scholarly journals Fractionation and characterization of oligomeric, protofibrillar and fibrillar forms of β-amyloid peptide

2000 ◽  
Vol 348 (1) ◽  
pp. 137-144 ◽  
Author(s):  
Robin V. WARD ◽  
Kevin H. JENNINGS ◽  
Robert JEPRAS ◽  
William NEVILLE ◽  
Davina E. OWEN ◽  
...  
Keyword(s):  
Cell Viability ◽  
Hippocampal Neurons ◽  
Senile Plaques ◽  
Gradient Centrifugation ◽  
Neuronal Cell ◽  
Amyloid Peptide ◽  
Aβ Peptide ◽  
Isolation And Characterization ◽  
Β Amyloid

The β-amyloid (Aβ) peptide, a major component of senile plaques in Alzheimer's disease brain, has been shown previously to undergo a process of polymerization to produce neurotoxic forms of amyloid. Recent literature has attempted to define precisely the form of Aβ responsible for its neurodegenerative properties. In the present study we describe a novel density-gradient centrifugation method for the isolation and characterization of structurally distinct polymerized forms of Aβ peptide. Fractions containing protofibrils, fibrils, sheet structures and low molecular mass oligomers were prepared. The fractionated forms of Aβ were characterized structurally by transmission electron microscopy. The effects on cell viability of these fractions was determined in the B12 neuronal cell line and hippocampal neurons. Marked effects on cell viability in the cells were found to correspond to the presence of protofibrillar and fibrillar structures, but not to monomeric peptide or sheet-like structures of polymerized Aβ. Biological activity correlated with a positive reaction in an immunoassay that specifically detects protofibrillar and fibrillar Aβ; those fractions that were immunoassay negative had no effect on cell viability. These data suggest that the effect of Aβ on cell viability is not confined to a single conformational form but that both fibrillar and protofibrillar species have the potential to be active in this assay.

The Isolation and Characterization of Platelet Fibringgen

1979 ◽  
Author(s):  
R.E Zimmermann
Keyword(s):  
Blood Clotting ◽  
Biochemical Analysis ◽  
Gradient Centrifugation ◽  
Physiological Role ◽  
Carbohydrate Content ◽  
Freezing And Thawing ◽  
Fibrin Formation ◽  
Isolation And Characterization ◽  
Controlled Precipitation

Since platelets contribute essentially to the blood clotting mechanism the question arises as to what extent their physiological role is expressed by a specified type of fibrinogen.Platelets are harvested , washed exhaustively and homogenated by freezing and thawing. After gradient centrifugation of the homogenate fibrinogen was isolated by controlled precipitation steps. According to biochemical analysis platelet fibrinogen is of a unique polypeptid-composition, differs in its carbohydrate content and reveals a different kinetic of fibrin formation.

Isolation and characterization of a mitochondrial RNA polymerase from Drosophila melanogaster

1987 ◽  
Vol 65 (2) ◽  
pp. 173-182 ◽  
Author(s):  
Michael Goldenthal ◽  
James T. Nishiura
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Polymerase ◽  
Nuclear Dna ◽  
Gradient Centrifugation ◽  
Mitochondrial Rna ◽  
Mitochondrial Rna Polymerase ◽  
Isolation And Characterization ◽  
Cscl Gradient ◽  
Nuclear Rna

A DNA-dependent RNA polymerase was solubilized from sucrose gradient isolated, DNase-treated mitochrondria of Drosophila melanogaster. The isolated mitochondria were not detectably contaminated with nuclear DNA as shown by CsCl gradient centrifugation and polylysine Kieselguhr chromatography. The detergent-solubilized RNA polymerase was sensitive to rifampicin, resistant to α-amanitin, had an apparent molecular mass of about 60 kilodaltons, and displayed a tendency to aggregate, both in crude extracts or when purified. The mitochondrial RNA polymerase could be distinguished from nuclear RNA polymerases on the basis of size, salt optima, rifampicin sensitivity, and α-amanitin resistance.

scholarly journals Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor α1 Expression

2009 ◽  
Vol 29 (8) ◽  
pp. 2264-2277 ◽  
Author(s):  
Mi-Hwa Kim ◽  
Hong-Beum Kim ◽  
Samudra Acharya ◽  
Hong-Moon Sohn ◽  
Jae Yeoul Jun ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Glial Cell ◽  
Cancer Progression ◽  
Neuronal Cell ◽  
Amyloid Peptide ◽  
Pancreatic Cancer Cells ◽  
Β Amyloid ◽  
Neurotropic Factor ◽  
Β Amyloid Peptide

ABSTRACT Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) dysregulation has been identified in several human tumors and in patients with a variety of neurodegenerative diseases. However, the function of Ape1/Ref-1 is unclear. We show here that Ape1/Ref-1 increases the expression of glial cell-derived neurotropic factor (GDNF) receptor α1 (GFRα1), a key receptor for GDNF. Expression of Ape1/Ref-1 led to an increase in the GDNF responsiveness in human fibroblast. Ape1/Ref-1 induced GFRα1 transcription through enhanced binding of NF-κB complexes to the GFRα1 promoter. GFRα1 levels correlate proportionally with Ape1/Ref-1 in cancer cells. The knockdown of endogenous Ape1/Ref-1 in pancreatic cancer cells markedly suppressed GFRα1 expression and invasion in response to GNDF, while overexpression of GFRα1 restored invasion. In neuronal cells, the Ape1/Ref-1-mediated increase in GDNF responsiveness not only stimulated neurite outgrowth but also protected the cells from β-amyloid peptide and oxidative stress. Our results show that Ape1/Ref-1 is a novel physiological regulator of GDNF responsiveness, and they also suggest that Ape1/Ref-1-induced GFRα1 expression may play important roles in pancreatic cancer progression and neuronal cell survival.

scholarly journals OCCURRENCE, ISOLATION, AND CHARACTERIZATION OF POLYRIBOSOMES IN YEAST

1967 ◽  
Vol 34 (2) ◽  
pp. 505-512 ◽  
Author(s):  
Leon Marcus ◽  
H. Ris ◽  
H. O. Halvorson ◽  
R. K. Bretthauer ◽  
R. M. Bock
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Nearest Neighbors ◽  
Amino Acid Incorporation ◽  
Size Classes ◽  
Isolation And Characterization ◽  
Acid Incorporation

This report details the procedural requirements for preparing cell-free extracts of yeast rich in polyribosomes. This enabled us to demonstrate the occurrence of polyribosomes in yeast, to show their role in protein synthesis, and to devise methods for their resolution and isolation. When certain precautions are met (the use of log phase cells, rapidly halting cell growth, gentle methods of disruption, sedimentation through exponential density gradients, etc.), individual polyribosome size classes ranging up to the heptosome can be fractionated and separated from their nearest neighbors. Larger size classes are resolved partially among themselves, free of smaller polyribosomes. This was confirmed by extensive electron micrographic studies of material from the various fractions obtained upon density gradient centrifugation of yeast extracts. Modifications of the gradients and procedure should allow fractionation and isolation of the larger polyribosomes, including those containing polycistronic messages. Yeast polyribosomes are disaggregated to single ribosomes by longer term grinding, cell disruption by the French pressure cell, the Hughes press, or by incubation with dilute RNAse. Yeast polyribosomes are active in the incorporation of amino acids into polypeptide; the single ribosomes exhibit only slight activity. The latter activity is probably due to the presence of a small fraction of monosomes still containing mRNA. Poly-U stimulates amino acid incorporation only in the single ribosomes.

Isolation and Characterization of the Yeast mRNA Capping Enzyme β Subunit Gene Encoding RNA 5′-Triphosphatase, Which Is Essential for Cell Viability

1997 ◽  
Vol 239 (1) ◽  
pp. 116-122 ◽  
Author(s):  
Toshihiko Tsukamoto ◽  
Yoshio Shibagaki ◽  
Shinobu Imajoh-Ohmi ◽  
Teruko Murakoshi ◽  
Masako Suzuki ◽  
...  
Keyword(s):  
Cell Viability ◽  
Β Subunit ◽  
Subunit Gene ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Mrna Capping ◽  
Capping Enzyme

scholarly journals Selective isolation and characterization of rare actinomycetes adopted in glacier soil of Manali ice point and its activity against Mycobacterium spp

2017 ◽  
Vol 7 (5) ◽  
pp. 1 ◽  
Author(s):  
RAJA A ◽  
P Gajalakshmi
Keyword(s):  
Active Compound ◽  
Gradient Centrifugation ◽  
Population Isolation ◽  
Isolation And Characterization ◽  
Novel Approach ◽  
Rare Actinomycetes ◽  
Nocardia Sp ◽  
Glacier Ice ◽  
Mycobacterium Spp

An exigent demand of antimicrobial agent against M. tuberculosis was lead to the isolation of novel rare actinomycetes from the unexplored cryophilic environment. Soil samples were collected from glacier ice point of Kullu Manali and processed for further studies. A novel approach was described for the isolation of rare actinomycetes from a heterogeneous population. Isolation was done by conventional and density gradient centrifugation. Sucrose gradient centrifugation showed a maximum of 24 actinomycetes isolates which belong to the genera of Streptomyces sp (12), Micromonospora sp (5) Planomonospora sp(2), Micropolyspora sp (2), Actinopolyspora sp (1) Nocardia sp (1) and Intrasporangium sp (1). Of these 24 actinomycetes, isolate Planomonospora sp (PL-2) showed potent anti-mycobacterial activity against Mycobacterium smegmatis (MTCC300) and M.tuberculosis (MTCC 6). Bioautography reveals that the Rf value of active compound was 0.75 and retains the antimicrobial activity at 75° C. Based on the C13 and H1 NMR the active compound was characterized as 2-(2-ethenylphenyl) heptane-1-ol. Phylogenetic analysis reveals active isolate was closely related to Planomonospora alba and the Genbank accession is JQ280498.

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