Antigenic studies on coronavirus. I. Identification of the structural antigens of human coronavirus, strain 229E

1981 ◽  
Vol 27 (3) ◽  
pp. 334-342 ◽  
Author(s):  
Shehab A. Yaseen ◽  
C. Margaret Johnson-Lussenburg
Keyword(s):  
Sodium Deoxycholate ◽  
Antigenic Analysis ◽  
Human Coronavirus ◽  
Specific Antigens ◽  
Integrated Or ◽  
Triton X ◽  
Rnp Antigen

Antigenic analysis of human coronavirus, strain 229E (HCV/229E), using a microimmunodiffusion technique, has resulted in the detection of six virion antigens. Comparison of the effect of several different virus-disrupting agents has shown that sodium deoxycholate or Triton X-100 were the best for HCV/229E disruption. Of the six coronavirion antigens, three were identified as virus specific and the remainder as host antigens, which were present as either integrated or nonspecifically adsorbed host components. One of the virus-specific antigens was identified as the internal nucleoprotein (229E/RNP). On the basis of immunodiffusion reactions with isolated 229E/RNP it was concluded that human convalescent sera reacted specifically with the 229E/RNP antigen.

scholarly journals Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

1985 ◽  
Vol 227 (2) ◽  
pp. 405-412 ◽  
Author(s):  
P W Cheng ◽  
W E Wingert ◽  
M R Little ◽  
R Wei
Keyword(s):  
Enzyme Activity ◽  
Freezing Point ◽  
Cetylpyridinium Chloride ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Gas Chromatography Mass Spectrometry ◽  
Tween 20 ◽  
Group A ◽  
Michaelis Constants ◽  
Triton X

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-β 1→3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1→3(Glc-NAc beta 1→6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.

scholarly journals Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system

1975 ◽  
Vol 150 (3) ◽  
pp. 537-551 ◽  
Author(s):  
P H Cooper ◽  
J N Hawthorne
Keyword(s):  
Phosphatase Activity ◽  
Metal Ion ◽  
Rat Kidney ◽  
Gradient Centrifugation ◽  
Sodium Deoxycholate ◽  
Subcellular Fractionation ◽  
Kidney Cortex ◽  
Ph Optimum ◽  
Triton X ◽  
Hydrolysis Of

Tthe properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergent sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the subtrates and in the presence Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and L+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and density-gradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that of the liver Golgimarker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble.

scholarly journals The effect of chemical agents on the turnover of the bound phosphate associated with the sodium-and-potassium ion-stimulated adenosine triphosphatase in ox brain microsomes

1970 ◽  
Vol 120 (1) ◽  
pp. 1-13 ◽  
Author(s):  
R. Rodnight
Keyword(s):  
Sodium Chloride ◽  
Atp Hydrolysis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Protein Ratio ◽  
Chemical Agents ◽  
Rapid Fall ◽  
Triton X ◽  
Hydrolysis Of ◽  
Sodium And Potassium

1. The effect of chemical agents on the turnover of the Na+-dependent bound phosphate and the simultaneous Na+-dependent hydrolysis of ATP by a membrane preparation from ox brain was studied at an ATP/protein ratio of 12.5pmol/μg. 2. The agents were added immediately after phosphorylation of the preparation in a medium containing 50mm-sodium chloride and 2.5μm-[γ-32P]ATP. 3. Concentrations of sodium chloride above 150mm, calcium chloride to 20mm and suramin to 1.4mm inhibited both phosphorylation and dephosphorylation and concomitantly slowed ATP hydrolysis. At 125mm-sodium chloride dephosphorylation and hydrolysis were slightly slowed without affecting phosphorylation. 4. Ethanol to 1.6m concentration inhibited dephosphorylation without affecting phosphorylation; the bound phosphate was increased and ATP hydrolysis slowed. 5. Ouabain to 4mm concentration partially inhibited ATP hydrolysis and caused a transient (1–2s) rise in bound phosphate followed by a rapid fall to a lower plateau value, which eventually declined to zero by the time ATP hydrolysis was complete. 6. Of the detergents examined Lubrol W, Triton X-100 and sodium deoxycholate had no significant effect on turnover. Sodium dodecyl sulphate and sodium decyl sulphate to 3.5mm and 20mm respectively completely inhibited turnover and ATP hydrolysis and stabilized the bound phosphate.

scholarly journals Metabolism of conjugated sterols in eggplant. Part 2. Phospholipid : steryl glucoside acyltransferase.

2008 ◽  
Vol 55 (1) ◽  
pp. 135-140 ◽  
Author(s):  
Anna Potocka ◽  
Jan Zimowski
Keyword(s):  
Divalent Cations ◽  
Solanum Melongena ◽  
Sodium Deoxycholate ◽  
Tween 20 ◽  
Acyl Donor ◽  
Kinetic Properties ◽  
Acyltransferase Activity ◽  
Steryl Glucoside ◽  
Membrane Bound ◽  
Triton X

A membrane-bound phospholipid : steryl glucoside acyltransferase from Solanum melongena leaves was partially purified and its specificity and molecular as well as kinetic properties were defined. Among the steryl glycosides tested (e.g. typical plant steryl glucosides, steryl galactosides and cholesteryl xyloside) the highest activity was found with cholesteryl glucoside, but some structurally related compounds such as sito- and stigmasteryl glucoside or galactoside as well as cholesteryl galactoside were also acylated, albeit at lower rates. The investigated enzyme was able to use all classes of phosphoglycerolipids (phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol) as an acyl source for biosynthesis of acylated steryl glucoside. Among them 1,2-dimirystoylphosphatidylic acid appeared to be the best acyl donor. Apart from phosphoglycerolipids, 1,2-diacylglycerols were also used as acyl donor for steryl glucoside acylation, although at a distinctly lower rate. The acyl moiety was transferred from the C-1 position of phospholipid molecule. The investigated acyltransferase activity was stimulated by 2-mercaptoethanol, Triton X-100, 1-monoacylglycerols and inhibited in the presence of divalent cations such as Ca(2+), Mn(2+), Zn(2+) or Co(2+), some lipids (MDGD, ceramide), detergents (Tween 20, 40, 60 and 80, Tyloxapol, sodium deoxycholate) and high ionic strength.

scholarly journals Towards uterus tissue engineering: a comparative study of sheep uterus decellularisation

2020 ◽  
Vol 26 (3) ◽  
pp. 167-178 ◽  
Author(s):  
T T Tiemann ◽  
A M Padma ◽  
E Sehic ◽  
H Bäckdahl ◽  
M Oltean ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Vascular Perfusion ◽  
Uterus Transplantation ◽  
Live Donors ◽  
Triton X

Abstract Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support foetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2 weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in vivo sheep uterus bioengineering experiments.

A simple method for ultrastructural visualization of cytoplasmic microfilament bundles (mfb) in rat hepatocytes

1978 ◽  
Vol 36 (2) ◽  
pp. 516-517
Author(s):  
S. Yokota ◽  
H.D. Fahimi
Keyword(s):  
Rat Hepatocytes ◽  
Sodium Deoxycholate ◽  
Uranyl Acetate ◽  
Cellular Motility ◽  
En Bloc ◽  
Adult Rats ◽  
Simple Method ◽  
Microfilament Bundles ◽  
Tris Maleate ◽  
Triton X

Microfilaments (MF) are present in a variety of animal and plant cells, and there is substantial evidence that they are involved in cellular motility and contractility (1). Although MF's were first discovered by electron microscopy (EM), they are not distinctly visible in routine EM preparations of whole tissue. In partially disrupted hepatocytes and in isolated bile canaliculi, however, the MF's are prominently contrasted with the uranyl acetate en bloc treatment (2). We now report our observations on the treatment of glutaraldehyde- (GA) fixed rat liver with various detergents. This procedure, followed by uranyl staining, increases markedly the electron density and the contrast of MFB's in tissue sections.Materials and Methods: Livers of adult rats were fixed by perfusion with 2.5% purified GA in 0.1M Na-cacodylate buffer, pH 7.2, and 0.05% CaCl2 for 10 min. 50-μm chopper sections were treated with 0.1-1% Triton X-100 or sodium deoxycholate (DOC) for 1 h at 25°C, rinsed briefly with the buffer, and then postfixed in 2% aqueous OsO4 for 60 min. at 4°C. This was followed by en bloc staining for 60 min. with 1% uranyl acetate in 0.2M Tris-maleate buffer, pH 5.2, and embedding in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate. In control preparations the detergent treatment was omitted, but all other procedures were identical.

scholarly journals Effect of detergent type on the performance of liver decellularized extracellular matrix-based bio-inks

2021 ◽  
Vol 12 ◽  
pp. 204173142199709
Author(s):  
Wonwoo Jeong ◽  
Min Kyeong Kim ◽  
Hyun-Wook Kang
Keyword(s):  
Extracellular Matrix ◽  
Sodium Dodecyl ◽  
Great Influence ◽  
Ammonium Hydroxide ◽  
Sodium Deoxycholate ◽  
Porcine Liver ◽  
Decellularized Extracellular Matrix ◽  
Cytochrome P450 Activity ◽  
Primary Mouse ◽  
Triton X

Decellularized extracellular matrix-based bio-inks (dECM bio-inks) for bioprinting technology have recently gained attention owing to their excellent ability to confer tissue-specific functions and 3D-printing capability. Although decellularization has led to a major advancement in bio-ink development, the effects of detergent type, the most important factor in decellularization, are still unclear. In this study, the effects of various detergent types on bio-ink performance were investigated. Porcine liver-derived dECM bio-inks prepared using widely used detergents, including sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), Triton X-100 (TX), and TX with ammonium hydroxide (TXA), were characterized in detail. SDS and SDC severely damaged glycosaminoglycan and elastin proteins, TX showed the lowest rate of decellularization, and TXA-based dECM bio-ink possessed the highest ECM content among all bio-inks. Differences in biochemical composition directly affected bio-ink performance, with TXA-dECM bio-ink showing the best performance with respect to gelation kinetics, intermolecular bonding, mechanical properties, and 2D/3D printability. More importantly, cytocompatibility tests using primary mouse hepatocytes also showed that the TXA-dECM bio-ink improved albumin secretion and cytochrome P450 activity by approximately 2.12- and 1.67-fold, respectively, compared with the observed values for other bio-inks. Our results indicate that the detergent type has a great influence on dECM damage and that the higher the dECM content, the better the performance of the bio-ink for 3D bioprinting.

scholarly journals Evaluation of Mycoplasma Inactivation during Production of Biologics: Egg-Based Viral Vaccines as a Model

2010 ◽  
Vol 76 (9) ◽  
pp. 2718-2728 ◽  
Author(s):  
Selwyn A. Wilson David ◽  
Dmitriy V. Volokhov ◽  
Zhiping Ye ◽  
Vladimir Chizhikov
Keyword(s):  
Cetyltrimethylammonium Bromide ◽  
Allantoic Fluid ◽  
Sodium Deoxycholate ◽  
Pharmaceutical Products ◽  
Mycoplasma Species ◽  
Viral Particles ◽  
Series Of Experiments ◽  
Influenza Vaccine Production ◽  
Triton X ◽  
Protein Rich

ABSTRACT Although mycoplasmas are generally considered to be harmless commensals, some mycoplasma species are able to cause infections in pediatric, geriatric, or immunocompromised patients. Thus, accidental contamination of biologics with mycoplasmas represents a potential risk for the health of individuals who receive cell-derived biological and pharmaceutical products. To assess the efficiency of inactivation of mycoplasmas by the agents used in the manufacture of egg-derived influenza vaccines, we carried out a series of experiments aimed at monitoring the viability of mycoplasmas spiked into both chicken allantoic fluid and protein-rich microbiological media and then treated with beta-propiolactone, formalin, cetyltrimethylammonium bromide, Triton X-100, and sodium deoxycholate, which are agents that are commonly used for virus inactivation and disruption of viral particles during influenza vaccine production. Twenty-two mycoplasma species (with one to four strains of each species) were exposed to these inactivating agents at different concentrations. The most efficient inactivation of the mycoplasmas evaluated was observed with either 0.5% Triton X-100 or 0.5% sodium deoxycholate. Cetyltrimethylammonium bromide at concentrations of ≥0.08% was also able to rapidly inactivate (in less than 30 min) all mycoplasmas tested. In contrast, negligible reductions in mycoplasma titers were observed with 0.0125 to 0.025% formaldehyde. However, increasing the concentration of formaldehyde to 0.1 to 0.2% improved the mycoplasmacidal effect. Incubation of mycoplasmas with 0.1% beta-propiolactone for 1 to 24 h had a marked mycoplasmacidal effect. A comparison of the mycoplasma inactivation profiles showed that strains of selected species (M ycoplasma synoviae, M ycoplasma gallisepticum, M ycoplasma orale, M ycoplasma pneumoniae, and A choleplasma laidlawii) represent a set of strains that can be utilized to validate the effectiveness of mycoplasma clearance obtained by inactivation and viral purification processes used for the manufacture of an inactivated egg-based vaccine.

On the Automatic Decellularisation of Porcine Aortae: A Repeatability Study Using a Non-Enzymatic Approach

2016 ◽  
Vol 201 (4) ◽  
pp. 299-318 ◽  
Author(s):  
Rory O'Connor Mooney ◽  
Niall Francis Davis ◽  
David Hoey ◽  
Lisa Hogan ◽  
Timothy M. McGloughlin ◽  
...  
Keyword(s):  
Gold Standard ◽  
Mechanical Characteristics ◽  
Sodium Deoxycholate ◽  
Future Research ◽  
Binding Assays ◽  
Control Groups ◽  
Axial Tensile ◽  
Group Protein ◽  
Triton X ◽  
Protein Preservation

Objective: To investigate the repeatability of automatic decellularisation of porcine aortae using a non-enzymatic approach, addressing current limitations associated with other automatic decellularisation processes. Materials and Methods: Individual porcine aortae (n = 3) were resected and every third segment (n = 4) was allocated to one of three different groups: a control or a manually or automatically decellularised group. Manual and automatic decellularisation was performed using Triton X-100 (2% v/v) and sodium deoxycholate. Protein preservation and the elimination of a galactosyl-α(1,3)galactose (GAL) epitope were measured using immunohistochemistry and protein binding assays. The presence of residual DNA was determined with gel electrophoresis and spectrophotometry. Scaffold integrity was characterised with scanning electron microscopy and uni-axial tensile testing. Manual and automatic results were compared to one another, to control groups and to current gold standards. Results: The results were comparable to those of current gold standard decellularisation techniques. Successful repeatability was achieved, both manually and automatically, with little effect on mechanical characteristics. Complete acellularity was not confirmed in either decellularisation group. Protein preservation was consistent in both the manually and automatically decellularised groups and between each individual aorta. Elimination of GAL was not achieved. Conclusion: Repeatable automatic decellularisation of porcine aortae is feasible using a Triton X-100-sodium deoxycholate protocol. Protein preservation was satisfactory; however, gold standard thresholds for permissible residual DNA levels were not achieved. Future research will focus on addressing this issue by optimisation of the existing protocol for thick tissues.

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