• Molecules and Cells
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• v.40 no.5
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• pp.339-345
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• 2017

Retroviral and lentiviral vectors are mostly pseudotyped and often purified and concentrated via ultracentrifugation. In this study, we quantified and compared the stabilities of retroviral [murine leukemia virus (MLV)-based] and lentiviral [human immunodeficiency virus (HIV)-1-based] vectors pseudotyped with relatively mechanically stable envelope proteins, vesicular stomatitis virus glycoproteins (VSVGs), and the influenza virus WSN strain envelope proteins against ultracentrifugation. Lentiviral genomic and functional particles were more stable than the corresponding retroviral particles against ultracentrifugation when pseudotyped with VSVGs. However, both retroviral and lentiviral particles were unstable when pseudotyped with the influenza virus WSN strain envelope proteins. Therefore, the stabilities of pseudotyped retroviral and lentiviral vectors against ultracentrifugation process are a function of not only the type of envelope proteins, but also the type of viral internal core (MLV or HIV-1 core). In addition, the fraction of functional viral particles among genomic viral particles greatly varied at times during packaging, depending on the type of envelope proteins used for pseudotyping and the viral internal core.

• Bulletin of the Korean Chemical Society
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• v.32 no.6
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• pp.2125-2128
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• 2011

• Journal of Animal Science and Technology
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• v.66 no.5
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• pp.1021-1033
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• 2024

Exosomes have been extensively studied as disease biomarker in humans, given their role in transporting bioactive molecules. However, despite the great potential of exosomes as noninvasive diagnostic markers and therapeutic nanocarriers for bovine diseases, few studies have been conducted on bovine exosome. Thus, this study aimed to quantitatively and qualitatively compare three isolation methods to identify a suitable method for bovine serum. Exosomes were isolated using ultracentrifugation alone (UC), a combination of ultracentrifugation and size exclusion chromatography (US), or membrane affinity-based exoEasy kit (EE). Isolated particles were evaluated using a range of complementary techniques. Transmission electron microscopy showed that all three isolation methods resulted in particles with a cup-shaped morphology. The particle concentration measured by nanoparticle trafficking analyzer of US was lower compared to those of UC and EE method. As a result of immunoblotting, exosome markers including TSG101, CD81, and HSP70 were detected in US particles, while in UC and EE, only TSG101 expression was confirmed. Particles isolated from UC and EE showed a contamination with the blood protein albumin, whereas particles from US did not show albumin contamination. In addition, to evaluate the possibility of using exosomes as biomarkers, the profiles of the small RNA in the exosomes were compared using the bioanalyzer 2100. As a result, in the EE method, the band of small RNA (25-200 nt) was most prominent, and in the US methods, a distinct band was observed in the small RNA range. Collectively, the purity of exosomes without non-exosomal contamination was highest in the US method. However, for the detection of small RNA, the EE method was found to be the most suitable. Therefore, the results suggest that the optimal isolation method varies depending on the specific purpose of exosome isolation.

• Annals of Laboratory Medicine
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• v.38 no.6
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• pp.518-523
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• 2018

Background: Lipemia, a significant source of analytical errors in clinical laboratory settings, should be removed prior to measuring biochemical parameters. We investigated whether lipemia in serum/plasma samples can be removed using a method that is easier and more practicable than ultracentrifugation, the current reference method. Methods: Seven hospital laboratories in Spain participated in this study. We first compared the effectiveness of ultracentrifugation ($108,200{\times}g$) and high-speed centrifugation ($10,000{\times}g$ for 15 minutes) in removing lipemia. Second, we compared high-speed centrifugation with two liquid-liquid extraction methods-LipoClear (StatSpin, Norwood, USA), and 1,1,2-trichlorotrifluoroethane (Merck, Darmstadt, Germany). We assessed 14 biochemical parameters: serum/plasma concentrations of sodium ion, potassium ion, chloride ion, glucose, total protein, albumin, creatinine, urea, alkaline phosphatase, gamma-glutamyl transferase, alanine aminotransferase, aspartate-aminotransferase, calcium, and bilirubin. We analyzed whether the differences between lipemia removal methods exceeded the limit for clinically significant interference (LCSI). Results: When ultracentrifugation and high-speed centrifugation were compared, no parameter had a difference that exceeded the LCSI. When high-speed centrifugation was compared with the two liquid-liquid extraction methods, we found differences exceeding the LCSI in protein, calcium, and aspartate aminotransferase in the comparison with 1,1,2-trichlorotrifluoroethane, and in protein, albumin, and calcium in the comparison with LipoClear. Differences in other parameters did not exceed the LCSI. Conclusions: High-speed centrifugation ($10,000{\times}g$ for 15 minutes) can be used instead of ultracentrifugation to remove lipemia in serum/plasma samples. LipoClear and 1,1,2-trichlorotrifluoroethane are unsuitable as they interfere with the measurement of certain parameters.

• Bulletin of the Korean Chemical Society
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• v.20 no.6
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• pp.636-638
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• 1999

• Food Science of Animal Resources
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• v.37 no.5
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• pp.654-662
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• 2017

Extracellular microvesicles are membranous nano-sized cellular organelles secreted by a variety of cells under normal and pathological conditions and heterogeneous in size ranging from 30 nm to $1{\mu}m$. They carry functional microRNAs that can influence immunity and development. For a particular application of microvesicles, choice of isolation method is particularly important; however, their isolation methods from colostrum in particular have not been described clearly. In this work, differential ultracentrifugation as a conventional method, ultracentrifugation with some modification such as additional precipitations, ultrafiltration, sucrose gradient separation and ExoQuick$^{TM}$ as a commercial reagent were compared. The goal was to compare mainly microvesicular total microRNA yield, distribution and purity among the methods then select the best isolation method for bovine colostrum microvesicles based largely on microRNA yield with the view of applying the vesicles in work where vesicular microRNA cargo is the target bioactive component. Highest yields for vesicular microRNA were obtained using conventional methods and among them, subsequent ultracentrifugation with 100,000 g and 135,000 g conventional method 2 was selected as it had the highest RNA to protein ratio indicating that it pelleted the least protein in relation to RNA an important factor for in vivo applications to assess microvesicle functionalities without risk of contaminating non-vesicular biomaterial. Microvesicles isolated using conventional method 2 were successfully internalized by cells in vitro showing their potential to deliver their cargo into cells in vitro and in vivo in case of functional studies.

• BMB Reports
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• v.34 no.4
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• pp.329-333
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• 2001

Active lipoprotein lipase (LPL) is known as a noncovalent homodimer of identical subunits, and dissociation of the dimer to a monomeric form renders the lipase inactive. In this study, the oligomerization status of LPL in human and rat plasma was investigated. The LPL activity was barely detectable in the control rat and human plasma. After the injection of heparin, the total lipolytic activity of plasma was rapidly increased, and reached its maximum in 30 min. Changes of the LPL protein correlated well with those of lipolytic activity. The LPL protein that is released by heparin into both human and rat plasma was active and dimeric in the sucrose density gradient ultracentrifugation. In control rat plasma, LPL was inactive, and a great fraction was present as an aggregate. However, the inactive LPL protein in the control human plasma retained the dimeric state, indicating that dimerization can be an entity independent of the catalytic activity of LPL. The released LPL is transported as a complex with lipoproteins in plasma. Lipoprotein profiles, determined by NaBr ultracentrifugation, exhibited typical LDL- and HDL-mammal patterns in humans and rats, respectively, with a smaller amount of the LDL fraction observed in rats. The difference in the lipoprotein profiles might influence the fate of the released LPL in plasma.

• BMB Reports
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• v.42 no.3
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• pp.166-171
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• 2009

Hsp70s assist in the process of protein folding through nucleotide-controlled cycles of substrate binding and release by alternating from an ATP-bound state in which the affinity for substrate is low to an ADP-bound state in which the affinity for substrate is high. It has been long recognized that the two-domain structure of Hsp70 is critical for these regulated interactions. Therefore, it is important to obtain information about conformational changes in the relative positions of Hsp70 domains caused by nucleotide binding. In this study, analytical ultracentrifugation and dynamic light scattering were used to evaluate the effect of ADP and ATP binding on the conformation of the human stress-induced Hsp70.1 protein. The results of these experiments showed that ATP had a larger effect on the conformation of Hsp70 than ADP. In agreement with previous biochemical experiments, our results suggest that conformational changes caused by nucleotide binding are a consequence of the movement in position of both nucleotide- and substrate-binding domains.

• The Korean Journal of Zoology
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• v.36 no.2
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• pp.231-237
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• 1993

Lipophorin (LP) was purified from hemolvmph of late last instar larvae of Hyphuntria cuneo D. by KBr density gradient ultracentrifugation. Chemical composition of LP was investigated by electrophoresis, thin laver chromatography and ryas chromatography. LP consisted of Apo-LP I and Apo-LP ll, and M.W. of them were 230 Kd and 80 Nd, respectivelv. Lipid of LP was mostly composed of neutral lipid including triacylglvcerol, diacvlslvcerol, monoacylglvcerol and free cholesterol, and phospholipid rich in phosphatidvlethanolamine and phosphatidvlcholine. Fatty acids present in these lipids were found to have be 14:0, 16:0, 18:1, and 20:1.

• The Korean Journal of Zoology
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• v.37 no.4
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• pp.488-494
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• 1994

Apolipophorin-III (ApoLp-III) was purified from adult haemolynph of Hyphantriu cuneo and their molecular weight and synthetic place were investigated. ApoLp-III purification was performed by KBr-density gradient ultracentrifugation followed by gel permeation chromatographv (Sephadex G-1001 and ion-exchange chromatography (CM-52) and their purity was confirmed on 10% SDS-PAGE. ApoLp-III has the molecular weight of 18 ItDa and is synthesized by fat body.