scholarly journals Circadian Analysis of the Mouse Cerebellum Proteome

2019 ◽  
Vol 20 (8) ◽  
pp. 1852 ◽  
Author(s):  
Marine Plumel ◽  
Stéphanie Dumont ◽  
Pauline Maes ◽  
Cristina Sandu ◽  
Marie-Paule Felder-Schmittbuhl ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Large Scale ◽  
Transcriptional Control ◽  
Triosephosphate Isomerase ◽  
Bimodal Distribution ◽  
Mitochondrial Proteins ◽  
Suprachiasmatic Nuclei ◽  
2D Dige ◽  
Rhythmic Expression ◽  
Dige Analysis

The cerebellum contains a circadian clock, generating internal temporal signals. The daily oscillations of cerebellar proteins were investigated in mice using a large-scale two-dimensional difference in gel electrophoresis (2D-DIGE). Analysis of 2D-DIGE gels highlighted the rhythmic variation in the intensity of 27/588 protein spots (5%) over 24 h based on cosinor regression. Notably, the rhythmic expression of most abundant cerebellar proteins was clustered in two main phases (i.e., midday and midnight), leading to bimodal distribution. Only six proteins identified here to be rhythmic in the cerebellum are also known to oscillate in the suprachiasmatic nuclei, including two proteins involved in the synapse activity (Synapsin 2 [SYN2] and vesicle-fusing ATPase [NSF]), two others participating in carbohydrate metabolism (triosephosphate isomerase (TPI1] and alpha-enolase [ENO1]), Glutamine synthetase (GLUL), as well as Tubulin alpha (TUBA4A). Most oscillating cerebellar proteins were not previously identified in circadian proteomic analyses of any tissue. Strikingly, the daily accumulation of mitochondrial proteins was clustered to the mid-resting phase, as previously observed for distinct mitochondrial proteins in the liver. Moreover, a number of rhythmic proteins, such as SYN2, NSF and TPI1, were associated with non-rhythmic mRNAs, indicating widespread post-transcriptional control in cerebellar oscillations. Thus, this study highlights extensive rhythmic aspects of the cerebellar proteome.

Two-Dimensional Differential in Gel Electrophoresis (2D-DIGE) Analysis of Grape Berry Proteome during Postharvest Withering

2011 ◽  
Vol 10 (2) ◽  
pp. 429-446 ◽  
Author(s):  
Mariasole Di Carli ◽  
Anita Zamboni ◽  
Mario Enrico Pè ◽  
Mario Pezzotti ◽  
Kathryn S. Lilley ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Grape Berry ◽  
Two Dimensional ◽  
2D Dige ◽  
Dige Analysis

scholarly journals The effect of pre-existing maternal obesity on the placental proteome: two-dimensional difference gel electrophoresis coupled with mass spectrometry

2012 ◽  
Vol 48 (2) ◽  
pp. 139-149 ◽  
Author(s):  
Karen Oliva ◽  
Gillian Barker ◽  
Clyde Riley ◽  
Mark J Bailey ◽  
Michael Permezel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Gel Electrophoresis ◽  
Soluble Protein ◽  
Obese Patients ◽  
Two Dimensional ◽  
2D Dige ◽  
Qrt Pcr ◽  
Difference Gel Electrophoresis ◽  
Dige Analysis

Our aim was to study the protein expression profiles of placenta obtained from lean and obese pregnant women with normal glucose tolerance at the time of term Caesarean section. We used two-dimensional difference gel electrophoresis (2D-DIGE), utilising narrow-range immobilised pH gradient strips that encompassed the broad pH range of 4–5 and 5–6, followed by MALDI-TOF mass spectrometry of selected protein spots. Western blot and quantitative RT-PCR (qRT-PCR) analyses were performed to validate representative findings from the 2D-DIGE analysis. Eight proteins were altered (six down-regulated and two up-regulated on obese placentas). Annexin A5 (ANXA5), ATP synthase subunit beta, mitochondria (ATPB), brain acid soluble protein 1 (BASP1), ferritin light chain (FTL), heterogeneous nuclear ribonucleoprotein C (HNRPC) and vimentin (VIME) were all lower in obese patients. Alpha-1-antitrypsin (A1AT) and stress-70 protein, mitochondrial (GRP75) were higher in obese patients. Western blot analysis of ANXA5, ATPB, FTL, VIME, A1AT and GRP75 confirmed the findings from the 2D-DIGE analysis. For brain acid soluble protein 1 and HNRPC, qRT-PCR analysis also confirmed the findings from the 2D-DIGE analysis. Immunohistochemical analysis was also used to determine the localisation of the proteins in human placenta. In conclusion, proteomic analysis of placenta reveals differential expression of several proteins in patients with pre-existing obesity. These proteins are implicated in a variety of cellular functions such as regulation of growth, cytoskeletal structure, oxidative stress, inflammation, coagulation and apoptosis. These disturbances may have significant implications for fetal growth and development.

2D-DIGE analysis of sera from transgenic mouse models reveals novel candidate protein biomarkers for human gastric cancer

2012 ◽  
Vol 77 ◽  
pp. 40-58 ◽  
Author(s):  
Megan A.S. Penno ◽  
Manuela Klingler-Hoffmann ◽  
Julie A. Brazzatti ◽  
Alex Boussioutas ◽  
Tracy Putoczki ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Transgenic Mouse ◽  
Mouse Models ◽  
Human Gastric Cancer ◽  
Transgenic Mouse Models ◽  
Protein Biomarkers ◽  
2D Dige ◽  
Candidate Protein ◽  
Dige Analysis

Optimized Large Scale Synthesis of Phosphorothioate Oligonucleotides on PS-PEG Supports

1997 ◽  
Vol 52 (1) ◽  
pp. 110-116
Author(s):  
Michael Gerster ◽  
Martin Maier ◽  
Nils Clausen ◽  
Jens Schewitz ◽  
Ernst Bayer
Keyword(s):  
Gel Electrophoresis ◽  
Continuous Flow ◽  
Large Scale ◽  
Electrospray Mass Spectrometry ◽  
Small Scale ◽  
Batch Mode ◽  
Crucial Step ◽  
Capillary Gel Electrophoresis ◽  
Phosphoramidite Chemistry ◽  
Large Scale Synthesis

Sulphurization is a crucial step during synthesis of phosphorothioate oligonucleotides. Insufficient reaction leads to inhomogeneous products with phosphodiester defects and subsequently to destabilization of the oligomers in biological media. To achieve a maximum extent of sulphur incorporation, various sulphurizing agents have been investigated. Solely, the use of Beaucage reagent provided satisfactory results on PS-PEG supports. Based on our investigations in small scale synthesis (1 μmol) with continuous-flow technique, upscaling to the 0.1-0.25 mmolar range has been achieved using a peptide synthesizer. The syntheses were performed in batch mode with standard phosphoramidite chemistry. Additionally, large scale synthesis of a phosphodiester oligonucleotide has been carried out on PS-PEG with optimized protocols and compared to small scale synthesis on different supports. Products were analysed by 31P NMR, capillary gel electrophoresis and electrospray mass spectrometry. An extent of sulphurization of 99% and coupling effiencies of more than 99% were obtained and the products proved to have similar purity compared to small scale syntheses on CPG

scholarly journals Cloud-to-ground lightning in Portugal: patterns and dynamical forcing

2012 ◽  
Vol 12 (3) ◽  
pp. 639-649 ◽  
Author(s):  
J. A. Santos ◽  
M. A. Reis ◽  
J. Sousa ◽  
S. M. Leite ◽  
S. Correia ◽  
...  
Keyword(s):  
Large Scale ◽  
Bimodal Distribution ◽  
Lightning Activity ◽  
Atmospheric Conditions ◽  
Remote Forcing ◽  
Cloud To Ground Lightning ◽  
Using Data ◽  
Number Of Discharges ◽  
Low Pressures ◽  
Large Scale Atmospheric Circulation

Abstract. An analysis of the cloud-to-ground discharges (CGD) over Portugal is carried out using data collected by a network of sensors maintained by the Portuguese Meteorological Institute for 2003–2009 (7 yr). Only cloud-to-ground flashes are considered and negative polarity CGD are largely dominant. The total number of discharges reveals a considerable interannual variability and a large irregularity in their distribution throughout the year. However, it is shown that a large number of discharges occur in the May–September period (71%), with a bimodal distribution that peaks in May and September, with most of the lightning activity recorded in the afternoon (from 16:00 to 18:00 UTC). In spring and autumn the lightning activity tends to be scattered throughout the country, whereas in summer it tends to be more concentrated over northeastern Portugal. Winter generally presents low lightning activity. Furthermore, two significant couplings between the monthly number of days with discharges and the large-scale atmospheric circulation are isolated: a regional forcing, predominantly in summer, and a remote forcing. In fact, the identification of daily lightning regimes revealed three important atmospheric conditions for triggering lightning activity: regional cut-off lows, cold troughs induced by remote low pressure systems and summertime regional low pressures at low-tropospheric levels combined with a mid-tropospheric cold trough.

Riboregulators in plant development

2007 ◽  
Vol 35 (6) ◽  
pp. 1638-1642 ◽  
Author(s):  
P. Laporte ◽  
F. Merchan ◽  
B.B. Amor ◽  
S. Wirth ◽  
M. Crespi
Keyword(s):  
Large Scale ◽  
Transcriptional Control ◽  
Rna Binding ◽  
Bioinformatic Analysis ◽  
Nodule Development ◽  
Small Interfering Rnas ◽  
Cortical Cells ◽  
Protein Coding ◽  
Early Nodulin ◽  
Model Plant Arabidopsis Thaliana

npcRNA (non-protein-coding RNAs) are an emerging class of regulators, so-called riboregulators, and include a large diversity of small RNAs [miRNAs (microRNAs)/siRNAs (small interfering RNAs)] that are involved in various developmental processes in plants and animals. In addition, several other npcRNAs encompassing various transcript sizes (up to several kilobases) have been identified using different genomic approaches. Much less is known about the mechanism of action of these other classes of riboregulators also present in the cell. The organogenesis of nitrogen-fixing nodules in legume plants is initiated in specific root cortical cells that express the npcRNA MtENOD40 (Medicago truncatula early nodulin 40). We have identified a novel RBP (RNA-binding protein), MtRBP1 (M. truncatula RBP 1), which interacts with the MtENOD40 RNA, and is exported into the cytoplasm during legume nodule development in the region expressing MtENOD40. A direct involvement of the MtENOD40 RNA in the relocalization of this RBP into cytoplasmic granules could be demonstrated, revealing a new RNA function in the cell. To extend these results, we searched for npcRNAs in the model plant Arabidopsis thaliana whose genome is completely known. We have identified 86 novel npcRNAs from which 27 corresponded to antisense RNAs of known coding regions. Using a dedicated ‘macroarray’ containing these npcRNAs and a collection of RBPs, we characterized their regulation in different tissues and plants subjected to environmental stresses. Most of the npcRNAs showed high variations in gene expression in contrast with the RBP genes. Recent large-scale analysis of the sRNA component of the transcriptome revealed an enormous diversity of siRNAs/miRNAs in the Arabidopsis genome. Bioinformatic analysis revealed that 34 large npcRNAs are precursors of siRNAs/miRNAs. npcRNAs, which are a sensitive component of the transcriptome, may reveal novel riboregulatory mechanisms involved in post-transcriptional control of differentiation or environmental responses.

2D-DIGE A Powerful Tool for Proteome Analysis

2020 ◽  
pp. 67-73
Keyword(s):  
Protein Expression ◽  
Gel Electrophoresis ◽  
Proteome Analysis ◽  
Great Sensitivity ◽  
Two Dimensional Gel Electrophoresis ◽  
2D Dige ◽  
Protein Sample ◽  
Different Types ◽  
Complex Protein ◽  
2D Gel

In the recent past, two dimensional gel electrophoresis has emerged as a powerful molecular biology tool for the comparative expression profiling of complex protein sample. It involves the separation as well as the resolution of diverse proteins sample on the basis of isoelectric points and molecular mass of protein in two dimension ways. In this way, it reflects the view of overall proteome status including differentiation in protein expression levels, post-translational modifications etc. Moreover, this allows the identification of novel biological signatures, which may give a particular identity of pathological background to cells or tissues associated with various types of cancers and neurological disorders. Therefore, by utilizing such tools, one can clearly investigate and compare the effects of particular drugs on cells of tissues and also one can analyze the effects of disease on the basis of variations in protein expression profile at broad spectrum. Recently, to get more error-less and accurate proteome profile, conventional 2-D gel electrophoresis has been enhanced with the inclusion of different types of protein labeling dyes which enables a more comparative analysis of diverse protein sample in a single 2-D gel. In this advanced technique (2-D-DIGE), protein samples are labeled with three different types of CyDyes (Cy2, Cy3, and Cy5) separately and combined and further resolved on the same gel. This will facilitate the more accurate spot matching on a single gel platform and will also minimize the experimental variations as commonly reported in the conventional 2D-gel electrophoresis. Therefore, in the present proteomic research era, 2D-DIGE has proved to be an extremely powerful tool with great sensitivity for up to 125 ng of proteins in clinical research volubility especially, neurological and cancer related disorders.

SV40-transformed hamster cells resistant to 100–250 microgram/ml of ethidium bromide

1978 ◽  
Vol 33 (1) ◽  
pp. 157-169
Author(s):  
G. Wolf ◽  
L. Tejmar ◽  
S. Borell ◽  
W. Klietman
Keyword(s):  
Mitochondrial Dna ◽  
Cell Lines ◽  
Ethidium Bromide ◽  
Gel Electrophoresis ◽  
Mitochondrial Proteins ◽  
Wild Type ◽  
Mitochondrial Ultrastructure ◽  
Gold Hamsters ◽  
Resistant Cells

SV40-transformed hamster cells were selected for resistance to ethidium bromide (EB). Several cell lines were established, which grew in the presence of up to 250 microgram/ml EB. The EB resistance is genetically stable. The cloned resistant cells show no difference in morphology, with the exception of the mitochondrial ultrastructure, which exhibits condensed cristae formation. The tumorigenicity of these cells in Syrian gold hamsters is considerably reduced. Incorporation of radioactive labelled thymidine into mitochondrial DNA is not influenced by the presence of the drug. Gel electrophoresis with mitochondrial proteins from wild-type and resistant cells reveals significantly different patterns. The mechanism of EB resistance is discussed.

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