scholarly journals Isolation and initial characterization of the mammalian midbody.

1982 ◽  
Vol 94 (3) ◽  
pp. 654-661 ◽  
Author(s):  
J M Mullins ◽  
J R McIntosh
Keyword(s):  
Cho Cells ◽  
Total Protein ◽  
Protein Extraction ◽  
Chinese Hamster ◽  
Protein Composition ◽  
Dense Matrix ◽  
Mitotic Spindles ◽  
Sds Page ◽  
The Matrix

Midbodies were isolated from synchronized cultures of Chinese hamster ovary (CHO) cells and their protein composition was studied by means of SDS PAGE. Gels of the midbodies included alpha and beta tubulins as major bands (approximately 30% of the total protein) and approximately 35 other bands, none of which constituted greater than 3.5% of the total protein. Extraction of the isolated midbodies with Sarkosyl NL-30- solubilized the midbody microtubules but left the central, dense matrix zone of the midbody intact. A protein doublet of approximately 115,000 mol wt was retained preferentially by the particulate fraction containing the matrix zones, indicating it to be a component of the matrix. The 115,000 mol wt doublet was also present in gels of isolated mitotic spindles from CHO cells. The overall protein composition of the isolated spindles was very similar to that of the isolated midbodies.

scholarly journals A Proteomic Approach to Identify Zein Proteins upon Eco-Friendly Ultrasound-Based Extraction

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1838
Author(s):  
Laura Darie-Ion ◽  
Madhuri Jayathirtha ◽  
Gabriela Elena Hitruc ◽  
Marius-Mihai Zaharia ◽  
Robert Vasile Gradinaru ◽  
...  
Keyword(s):  
Protein Extraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Industrial Applications ◽  
Experimental Conditions ◽  
Proteomic Approach ◽  
Sds Page ◽  
Pharmaceutical Industries

Zein is a type of prolamin storage protein that has a variety of biomedical and industrial applications. Due to the considerable genetic variability and polyploidity of the starting material, as well as the extraction methods used, the characterization of the protein composition of zein requires a combination of different analytical processes. Therefore, we combined modern analytical methods such as mass spectrometry (MS), Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), atomic force microscopy (AFM), or Fourier transform infrared spectroscopy–attenuated total reflectance (FTIR-ATR) for a better characterization of the extracted zein. In this study, we present an enhanced eco-friendly extraction method, including grinding and sieving corn seeds, for prolamins proteins using an ultrasonic extraction methodology. The use of an ultrasonic homogenizer, 65% ethanol extraction buffer, and 710 µm maize granulation yielded the highest protein extraction from all experimental conditions we employed. An SDS PAGE analysis of the extracted zein protein mainly revealed two intense bands of approximatively 20 and 23 kDa, suggesting that the extracted zein was mostly α-zein monomer. Additionally, MS analysis revealed as a main component the α-zein PMS2 (Uniprot accession no. P24450) type protein in the maize flour extract. Moreover, AFM studies show that extracting zein with a 65% ethanol and a 710 µm granulation yields a homogeneous content that could allow these proteins to be employed in future medical applications. This research leads to a better understanding of zeins content critical for developing new applications of zein in food and pharmaceutical industries, such as biocompatible medical vehicles based on polyplexes complex nanoparticles of zein with antimicrobial or drug delivery properties.

scholarly journals Cloning and characterization of small circular DNA from Chinese hamster ovary cells.

1984 ◽  
Vol 4 (1) ◽  
pp. 173-180 ◽  
Author(s):  
S W Stanfield ◽  
D R Helinski
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Copy ◽  
Chromosomal Dna ◽  
Cho Cell ◽  
Ovary Cells ◽  
Chromosomal Sequences

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.

scholarly journals Isolation and characterization of Chinese hamster ovary cell variants defective in adhesion to fibronectin-coated collagen.

1980 ◽  
Vol 87 (3) ◽  
pp. 755-763 ◽  
Author(s):  
P A Harper ◽  
R L Juliano
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Divalent Cations ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Human Diploid Fibroblasts ◽  
Isolation And Characterization ◽  
A Cell ◽  
Cold Insoluble Globulin

Variant clones of Chinese hamster ovary (CHO) cells were selected for reduced adhesion to serum-coated tissue culture plates. These clones also displayed reduced adhesion to substrata composed of collagen layers coated with bovine serum or with fibronectin (cold-insoluble globulin). Wild-type (WT) and adhesion variant (ADv) cells grew at comparable rates in suspension culture, but the adhesion variants could not be grown in monolayer culture because of their inability to attach to the substratum. The adhesion deficit in these cells was not corrected by raising the concentration of divalent cations or of serum to levels 10-fold greater than those normally utilized in cell culture. However, both WT and ADv clones could adhere, spread, and attain a normal CHO morphology on substrata coated with concanavalin A or poly-L-lysine. In addition, the adhesion variants could attach to substrata coated with "footpad" material (substratum-attached material) derived from monolayers of human diploid fibroblasts or WT CHO cells. These observations suggest that the variant clones may have a cell surface defect that prevents them from utilizing exogeneous fibronectin as an adhesion-promoting ligand; however the variants seem to have normal cytoskeletal and metabolic capacities that allow them to attach and spread on substrata coated with alternative ligands. These variants should be extremely useful in studying the molecular basis of cell adhesion.

scholarly journals Development of a Microfluidic GHz Impedance Cytometer

2013 ◽  
Vol 80 (12) ◽  
Author(s):  
Niels Haandbæk ◽  
Sebastian C. Bürgel ◽  
Flavio Heer ◽  
Andreas Hierlemann
Keyword(s):  
Dielectric Properties ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Single Cells ◽  
Chinese Hamster ◽  
Frequency Range ◽  
Cell Nuclei ◽  
Dielectric Characterization ◽  
The Difference

AbstractThis article presents a novel microfluidic impedance cytometer enabling dielectric characterization of single cells at frequencies up to 500 MHz. The dielectric properties of cells at lower frequencies contain information about their size and membrane capacitance. The increased frequency range of the presented cytometer potentially allows for characterization of intracellular components, such as vacuoles or the cell nuclei. We demonstrate the overall capabilities of the cytometer through discrimination of polystyrene beads from Chinese hamster ovary (CHO) cells. The discrimination is based on the difference in dielectric properties at frequencies up to 500 MHz.

A Method Suitable for Total Protein Extraction from Wood Forming Tissue of Pinus koraiensis

2011 ◽  
Vol 179-180 ◽  
pp. 1199-1202
Author(s):  
Jiang Tao Shi ◽  
Jian Li
Keyword(s):  
Total Protein ◽  
Extraction Method ◽  
Protein Extraction ◽  
Pinus Koraiensis ◽  
Total Proteins ◽  
Sds Page ◽  
Quality Product

Total protein extraction method should be adjusted for get higher quality product for special material. We optimized the protocols and extracted total proteins from P. koraiensis adopt TCA-acetone, TCA-acetone contains β-mercaptoethanol, methanol and protein extraction kit respectively. Then we compared extracts from these methods using SDS-PAGE. The results show that there are no obvious differences between TCA, TCA-2ME and Methanol, which all have better bands. In contrast, extracts from protein extraction kit had ambiguous bands. We also found that it should take about eight hours at least for precipitate of mixtures of samples and extraction solutions. We think optimized TCA-acetone is an idea method for protein extraction from P. koraiensis.

scholarly journals Proteomic characterization of pilot scale hot-water extracts from the industrial carrageenan red seaweed Eucheuma denticulatum

2020 ◽  
Author(s):  
Simon Gregersen ◽  
Margarita Pertseva ◽  
Paolo Marcatili ◽  
Susan Løvstad Holdt ◽  
Charlotte Jacobsen ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
De Novo ◽  
Protein Extraction ◽  
Transcriptome Assembly ◽  
Protein Composition ◽  
Pilot Scale ◽  
Extracellular Proteins ◽  
Hot Water ◽  
Label Free

AbstractSeaweeds have a long history as a resource for polysaccharides/hydrocolloids extraction for use in the food industry due to their functionality as stabilizing agents. In addition to the carbohydrate content, seaweeds also contains a significant amount of protein, which may find application in food and feed. Here, we present a novel combination of transcriptomics, proteomics, and bioinformatics to determine the protein composition in two pilot-scale extracts from Eucheuma denticilatum (Spinosum) obtained via hot-water extraction. The extracts were characterized by qualitative and quantitative proteomics using LC-MS/MS and a de-novo transcriptome assembly for construction of a novel proteome. Using label-free, relative quantification, we were able to identify the most abundant proteins in the extracts and determined that the majority of quantified protein in the extracts (>75%) is constituted by merely three previously uncharacterized proteins. Putative subcellular localization for the quantified proteins was determined by bioinformatic prediction, and by correlating with the expected copy number from the transcriptome analysis, we determined that the extracts were highly enriched in extracellular proteins. This implies that the method predominantly extracts extracellular proteins, and thus appear ineffective for cellular disruption and subsequent release of intracellular proteins. Ultimately, this study highlight the power of quantitative proteomics as a novel tool for characterization of alternative protein sources intended for use in foods. Additionally, the study showcases the potential of proteomics for evaluation of protein extraction methods and as powerful tool in the development of an efficient extraction process.

scholarly journals Physicochemical and Biological Characterization of rhC1INH Expressed in CHO Cells

2021 ◽  
Vol 14 (11) ◽  
pp. 1180
Author(s):  
Ekaterina Zubareva ◽  
Maksim Degterev ◽  
Alexander Kazarov ◽  
Maria Zhiliaeva ◽  
Ksenia Ulyanova ◽  
...  
Keyword(s):  
Cho Cells ◽  
Specific Activity ◽  
Therapeutic Option ◽  
Chinese Hamster ◽  
Biological Characterization ◽  
Acquired Angioedema ◽  
C1 Esterase Inhibitor ◽  
Rabbit Milk ◽  
Esterase Inhibitor

The disfunction or deficiency of the C1 esterase inhibitor (C1INH) is associated with hereditary or acquired angioedema (HAE/AAE), a rare life-threatening condition characterized by swelling in the skin, respiratory and gastrointestinal tracts. The current treatment options may carry the risks of either viral infection (plasma-derived Berinert®) or immune reaction (human recombinant C1INH from rabbit milk, Ruconest®). This study describes the physicochemical and biological characterization of a novel recombinant human C1 esterase inhibitor (rhC1INH) from Chinese hamster ovary (CHO) cells for the treatment of hereditary angioedema compared to the marketed products Berinert® and Ruconest®. The mass spectrometry results of total deglycosylated rhC1INH revealed a protein with a molecular mass of 52,846 Da. Almost full sequence coverage (98.6%) by nanoLC-MS/MS peptide mapping was achieved. The purity and C1s inhibitory activity of rhC1INH from CHO cells are comparable with Ruconest®, although we found differences in charge isoforms distribution, intact mass values, and N-glycans profile. Comparison of the specific activity (IC50 value) of the rhC1INH with human C1 esterase inhibitor from blood serum showed similar inhibitory properties. These data allow us to conclude that the novel rhC1INH molecule could become a potential therapeutic option for patients with HAE/AAE.

scholarly journals Morphological, Molecular, Biochemical and Nutritional Characterization of Three Major Thais Species from the Sindh Coast of Pakistan

2018 ◽  
Vol 10 (1) ◽  
pp. 33-45
Author(s):  
Syed Abid Ali ◽  
Fozia Humayun ◽  
Iqra Munir ◽  
Shakil Ahmad ◽  
Zarrien Ayub ◽  
...  
Keyword(s):  
Total Protein ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Nutritional Composition ◽  
The Body ◽  
Size Exclusion ◽  
High Concentration ◽  
Sds Page

Objective: The present study was conducted to investigate the biomass assessment, morphological and molecular identification, nutritive status and biochemical characterization of three major Thais species (T. bufo, T. hippocastanum and T. rudolphi) from the Sindh Coast, Pakistan. Methods: Samples were collected from Buleji and Paradise Point at the Sindh Coast. Species were identified morphologically as well as genetically by amplifying two mitochondrial 16S rDNA & Cytochrome Oxidase I (COI) and one nuclear (Histone H3) genes. Shell microstructure and chemistry were also studied by scanning electron microscopy and Energy Dispersive X-ray spectrometry (EDX). The body muscle was dissected and used for nutritional composition determination such as estimation of total protein, carbohydrates, lipids, protein fingerprinting by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Size-Exclusion - Fast Protein Liquid Chromatography (SEC-FPLC), amino acid and fatty acid analysis. Results: Nutritionally, the total protein was found to be the major content followed by carbohydrate and lipid in the three Thais sp. The presence of medicinally important hemocyanin as abundant hemolymph protein was confirmed via SDS-PAGE and SEC FPLC. Nine different types of fatty acids and a high concentration of essential amino acids were also determined. Conclusion: Our findings suggest that Thais sp. are nutritionally rich and can be consumed as a valuable marine resource to overcome the malnutrition problem in developing countries.

Purification and partial characterization of a cytotonic enterotoxin produced by Aeromonas hydrophila

1989 ◽  
Vol 35 (7) ◽  
pp. 719-727 ◽  
Author(s):  
A. K. Chopra ◽  
C. W. Houston
Keyword(s):  
Cholera Toxin ◽  
Cho Cells ◽  
Aeromonas Hydrophila ◽  
Chinese Hamster ◽  
Biologically Active ◽  
Partial Characterization ◽  
Cytotoxic Activities ◽  
Ileal Loop

This report describes the purification and partial characterization of a cytotonic enterotoxin produced by a human diarrheal isolate (SSU) of Aeromonas hydrophila. The extracellular enterotoxin was purified by (NH4)2SO4 precipitation, hydrophobic column chromatography, and chromatofocusing. The highly purified enterotoxin exhibited a molecular mass of 44 kDa and an isoelectric point in the range of 4.3–5.5 as determined by chromatofocusing. Western blot analysis using Aeromonas anti-enterotoxin revealed a single band at 44 kDa; however, cholera antitoxin failed to detect the enterotoxin antigen. This non-cholera toxin cross-reactive (non-CTC) enterotoxin was biologically active in vivo as determined by rabbit ligated ileal loop and rabbit skin vascular permeability assays. Biological activity also was expressed in vitro by this toxin as measured by the elongation of Chinese hamster ovary (CHO) cells. The enterotoxic activity associated with this molecule was neutralized completely by homologous antibodies but not by cholera antitoxin. The purified toxin preparation was free of hemolytic and cytotoxic activities as determined by its inability to lyse rabbit red blood cells or damage CHO cells, respectively. Furthermore, this toxin induced the elevation of cAMP in CHO cells suggesting thereby that the mechanism of action of Aeromonas non-CTC enterotoxin may be similar to heat-labile enterotoxins of Escherichia coli and Vibrio cholerae.Key words: Aeromonas hydrophila, cytotonic enterotoxin, cholera toxin, cAMP

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