulin and prolactin synergistically stimulate â-casein mRNA translation by cytoplasmatic polyadenylation. The interaction between insulin and prolactin was demonstrated two decades ago as crucial for milk-protein synthesis, but the molecular mechanisms involved were not elucidated. We found in differentiated CID 9 mouse mammary epithelial cells line that insulin and prolactin synergistically increases the rate of milk protein mRNA translation. We focused on â-casein, the major milk protein, and found that the increase in â-casein mRNA translation was reflected in a shift to larger polysomes, indicating an effect on translational initiation. Inhibitors of the PI3K, mTOR, and MAPK pathways blocked insulin-stimulated total protein and â-casein synthesis but not the synergistic stimulation. Conversely, cordycepin, a polyadenylation inhibitor, abolished synergistic stimulation of protein synthesis without affecting insulin-stimulated translation. The poly(A) tract of â-casein mRNA progressively increased over 30 min of treatment with insulin plus prolactin. The 3’-untranslated region of â-casein mRNA was found to contain a cytoplasmic polyadenylation element (CPE), and in reporter constructs, this was sufficient for the translational enhancement and mRNA-specific polyadenylation. Furthermore, insulin and prolactin stimulated phosphorylation of cytoplasmic polyadenylation element binding protein (CPEB) but did not increase cytoplasmic polyadenylation.

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Barash, Itamar, and Robert Rhoads. Translational Mechanisms Governing Milk Protein Levels and Composition. United States Department of Agriculture, 2006. http://dx.doi.org/10.32747/2006.7696526.bard.

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Original objectives: The long-term goal of the research is to achieve higher protein content in the milk of ruminants by modulating the translational apparatus of the mammary gland genetically, nutritionally, or pharmacologically. The short-term objectives are to obtain a better understanding of 1) the role of amino acids (AA) as regulators of translation in bovine and mouse mammary epithelial cells and 2) the mechanism responsible for the synergistic enhancement of milk-protein mRNA polyadenylation by insulin and prolactin. Background of the topic: In many cell types and tissues, individual AA affect a signaling pathway which parallels the insulin pathway to modulate rates and levels of protein synthesis. Diverse nutritional and hormonal conditions are funneled to mTOR, a multidomain serine/threonine kinase that regulates a number of components in the initiation and elongation stages of translation. The mechanism by which AA signal mTOR is largely unknown. During the current grant period, we have studied the effect of essential AA on mechanisms involved in protein synthesis in differentiated mammary epithelial cells cultured under lactogenic conditions. We also studied lactogenic hormone regulation of milk protein synthesis in differentiated mammary epithelial cells. In the first BARD grant (2000-03), we discovered a novel mechanism for mRNA-specific hormone-regulated translation, namely, that the combination of insulin plus prolactin causes cytoplasmic polyadenylation of milk protein mRNAs, which leads to their efficient translation. In the current BARD grant, we have pursued the signaling pathways of this novel hormone action. Major conclusions/solutions/achievements: The positive and negative signaling from AA to the mTOR pathway, combined with modulation of insulin sensitization, mediates the synthesis rates of total and specific milk proteins in mammary epithelial cells. The current in vitro study revealed cryptic negative effects of Lys, His, and Thr on cellular mechanisms regulating translation initiation and protein synthesis in mammary epithelial cells that could not be detected by conventional in vivo analyses. We also showed that a signaling pathway involving Jak2 and Stat5, previously shown to lead from the prolactin receptor to transcription of milk protein genes, is also used for cytoplasmic polyadenylation of milk protein mRNAs, thereby stabilizing these mRNAs and activating them for translation. Implications: In vivo, plasma AA levels are affected by nutritional and hormonal effects as well as by conditions of exercise and stress. The amplitude in plasma AA levels resembles that applied in the current in vitro study. Thus, by changing plasma AA levels in the epithelial cell microenvironment or by sensitizing the mTOR pathway to their presence, it should be possible to modulate the rate of milk protein synthesis. Furthermore, knowledge that phosphorylation of Stat5 is required for enhanced milk protein synthesis in response to lactogenic opens the possibility for pharmacologic approaches to increase the phosphorylation of Stat5 and, thereby, milk protein production.

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Young, Erin, Cem Kuscu, Christine Watkins, and Murat Dogan. Using CRISPR Gene Editing to Prevent Accumulation of Lipids in Hepatocytes. University of Tennessee Health Science Center, January 2022. http://dx.doi.org/10.21007/com.lsp.2022.0007.

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CRISPR gene editing is a molecular technology that can be used to silence gene expression. In this experiment, genes that are known to play a role in lipid accumulation in hepatocytes were targeted. Specifically, levels of fatty acid transport proteins 2 and 5 (FATP2 & 5) have been shown to be elevated in cases of non-alcoholic fatty liver disease. The goal of this experiment was to reduce expression of these genes by using a dead Cas9 (dCas9) protein with an attached inhibitory domain (KRAB) that acts on the promotor region. When measuring the mRNA expression, it was determined that the levels of the CRISPR-modified gene products were significantly reduced compared to the control. However, the same extent of inhibition was not consistently observed when conducting flow cytometry. Current work is aimed at discovering why lipid accumulation is not inhibited to the expected degree based on the results of mRNA expression.

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Stern, David, and Gadi Schuster. Manipulation of Gene Expression in the Chloroplast. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575289.bard.

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The steady-state level of a given mRNA is determined by its rates of transcription and degradation. The stabilities of chloroplast mRNAs vary during plant development, in part regulating gene expression. Furthermore, the fitness of the organelle depends on its ability to destroy non-functional transcripts. In addition, there is a resurgent interest by the biotechnology community in chloroplast transformation due to the public concerns over pollen transmission of introduced traits or foreign proteins. Therefore, studies into basic gene expression mechanisms in the chloroplast will open the door to take advantage of these opportunities. This project was aimed at gaining mechanistic insights into mRNA processing and degradation in the chloroplast and to engineer transcripts of varying stability in Chlamydomonas reinhardtii cells. This research uncovered new and important information on chloroplast mRNA stability, processing, degradation and translation. In particular, the processing of the 3' untranslated regions of chloroplast mRNAs was shown to be important determinants in translation. The endonucleolytic site in the 3' untranslated region was characterized by site directed mutagensis. RNA polyadenylation has been characterized in the chloroplast of Chlamydomonas reinhardtii and chloroplast transformants carrying polyadenylated sequences were constructed and analyzed. Data obtained to date suggest that chloroplasts have gene regulatory mechanisms which are uniquely adapted to their post-endosymbiotic environment, including those that regulate RNA stability. An exciting point has been reached, because molecular genetic studies have defined critical RNA-protein interactions that participate in these processes. However, much remains to be learned about these multiple pathways, how they interact with each other, and how many nuclear genes are consecrated to overseeing them. Chlamydomonas is an ideal model system to extend our understanding of these areas, given its ease of manipulation and the existing knowledge base, some of which we have generated.

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Meidan, Rina, and Robert Milvae. Regulation of Bovine Corpus Luteum Function. United States Department of Agriculture, March 1995. http://dx.doi.org/10.32747/1995.7604935.bard.

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The main goal of this research plan was to elucidate regulatory mechanisms controlling the development, function of the bovine corpus luteum (CL). The CL contains two different sterodigenic cell types and therefore it was necessary to obtain pure cell population. A system was developed in which granulosa and theca interna cells, isolated from a preovulatory follicle, acquired characteristics typical of large (LL) and small (SL) luteal cells, respectively, as judged by several biochemical and morphological criteria. Experiments were conducted to determine the effects of granulosa cells removal on subsequent CL function, the results obtained support the concept that granulosa cells make a substaintial contribution to the output of progesterone by the cyclic CL but may have a limited role in determining the functional lifespan of the CL. This experimental model was also used to better understand the contribution of follicular granulosa cells to subsequent luteal SCC mRNA expression. The mitochondrial cytochrome side-chain cleavage enzyme (SCC), which converts cholesterol to pregnenolone, is the first and rate-limiting enzyme of the steroidogenic pathway. Experiments were conducted to characterize the gene expression of P450scc in bovine CL. Levels of P450scc mRNA were higher during mid-luteal phase than in either the early or late luteal phases. PGF 2a injection decreased luteal P450scc mRNA in a time-dependent manner; levels were significantly reduced by 2h after treatment. CLs obtained from heifers on day 8 of the estrous cycle which had granulosa cells removed had a 45% reduction in the levels of mRNA for SCC enzymes as well as a 78% reduction in the numbers of LL cells. To characterize SCC expression in each steroidogenic cell type we utilized pure cell populations. Upon luteinization, LL expressed 2-3 fold higher amounts of both SCC enzymes mRNAs than SL. Moreover, eight days after stimulant removal, LL retained their P4 production capacity, expressed P450scc mRNA and contained this protein. In our attempts to establish the in vitro luteinization model, we had to select the prevulatory and pre-gonadotropin surge follicles. The ratio of estradiol:P4 which is often used was unreliable since P4 levels are high in atretic follicles and also in preovulatory post-gonadotropin follicles. We have therefore examined whether oxytocin (OT) levels in follicular fluids could enhance our ability to correctly and easily define follicular status. Based on E2 and OT concentrations in follicular fluids we could more accurately identify follicles that are preovulatory and post gonadotropin surge. Next we studied OT biosynthesis in granulosa cells, cells which were incubated with forskolin contained stores of the precursor indicating that forskolin (which mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release. While studying in vitro luteinization, we noticed that IGF-I induced effects were not identical to those induced by insulin despite the fact that megadoses of insulin were used. This was the first indication that the cells may secrete IGF binding protein(s) which regonize IGFs and not insulin. In a detailed study involving several techniques, we characterized the species of IGF binding proteins secreted by luteal cells. The effects of exogenous polyunsaturated fatty acids and arachidonic acid on the production of P4 and prostanoids by dispersed bovine luteal cells was examined. The addition of eicosapentaenoic acid and arachidonic acid resulted in a dose-dependent reduction in basal and LH-stimulated biosynthesis of P4 and PGI2 and an increase in production of PGF 2a and 5-HETE production. Indomethacin, an inhibitor of arachidonic acid metabolism via the production of 5-HETE was unaffected. Results of these experiments suggest that the inhibitory effect of arachidonic acid on the biosynthesis of luteal P4 is due to either a direct action of arachidonic acid, or its conversion to 5-HETE via the lipoxgenase pathway of metabolism. The detailed and important information gained by the two labs elucidated the mode of action of factors crucially important to the function of the bovine CL. The data indicate that follicular granulosa cells make a major contribution to numbers of large luteal cells, OT and basal P4 production, as well as the content of cytochrome P450 scc. Granulosa-derived large luteal cells have distinct features: when luteinized, the cell no longer possesses LH receptors, its cAMP response is diminished yet P4 synthesis is sustained. This may imply that maintenance of P4 (even in the absence of a Luteotropic signal) during critical periods such as pregnancy recognition, is dependent on the proper luteinization and function of the large luteal cell.

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Christopher, David A., and Avihai Danon. Plant Adaptation to Light Stress: Genetic Regulatory Mechanisms. United States Department of Agriculture, May 2004. http://dx.doi.org/10.32747/2004.7586534.bard.

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Original Objectives: 1. Purify and biochemically characterize RB60 orthologs in higher plant chloroplasts; 2. Clone the gene(s) encoding plant RB60 orthologs and determine their structure and expression; 3. Manipulate the expression of RB60; 4. Assay the effects of altered RB60 expression on thylakoid biogenesis and photosynthetic function in plants exposed to different light conditions. In addition, we also examined the gene structure and expression of RB60 orthologs in the non-vascular plant, Physcomitrella patens and cloned the poly(A)-binding protein orthologue (43 kDa RB47-like protein). This protein is believed to a partner that interacts with RB60 to bind to the psbA5' UTR. Thus, to obtain a comprehensive view of RB60 function requires analysis of its biochemical partners such as RB43. Background & Achievements: High levels of sunlight reduce photosynthesis in plants by damaging the photo system II reaction center (PSII) subunits, such as D1 (encoded by the chloroplast tpsbAgene). When the rate of D1 synthesis is less than the rate of photo damage, photo inhibition occurs and plant growth is decreased. Plants use light-activated translation and enhanced psbAmRNA stability to maintain D1 synthesis and replace the photo damaged 01. Despite the importance to photosynthetic capacity, these mechanisms are poorly understood in plants. One intriguing model derived from the algal chloroplast system, Chlamydomonas, implicates the role of three proteins (RB60, RB47, RB38) that bind to the psbAmRNA 5' untranslated leader (5' UTR) in the light to activate translation or enhance mRNA stability. RB60 is the key enzyme, protein D1sulfide isomerase (Pill), that regulates the psbA-RN :Binding proteins (RB's) by way of light-mediated redox potentials generated by the photosystems. However, proteins with these functions have not been described from higher plants. We provided compelling evidence for the existence of RB60, RB47 and RB38 orthologs in the vascular plant, Arabidopsis. Using gel mobility shift, Rnase protection and UV-crosslinking assays, we have shown that a dithiol redox mechanism which resembles a Pill (RB60) activity regulates the interaction of 43- and 30-kDa proteins with a thermolabile stem-loop in the 5' UTR of the psbAmRNA from Arabidopsis. We discovered, in Arabidopsis, the PD1 gene family consists of II members that differ in polypeptide length from 361 to 566 amino acids, presence of signal peptides, KDEL motifs, and the number and positions of thioredoxin domains. PD1's catalyze the reversible formation an disomerization of disulfide bonds necessary for the proper folding, assembly, activity, and secretion of numerous enzymes and structural proteins. PD1's have also evolved novel cellular redox functions, as single enzymes and as subunits of protein complexes in organelles. We provide evidence that at least one Pill is localized to the chloroplast. We have used PDI-specific polyclonal and monoclonal antisera to characterize the PD1 (55 kDa) in the chloroplast that is unevenly distributed between the stroma and pellet (containing membranes, DNA, polysomes, starch), being three-fold more abundant in the pellet phase. PD1-55 levels increase with light intensity and it assembles into a high molecular weight complex of ~230 kDa as determined on native blue gels. In vitro translation of all 11 different Pill's followed by microsomal membrane processing reactions were used to differentiate among PD1's localized in the endoplasmic reticulum or other organelles. These results will provide.1e insights into redox regulatory mechanisms involved in adaptation of the photosynthetic apparatus to light stress. Elucidating the genetic mechanisms and factors regulating chloroplast photosynthetic genes is important for developing strategies to improve photosynthetic efficiency, crop productivity and adaptation to high light environments.

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Abcouwer, Steve P. Demonstration that a mRNA Binding Protein is Responsible for GADD45 mRNA Destabilization. Fort Belvoir, VA: Defense Technical Information Center, May 2003. http://dx.doi.org/10.21236/ada418512.

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Woodson, William, Shimon Mayak, and Haim Rabinowitch. Physiological and Molecular Characterization of the Response to Ethylene during Senescence of Carnation Genotypic Variants. United States Department of Agriculture, July 1995. http://dx.doi.org/10.32747/1995.7613011.bard.

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The senescence of carnation (Dianthus caryophyllus L.) flowers is associated with increased production of the phytohormone ethylene, which in turn serves to initiate and regulate the processes involved in programmed petal death. We investigated the regulation of ethylene production and petal senescence in carnation. Several carnation genotypes were identified that exhibited extended vase-life in comparison to flowers from typical commercial cultivars. The capacity of these genotypes to produce ethylene during postharvest vase-life and to respond to exogenous ethylene was investigated. Several genotypes, represented by 'Sandrosa' and 87-37G produced little ethylene durig their postharvest vase-life and as a result failed to exhibit the symptoms (in-rolling and wilting) typical of flowers producing elevated levels of ethylene. These genotypes were further separated by their capacity to respond to exogenous ethylene by both increased ethylene synthesis and premature petal senescence. In one case a genotype (799) was identified that was not capable of responding to exogenous ethylene by either increased ethylene production or premature petal senescence. The regulation of ethylene production during petal senescence was investigated both at the enzyme and gene levels. A full length cDNA was identified for the petal senescence-related ACC synthase gene. Utilizing this, and other ethylene biosynthetic pathway cDNA probes, an increase in both ACC synthase and ACC oxidase mRNAs were detected following ethylene treatment. An increase in ACC oxidase mRNA and enzyme activity was detected within 2-3 h following ethylene treatment, indicating the expression of this gene is an early response to ethylene. An investigation into the expression of novel proteins during petal senescence revealed a number of polypeptides increased in abundance and possibly play a role in the regulation or biochemical processes of senescence. One polypeptide of 70 kDa was identified as being encoded by the previously characterized gene SR12 and possibly represents a b-galactosidase involved in the remobilization of carbohydrates during senescence.

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Funkenstein, Bruria, and Cunming Duan. GH-IGF Axis in Sparus aurata: Possible Applications to Genetic Selection. United States Department of Agriculture, November 2000. http://dx.doi.org/10.32747/2000.7580665.bard.

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Many factors affect growth rate in fish: environmental, nutritional, genetics and endogenous (physiological) factors. Endogenous control of growth is very complex and many hormone systems are involved. Nevertheless, it is well accepted that growth hormone (GH) plays a major role in stimulating somatic growth. Although it is now clear that most, if not all, components of the GH-IGF axis exist in fish, we are still far from understanding how fish grow. In our project we used as the experimental system a marine fish, the gilthead sea bream (Sparus aurata), which inhabits lagoons along the Mediterranean and Atlantic coasts of Europe, and represents one of the most important fish species used in the mariculture industry in the Mediterranean region, including Israel. Production of Sparus is rapidly growing, however, in order for this production to stay competitive, the farming of this fish species has to intensify and become more efficient. One drawback, still, in Sparus extensive culture is that it grows relatively slow. In addition, it is now clear that growth and reproduction are physiological interrelated processes that affect each other. In particular sexual maturation (puberty) is known to be closely related to growth rate in fish as it is in mammals, indicating interactions between the somatotropic and gonadotropic axes. The goal of our project was to try to identify the rate-limiting components(s) in Sparus aurata GH-IGF system which might explain its slow growth by studying the ontogeny of growth-related genes: GH, GH receptor, IGF-I, IGF-II, IGF receptor, IGF-binding proteins (IGFBPs) and Pit-1 during early stages of development of Sparus aurata larvae from slow and fast growing lines. Our project was a continuation of a previous BARD project and could be divided into five major parts: i) obtaining additional tools to those obtained in the previous project that are necessary to carry out the developmental study; ii) the developmental expression of growth-related genes and their cellular localization; iii) tissue-specific expression and effect of GH on expression of growth-related genes; iv) possible relationship between GH gene structure, growth rate and genetic selection; v) the possible role of the IGF system in gonadal development. The major findings of our research can be summarized as follows: 1) The cDNAs (complete or partial) coding for Sparus IGFBP-2, GH receptor and Pit-1 were cloned. Sequence comparison reveals that the primary structure of IGFBP-2 protein is 43-49% identical to that of zebrafish and other vertebrates. Intensive efforts resulted in cloning a fragment of 138 nucleotides, coding for 46 amino acids in the proximal end of the intracellular domain of GH receptor. This is the first fish GH receptor cDNA that had been cloned to date. The cloned fragment will enable us to complete the GH - receptor cloning. 2) IGF-I, IGF-II, IGFBP-2, and IGF receptor transcripts were detected by RT-PCR method throughout development in unfertilized eggs, embryos, and larvae suggesting that these mRNAs are products of both the maternal and the embryonic genomes. Preliminary RT-PCR analysis suggest that GH receptor transcript is present in post-hatching larvae already on day 1. 3) IGF-1R transcripts were detected in all tissues tested by RT-PCR with highest levels in gill cartilage, skin, kidney, heart, pyloric caeca, and brain. Northern blot analysis detected IGF receptor only in gonads, brain and gill cartilage but not in muscle; GH increased slightly brain and gill cartilage IGF-1R mRNA levels. 4) IGFBP-2 transcript were detected only in liver and gonads, when analyzed by Northern blots; RT-PCR analysis revealed expression in all tissues studied, with the highest levels found in liver, skin, gonad and pyloric caeca. 5) Expression of IGF-I, IGF-II, IGF-1R and IGFBP-2 was analyzed during gonadal development. High levels of IGF-I and IGFBP-2 expression were found in bisexual young gonads, which decreased during gonadal development. Regardless of maturational stage, IGF-II levels were higher than those of IGF-L 6) The GH gene was cloned and its structure was characterized. It contains minisatellites of tandem repeats in the first and third introns that result in high level of genetic polymorphism. 7) Analysis of the presence of IGF-I and two types of IGF receptor by immunohistochemistry revealed tissue- and stage-specific expression during larval development. Immunohistochemistry also showed that IGF-I and its receptors are present in both testicular and ovarian cells. Although at this stage we are not able to pinpoint which is the rate-limiting step causing the slow growth of Sparus aurata, our project (together with the previous BARD) yielded a great number of experimental tools both DNA probes and antibodies that will enable further studies on the factors regulating growth in Sparus aurata. Our expression studies and cellular localization shed new light on the tissue and developmental expression of growth-related genes in fish.

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Yaron, Zvi, Martin P. Schreibman, Abigail Elizur, and Yonathan Zohar. Advancing Puberty in the Black Carp (Mylopharyngodon Piceus) and the Striped Bass (Morone Saxatilis). United States Department of Agriculture, August 1993. http://dx.doi.org/10.32747/1993.7568102.bard.

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The black carp (bc)GtH IIb cDNA was amplified and isolated, cloned and sequenced. Comparison of the bcGtH IIb deduced a.a. sequence with that of GtH IIb from other teleosts revealed high homology to cyprinid species and a lower homology to salmonid or perciform fish. The gene coding for the GtH IIb was isolated and sequenced. Three bc recombinant phages which hybridized to the goldfish GtH Ib cDNA probe were isolated and are currently being characterized. The region coding for the mature GtH IIb was expressed in a bacterial expression vector resulting in the production of a recombinant protein. In vitro folding resulted in a protein only 1.3% of which displaced the native common carp GtH II in a RIA. Therefore, the common carp GtH RIA was utilized for the physiological studies at the current phase of the project. Two non-functional sites were identified along the brain-pituitary gonadal axis in the immature black carp. The pituitary is refractory to GnRH stimulation due to a block proximal to the activation of PKA and PKC probably at the level of GnRH receptors. The gonads, although capable of producing steroids, are refractory to gonadotropic stimulation but do respond to cAMP antagonists, indicating a block at the GtH receptor level. Attempts to advance puberty in 2 and 3 y old black carp showed that testosterone (T) stimulates GtH synthesis in the pituitary and increases its sensitivity to GnRh. A 2 month treatment combining T+GnRH increased the circulating GFtH level in 3 y old fish. Addition of domperidone to such a treatment facilitated both the accumulation of GtH in the pituitary and its response to GnRH. The cDNA of striped bass GtH a, Ib and IIb subunits were amplified, isolated, cloned and sequenced, and their deduced a.a. sequences were compared with those of other teleosts. A ribonuclease protection assay was developed for a sensitive and simultaneous determination of all GtH subunits, and of b-actin mRNAs of the striped bass. GnRH stimulated dramatically the expression of the a and GtH IIb subunits but the level of GtH Ib mRNA increased only moderately. These findings suggest that GtH-II, considered in salmonids to be involved only in final stages of gametogenesis, can be induced by GnRH to a higher extent than GtH-I in juvenile striped bass. The native GtH II of the striped bass was isolated and purified, and an ELISA for its determination was developed. The production of all recombinant striped bass GtH subunits is in progress using the insect cell (Sf9) culture and the BAC-TO-BAC baculovirus expression system. A recombinant GtH IIb subunit has been produced already, and its similarity to the native subunit was confirmed. The yield of the recombinant glycoprotein can reach 3.5 mg/ml after 3 days culture. All male striped bass reach puberty after 3 y. However, precocious puberty was discovered in 1 and 2 y old males. Females become vitellogenic during their 4th year. In immature 2 y old females, T treatment elevates the pituitary GtH II content while GnRH only potentiates the effect. However, in males GnRH and not T affects GtH accumulation in the pituitary. Neither GnRH, nor T treatment resulted in gonadal growth in 2 y old striped bass, indicating that either the accumulated GtH II was not released, or if released, the gonads were refractory to GtH stimulation, similar to the situation in the immature black carp. In 3 y old female striped bass, 150 day GnRHa treatment resulted in an increase in GSI, while T treatment, with or without GnRHa, resulted in a decrease in oocyte diameter, similar to the effect seen in the black carp. Further attempts to advance puberty in both fish species should take into account the positive effect of T on pituitary GtH and its negative effect of ovarian growth.

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