scholarly journals Neurofilament proteins of rat peripheral nerve and spinal cord.

1978 ◽  
Vol 78 (3) ◽  
pp. 653-662 ◽  
Author(s):  
W W Schlaepfer ◽  
L A Freeman
Keyword(s):  
Spinal Cord ◽  
Peripheral Nerve ◽  
Osmotic Shock ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Tissue Homogenates ◽  
Central Nervous Systems ◽  
Rat Peripheral Nerve ◽  
Staining Techniques ◽  
A Minor

Intact neurofilaments were isolated in parallel from rat peripheral nerve and spinal cord by osmotic shock into hypotonic media containing divalent cation chelators. Isolated neurofilaments were washed and separated by multiple centrifugations in 0.1 M NaCl. Abundant intact neurofilaments were identified in the washed pellets by negative staining techniques. Their origin from neurofilaments was confirmed by immune electron microscopy. Washed neurofilaments were extracted from lipid and membranous components with 8 M urea. Analyses of neurofilament isolates on sodium dodecyl sulfate gels showed that proteins of 200,000, 150,000, and 69,000 mol wt were the major components of intact neurofilaments derived from rat peripheral and central nervous systems. These same proteins were identified in whole tissue homogenates of both sources and became enriched during the isolation of intact neurofilaments. A minor component of 64,000 mol wt arose during isolation. Other proteins were identified as contaminants. Small amounts of proteins with electrophoretic migration of tubulin and actin remain in neurofilament isolates.

Herpes simplex virus type 2 UL14 gene product has heat shock protein(HSP)-like functions

2002 ◽  
Vol 115 (12) ◽  
pp. 2517-2527
Author(s):  
Yohei Yamauchi ◽  
Kaoru Wada ◽  
Fumi Goshima ◽  
Tohru Daikoku ◽  
Kenzo Ohtsuka ◽  
...  
Keyword(s):  
Heat Shock ◽  
Nuclear Translocation ◽  
Osmotic Shock ◽  
Minor Component ◽  
Firefly Luciferase ◽  
Hsp70 Family ◽  
Arginine Residues ◽  
Simplex Virus ◽  
A Minor ◽  
Shock Proteins

The HSV-2 UL14 gene encodes a 32 kDa protein that is a minor component of the viral tegument. The protein relocates other viral proteins such as VP26 and UL33 protein into the nuclei of transiently coexpressing cells(Yamauchi et al., 2001). We found that the protein shared some characteristics of heat shock proteins(HSPs) or molecular chaperones, such as nuclear translocation upon heat shock,ATP deprivation and osmotic shock. Interestingly, a significant homology over a stretch of 15 amino acids was found between an N-terminal region of HSV UL14 protein and the substrate-binding domain of Hsp70 family proteins. Two arginine residues in this region were important for nuclear translocation of VP26. In addition, overexpression of UL14 protein increased the activity of coexpressed firefly luciferase, which suggested that the protein functioned in the folding of newly synthesized luciferase. We thus conclude that UL14 protein can act as a chaperone-like protein in a singly expressed state.

scholarly journals Spray-Dried Amorphous Solid Dispersions of Griseofulvin in HPC/Soluplus/SDS: Elucidating the Multifaceted Impact of SDS as a Minor Component

Pharmaceutics ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 197 ◽  
Author(s):  
Mahbubur Rahman ◽  
Stephanie Ahmad ◽  
James Tarabokija ◽  
Nathaniel Parker ◽  
Ecevit Bilgili
Keyword(s):  
Solid Dispersions ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Amorphous Solid ◽  
Amorphous Solid Dispersions ◽  
Acetone Water ◽  
Poorly Soluble ◽  
A Minor ◽  
The Impact ◽  
Spray Dried

This study aimed to elucidate the impact of a common anionic surfactant, sodium dodecyl sulfate (SDS), along with hydroxypropyl cellulose (HPC) and Soluplus (Sol) on the release of griseofulvin (GF), a poorly soluble drug, from amorphous solid dispersions (ASDs). Solutions of 2.5% GF and 2.5%–12.5% HPC/Sol with 0.125% SDS/without SDS were prepared in acetone–water and spray-dried. The solid-state characterization of the ASDs suggests that GF–Sol had better miscibility and stronger interactions than GF–HPC and formed XRPD-amorphous GF, whereas HPC-based ASDs, especially the ones with a lower HPC loading, had crystalline GF. The dissolution tests show that without SDS, ASDs provided limited GF supersaturation (max. 250%) due to poor wettability of Sol-based ASDs and extensive GF recrystallization in HPC-based ASDs (max. 50%). Sol-based ASDs with SDS exhibited a dramatic increase in supersaturation (max. 570%), especially at a higher Sol loading, whereas HPC-based ASDs with SDS did not. SDS did not interfere with Sol’s ability to inhibit GF recrystallization, as confirmed by the precipitation from the supersaturated state and PLM imaging. The favorable use of SDS in a ternary ASD was attributed to both the wettability enhancement and its inability to promote GF recrystallization when used as a minor component along with Sol.

scholarly journals The lanthanide-enhanced affinity chromatography of clostridial collagenase

1985 ◽  
Vol 225 (2) ◽  
pp. 553-556 ◽  
Author(s):  
C H Evans
Keyword(s):  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Electrophoretic Analysis ◽  
Lanthanide Ions ◽  
Affinity Column ◽  
Major Protein ◽  
Periodic Acid Schiff ◽  
Highly Active ◽  
A Minor

Clostridiopeptidase A (EC 3.4.24.3) did not bind to a collagen affinity column in the absence of Ca2+, but did so in the presence of lanthanide ions (Ln3+). The sequestered enzyme could be eluted with EGTA. For the four Ln3+ ions tested, the order of efficiency in promoting enzyme binding, Sm3+ greater than Lu3+ greater than Er3+ much greater than La3+, reflected their relative abilities to inhibit clostridiopeptidase A. By using Sm3+ as an adjunct, it proved possible to separate a highly active preparation of collagenase from crude clostridial collagenase. Sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis of the preparation revealed a major protein of Mr 95000 and a minor component of Mr 82000. As both were stained by periodic acid/Schiff reagent, they were probably glycoproteins.

scholarly journals Immunological and ultrastructural studies of neurofilaments isolated from rat peripheral nerve

1977 ◽  
Vol 74 (1) ◽  
pp. 226-240 ◽  
Author(s):  
WW Schlaepfer
Keyword(s):  
Electron Microscopy ◽  
Peripheral Nerve ◽  
High Speed ◽  
Osmotic Shock ◽  
Antigenic Determinants ◽  
Immune Electron Microscopy ◽  
Ultrastructural Studies ◽  
Tissue Extracts ◽  
Carbon Coated ◽  
Rat Peripheral Nerve

Neurofilaments were isolated from desheathed and minced segments of rat peripheral nerve by osmotic shock into 0.01 M Tris-HCI buffer, pH 7.2. Freshly isolated neurofilaments were observed to undergo disassembly by progressive fragmentation upon exposure of dilute tissue extracts to this buffer. Low- and high-speed centrifugations of these tissue extracts separated membranous and particulate constituents and produced a progressive enrichment of 68,000-dalton polypeptide band in successive supernates, as determined by analyses of soluble proteins by SDS-polyacrylamide electrophoresis. The final high-speed supernatant fractions (S3) of nerve extracts, which were predominantly composed of 68,000-dalton polypeptide, were used to raise a specific experimental antisera in rabbits. Utilizing techniques of immune electron microscopy, experimental rabbit antisear was shown to contain antibodies against neurofilaments. Intact neurofilaments isolated from rat nerves and attached to carbon-coated grids became decorated when exposed to experimental rabbit antisera or purified gamma globulin (IgG) derivatives. The decoration of neurofilaments closely resembled the IgG coating seen in immune electron microscopy. Antibody absorption techniques were used to identify the biochemical constituency of neurofilamentous antigenic determinants. The decoration of neurofilament by experimental IgG was not altered by additions of tubulin or bovine serum albumin, but was prevented by additions of S3 fractions as well as the 68,000-dalton polypeptide of this fraction which was eluted and recovered from polyacrylamide gels. These findings are indicative that a 68,000-dalton polypeptide is a constituent subunit of rat peripheral nerve neurofilaments.

scholarly journals Heterogeneity in human prothrombin: analysis of cause

Blood ◽  
1981 ◽  
Vol 58 (5) ◽  
pp. 886-891 ◽  
Author(s):  
SP Bajaj ◽  
SI Rapaport ◽  
C Prodonos ◽  
WA Russell
Keyword(s):  
Sialic Acid ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Neutral Sugar ◽  
Citrate Buffer ◽  
Amino Terminal ◽  
Fraction H ◽  
Single Donor ◽  
Normal Human ◽  
A Minor

Abstract Two fractions of human prothrombin can be isolated from single donor plasma by the technique of heparin-agarose chromatography in (sodium) citrate buffer, pH 7.5, as previously reported for pooled plasma. The two fractions, designated H-II1 and H-II2, are found in a ratio of approximately 4:1. Both forms comigrate in sodium dodecyl sulfate gel electrophoresis; however, under nondenaturing electrophoretic conditions, each fraction migrates as a discrete entity with a different mobility. The larger fraction (H-II1) has a faster mobility towards the anode. Isoelectric focusing in urea of H-II1 reveals that it has two components, a minor component with a pl of 5.25 (H-II1a) and a major component with a pl of 5.40 (H-II1b). H-II2 has a pl of 5.6 H- II1 and H-II2 possess the same amino terminal residue (alanine, 0.87- 0.92 mole/mole) and the same number of gamma -carboxyglutamic acid residues (9.8–10.5). Their amino acid composition is indistinguishable. However, the two fractions of prothrombin differ in their content of neutral sugar and of sialic acid residues. Removal of sialic acid with neuraminidase abolishes the electrophoretic heterogeneity. Thus, the charge heterogneity of the three variants of prothrombin found in normal human plasma appears to result exclusively from differences in the number of sialic acid residues attached to the protein moiety of the molecule.

scholarly journals Immunofluorescence studies of neurofilaments in the rat and human peripheral and central nervous system

1977 ◽  
Vol 74 (1) ◽  
pp. 241-250 ◽  
Author(s):  
WW Schlaepfer ◽  
RG Lynch
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Peripheral Nerve ◽  
Neuronal Cell ◽  
Fluorescein Isothiocyanate ◽  
Linear Processes ◽  
Nervous Systems ◽  
Rabbit Igg ◽  
Central Nervous Systems ◽  
Rat Peripheral Nerve

Localization of antisera to neurofilament antigens derived from rat peripheral nerve was carried out in tissues of rat and human peripheral and central nervous systems by indirect immunofluorescence. Unfixed and chloroform-methanol-fixed frozen sections of tissues were incubated in purified IgG of the experimental rabbit antisera and subsequently exposed to goat anti-rabbit IgG conjugated with fluorescein isothiocyanate. Control studies were conducted on identical tissue preparations incubated in the same concentrations of nonspecific rabbit IgG or in experimental rabbit IgG absorbed with extracts of rat peripheral nerve containing neurofilament antigen. Extensive immunofluorescence was observed in rat and human peripheral and central nervous systems. The distribution and configuration of immunofluorescence corresponded to neurofilament-rich structural components of these tissues. Prominent immunofluorescence was also noted in neuronal cell bodies of spinal sensory ganglia, especially in perikarya of the large neuronal type. Immunofluorescence of the central nervous system was located predominantly in myelinated axons of the white matter in cerebrum, cerebellum, brain stem, and spinal cord. Less intense immunofluorescence was also seen in neuronal perikarya and in short thin linear processes of grey matter.

scholarly journals The isolation of an o-diphenal oxidase from third-instar larvae of the blowfly Calliphora erythrocephala

1975 ◽  
Vol 149 (3) ◽  
pp. 707-712 ◽  
Author(s):  
R N Pau ◽  
P A M. Eagles
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Sucrose Density Gradient ◽  
Crystalline Fraction ◽  
Calliphora Erythrocephala ◽  
A Minor ◽  
Third Instar Larvae

1. The isolation of an o-diphenol oxidase from an acetone-dried powder of late-third-instar larvae of Calliphora erythrocephala was investigated. An insoluble and micro-crystalline fraction containing the enzyme activity was obtained after fractionating extracts of the acetone-dried powder with (NH4)2SO4 and acetone. 2. This fraction can be solubilized in 0.1% sodium dodecyl sulphate without loss of activity. 3. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate shows that the o-diphenol oxidase is a minor component of the extracts from the acetone-dried powder. 4. The o-diphenol oxidase was purified by zonal centrifugation on a sucrose density gradient in the presence of sodium dodecyl sulphate. 5. The amino acid composition of the purified enzyme resembles that of some other o-diphenol oxidases. 6. The subunit composition of the o-diphenol oxidase is discussed.

scholarly journals Observations on the disassembly of isolated mammalian neurofilaments.

1978 ◽  
Vol 76 (1) ◽  
pp. 50-56 ◽  
Author(s):  
W W Schlaepfer
Keyword(s):  
Peripheral Nerve ◽  
Hypertonic Saline ◽  
Structural Stability ◽  
Mitotic Spindle ◽  
Reducing Agents ◽  
Negative Staining ◽  
Rat Peripheral Nerve ◽  
Staining Techniques ◽  
Saline Solutions

Intact mammalian neurofilaments were separated by centrifugation of osmotically shocked, desheathed segments of rat peripheral nerve. Neurofilament-rich supernates were incubated in different media with varying dilutions or dialysis of samples. Neurofilaments attached to carbon-Formvar-coated grids were exposed to similar incubations. The relative preservation or disruption of neurofilaments during different incubational conditions was monitored through periodic examinations of neurofilaments by negative staining techniques. Maximum structural stability of neurofilaments was manifested during incubation in isotonic NaCl or KCl. Decreasing salinity of incubational media led to increasing disruption of neurofilaments, especially in solutions less than 0.01 M. Hypertonic saline solutions were found to be less disruptive to mammalian neurofilaments. Additional studies examined neurofilamentous alterations effected by pH, protein denaturants, mitotic spindle inhibitors, reducing agents, and freeze-thawing procedures.

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