scholarly journals A polypeptide of 59 kDa is associated with bundles of cytoplasmic filaments in Neurospora crassa

1990 ◽  
Vol 268 (3) ◽  
pp. 649-655 ◽  
Author(s):  
A L Rosa ◽  
M E Alvarez ◽  
D Lawson ◽  
H J F Maccioni
Keyword(s):  
Monoclonal Antibody ◽  
Ionic Strength ◽  
Neurospora Crassa ◽  
High Ionic Strength ◽  
Differential Centrifugation ◽  
Main Constituent ◽  
Total N ◽  
Cytoplasmic Filaments ◽  
Triton X ◽  
Isolation Buffer

Complex arrangements of filamentous structures have been isolated from vegetative cells of the fungus Neurospora crassa. They were enriched by differential centrifugation and purified by permeation chromatography. The filamentous structures are made up of units of 8-10 nm diameter and were isolated in bundles of up to six to nine units. The main constituent of these structures is a polypeptide with an apparent molecular mass of 59 kDa (P59Nc), which represents 4-5% of the total N. crassa proteins. The filamentous structures are cold-stable and are not affected by high-ionic-strength solutions or by the presence of 10 mM-EDTA or 1% (w/v) Triton X-100; they were disassembled by raising the pH of the solution or by using Tris-based buffers. The disassembled form assembled into structures sedimentable at 105,000 g after dialysis against the isolation buffer. The sedimentable structures were organized in the form of regular aggregates of 42-45 nm polypeptides and reacted weakly with anti-IFA, a monoclonal antibody which recognizes an epitope common to many of the higher-eukaryote intermediate-filament polypeptides. Immunofluorescence examination of wall-digested hyphae of N. crassa using affinity-purified antibodies prepared against P59Nc showed immunostaining of abundant filamentous and dot-shaped structures distributed in the cytoplasm.

Identification and localization of major cortical proteins in the ciliated protozoan, Euplotes eurystomus

1989 ◽  
Vol 92 (3) ◽  
pp. 433-439 ◽  
Author(s):  
N.E. Williams ◽  
J.E. Honts ◽  
Q. Lu ◽  
C.L. Olson ◽  
K.C. Moore
Keyword(s):  
Ionic Strength ◽  
High Ionic Strength ◽  
Basal Bodies ◽  
Cell Type ◽  
Ciliated Protozoan ◽  
Shape Preserving ◽  
Sds Page ◽  
Triton X ◽  
Development And Evolution ◽  
Integrated Structures

Shape-preserving cortical residues have been isolated from Euplotes eurystomus cells by the application of Triton X-100 at high ionic strength. These integrated structures consist of articulated plates and widely interspersed cages that formerly contained basal bodies associated with the clusters of cilia characteristic of this cell type. Using SDS-PAGE and immunolocalization procedures, we have identified major subunit proteins of both the plates (116, 110 (X10(3] Mr, and the basal body cages (86 X 10(3) Mr). The potential for studies of these proteins in contributing to our understanding of cortical development and evolution in Euplotes is discussed.

The Thrombin Catalyzed Activation Of Human Factor V Probed With A Hybridoma Antibody

1981 ◽  
Author(s):  
J A Katzmann ◽  
M E Nesheim ◽  
K G Mann
Keyword(s):  
Monoclonal Antibody ◽  
Ionic Strength ◽  
Human Factor ◽  
Antigenic Site ◽  
High Ionic Strength ◽  
Factor V ◽  
Single Chain ◽  
End Products ◽  
Affinity Resin ◽  
Dependent Interaction

The peptides produced during the thrombin (IIa) catalyzed activation of human Factor V (HFV) were investigated using a murine monoclonal antibody. This antibody (HFV-1) binds HFV and activated HFV (HFVa) and releases them at high ionic strength. HFV-1 coupled to sepharose was used to purify the single chain, 330,000 apparent MW procofactor molecule. Gel electrophoresis in SDS indicated that IIa- mediated activation proceeded with the appearance of intermediates with apparent MW of 270K, 205K and 150K and resulted in at least 3 end products with apparent MW of 110K, 93K and 70K. Aliquots of partially and fully activated HFV were chromatographed on the HFV-1 affinity resin. The peptides not associated with HFVa were obtained by washing the column with 20 mM imidazole, 0.154 M NaCl, 5 mM CaCl2, pH 6.5; the peptides noncovalently bound to the epitope-containing peptides were obtained by elution in the above buffer made 5 mM EDTA in place of Ca++ and the epitope-containing peptides were eluted in buffer made 1.2 M in NaCl. The 150K intermediate was released in EDTA and the 205K and 270K peptides were eluted in high salt. Of the end products of activaion, the 110K peptide was not retained by the coliman, the 93K peptide was released in EDTA, and the 70K peptide was released only at high ionic strength. These data indicate that the 270K and 205K intermediates, and the 70K end product share the antigenic site involved in the interaction with the monoclonal antibody. In addition a Ca++- dependent interaction between the epitope-containing domain and the 150K intermediate and 93K end product is suggested. From this data and by analogy with the bovine protein, we suggest that HFVa is a Ca++-dependent multiple subunit protein consisting minimally of two peptides of apparent MW of 93,000 and 70,000.

Importance of Protease Inhibition in Studies on Purified Factor VIII (Antihaemophilic Factor)

1976 ◽  
Vol 35 (01) ◽  
pp. 186-190 ◽  
Author(s):  
Eugen A. Beck ◽  
Peter Bachmann ◽  
Peter Barbier ◽  
Miha Furlan
Keyword(s):  
Ionic Strength ◽  
Factor Viii ◽  
Gel Filtration ◽  
High Ionic Strength ◽  
Procoagulant Activity ◽  
Carrier Protein ◽  
Protease Inhibition ◽  
Functional Sites ◽  
Molecular Weight Peak ◽  
Von Willebrand

SummaryAccording to some authors factor VIII procoagulant activity may be dissociable from carrier protein (MW~ 2 × 106) by agarose gel filtration, e.g. at high ionic strength. We were able to reproduce this phenomenon. However, addition of protease inhibitor (Trasylol) prevented the appearance of low molecular weight peak of factor VIII procoagulant activity both at high ionic strength and elevated temperature (37°C). We conclude from our results that procoagulant activity and carrier protein (von Willebrand factor, factor VIII antigen) are closely associated functional sites of native factor VIII macro molecule. Consequently, proteolytic degradation should be avoided in functional and structural studies on factor VIII and especially in preparing factor VIII concentrate for therapeutic use.

THE RELATIVE DISTRIBUTION OF THYROXINE, TRIIODOTHYRONINE AND 3,3′,5′-(REVERSE)-TRIIODOTHYRONINE IN VARIOUS FRACTIONS OF THYROGLOBULIN

1978 ◽  
Vol 88 (2) ◽  
pp. 298-305 ◽  
Author(s):  
Peter Laurberg
Keyword(s):  
Ionic Strength ◽  
Guinea Pig ◽  
Sucrose Gradient ◽  
Deae Cellulose ◽  
High Ionic Strength ◽  
Relative Distribution ◽  
Thyroid Extract ◽  
Reverse Triiodothyronine ◽  
Thyroid Secretion ◽  
Stable Iodine

ABSTRACT Thyroglobulin fractions rich and poor in new thyroglobulin were separated by means of DEAE-cellulose chromatography of dog thyroid extracts and by zonal ultracentrifugation in a sucrose gradient of guinea pig thyroid extract incubated at low temperature. The distribution of thyroxine, triiodothyronine and 3,3′,5′-(reverse)-triiodothyronine in hydrolysates of the different fractions was estimated by radioimmunoassays. Following DEAE-cellulose chromatography there was a small but statistically significant increase in the T4/T3 ratio in thyroglobulin fractions eluted at high ionic strength - that is fractions relatively rich in stable iodine but poor in fresh thyroglobulin. There were no differences in the T4/rT3 ratios between the different fractions. The ratios between iodothyronines were almost identical in the various thyroglobulin fractions following zonal ultracentrifugation in a sucrose gradient of cold treated guinea pig thyroid extract. These findings lend no support to the possibility that a relatively high content of triiodothyronines in freshly synthesized thyroglobulin modulates the thyroid secretion towards a preferential secretion of triiodothyronine and 3,3′,5′-(reverse)-triiodothyronine at the expense of the secretion of thyroxine.

scholarly journals Kaolin based protective barrier in municipal landfills against adverse chemo-mechanical loadings

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Partha Das ◽  
Tadikonda Venkata Bharat
Keyword(s):  
Ionic Strength ◽  
High Ionic Strength ◽  
The Self ◽  
Design Parameters ◽  
Layered System ◽  
Geosynthetic Clay Liners ◽  
Clay Liners ◽  
Landfill Liner ◽  
First Time ◽  
Applied Stresses

AbstractIn this work, we assess the self-sealing and swelling ability of the compacted granular bentonite (GB) under an inorganic salt environment and induced overburden stresses from the landfill waste. The laboratory permeation tests with high ionic strength salt solutions reveal that the GB fails to seal and exhibits a significant mechanical collapse under different applied stresses. The applicability of GB in the form of geosynthetic clay liners as the bottom liner facilities in landfills that produce high ionic strength salt leachates, therefore, remains a serious concern. We propose an additional barrier system based on kaolin, for the first time, to address this problem. The proposed kaolin-GB layered system performs satisfactorily in terms of its sealing and swelling ability even in adverse saline conditions and low overburden stresses. The kaolin improves the osmotic efficiency of the self and also helps the underlying GB layer to seal the inter-granular voids. The estimated design parameters by through-diffusion test suggest that the kaolin-GB layered system effectively attenuates the permeant flux and suitable as a landfill liner.

scholarly journals High ionic strength dissociation assay (HISDA) for high drug tolerant immunogenicity testing

Bioanalysis ◽  
2020 ◽  
Author(s):  
Gregor Jordan ◽  
Alexander Pöhler ◽  
Florence Guilhot ◽  
Meike Zaspel ◽  
Roland F Staack
Keyword(s):  
Ionic Strength ◽  
Acid Treatment ◽  
Structural Integrity ◽  
Drug Tolerance ◽  
High Ionic Strength ◽  
Acid Dissociation ◽  
High Drug ◽  
Overnight Incubation ◽  
Antidrug Antibody ◽  
High Doses

Aim: Antidrug antibody (ADA) assessment may be challenged in studies that involve the administration of high doses of biotherapeutics and/or with long half-lives. In such cases, ADA assays with optimized drug tolerance are desired. Material & Methods: We evaluated the use of MgCl2 to develop high ionic strength dissociation assays in two investigational examples (bridging enzyme-linked immunosorbent ADA assays) to attain high drug tolerance while maintaining best possible structural integrity of ADAs. Results: Both ADA-bridging assays treated with MgCl2 showed improved drug tolerance and higher signal-to-blank values compared with overnight incubation or acid treatment. Conclusion: The use of MgCl2 treatment in ADA-bridging assays provides a sensitive, drug tolerant and easy-to-use alternative in cases where acid dissociation is not possible or unwanted.

scholarly journals High salt compatible oxyanion receptors by dual ion imprinting

2020 ◽  
Vol 11 (16) ◽  
pp. 4246-4250 ◽  
Author(s):  
Sudhirkumar Shinde ◽  
Mona Mansour ◽  
Anil Incel ◽  
Liliia Mavliutova ◽  
Celina Wierzbicka ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Ion Pair ◽  
High Ionic Strength ◽  
High Salt ◽  
Ion Uptake ◽  
Ion Imprinting

Imprinting of an ion-pair in presence of mutually compatible anion and cation host monomers leads to polymers showing enhanced ion uptake in competitive high ionic strength buffers.

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