scholarly journals PURIFICATION OF INTACT MICROTUBULES FROM BRAIN

1970 ◽  
Vol 47 (2) ◽  
pp. 384-394 ◽  
Author(s):  
Joel B. Kirkpatrick ◽  
Lyon Hyams ◽  
Virginia L. Thomas ◽  
Peter M. Howley
Keyword(s):  
Low Temperature ◽  
Gel Electrophoresis ◽  
Combined Effects ◽  
Acidic Ph ◽  
Electron Microscopic ◽  
Major Band ◽  
Negative Stain ◽  
Hexylene Glycol ◽  
Electrophoresis System ◽  
Microtubule Structure

Hundred-fold purification of intact microtubules from homogenates of rat brain is reported. The success of purification depends on stabilizing the microtubule structure by the combined effects of hexylene glycol, acidic Ph, and low temperature. A practical, negative stain, electron microscopic assay is used to study purity and stability of microtubule fractions. The purified fractions show a major band which migrates like purified tubulin in the SDS gel electrophoresis system.

Molecular Structure of the Outermost Cell Wall Component of Spirillum Serpens

1977 ◽  
Vol 35 ◽  
pp. 342-343
Author(s):  
Wah Chiu ◽  
David Grano
Keyword(s):  
Molecular Structure ◽  
Cell Wall ◽  
Outer Membrane ◽  
Gel Electrophoresis ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Cell Wall Component ◽  
Protein Components ◽  
Exposure Technique ◽  
Structural Aspects

The periodic structure external to the outer membrane of Spirillum serpens VHA has been isolated by similar procedures to those used by Buckmire and Murray (1). From SDS gel electrophoresis, we have found that the isolated fragments contain several protein components, and that the crystalline structure is composed of a glycoprotein component with a molecular weight of ∽ 140,000 daltons (2). Under an electron microscopic examination, we have visualized the hexagonally-packed glycoprotein subunits, as well as the bilayer profile of the outer membrane. In this paper, we will discuss some structural aspects of the crystalline glycoproteins, based on computer-reconstructed images of the external cell wall fragments.The specimens were prepared for electron microscopy in two ways: negatively stained with 1% PTA, and maintained in a frozen-hydrated state (3). The micrographs were taken with a JEM-100B electron microscope with a field emission gun. The minimum exposure technique was essential for imaging the frozen- hydrated specimens.

Electron microscopic assays of restriction endonuclease cutting, exonuclease digestion, and hybridization

1984 ◽  
Vol 42 ◽  
pp. 686-687
Author(s):  
Mark Hannibal ◽  
Jacob Varkey ◽  
Michael Beer
Keyword(s):  
Electron Microscope ◽  
Low Temperature ◽  
Restriction Endonuclease ◽  
Double Helix ◽  
Test Material ◽  
Electron Microscopic ◽  
Exonuclease Iii ◽  
Dna Molecule ◽  
Linear Molecules ◽  
Exonuclease Digestion

Workman and Langmore have recently proposed a procedure for isolating particular chromatin fragments. The method requires restriction endonuclease cutting of the chromatin and a probe, their digestion with two exonucleases which leave complimentary single strand termini and low temperature hybridization of these. We here report simple electron microscopic monitoring of the four reactions involved.Our test material was ϕX-174 RF DNA which is cut once by restriction endonuclease Xho I. The conversion of circles to linear molecules was followed in Kleinschmidt spreads. Plate I shows a circular and a linear DNA molecule. The rate of cutting is shown in Figure 1.After completion of the endonuclease cutting, one portion of the DNA was treated with exonuclease III, an enzyme known to digest the 3' terminals of double helical DNA. Aliquots when examined in the electron microscope reveal a decreasing length of double helix and increasing bushes at the ends.

Structural Studies on the Eukaryotic Ribosome

1990 ◽  
Vol 48 (1) ◽  
pp. 256-257
Author(s):  
Adriana Verschoor ◽  
Ronald Milligan ◽  
Suman Srivastava ◽  
Joachim Frank
Keyword(s):  
Chick Embryo ◽  
3D Reconstruction ◽  
Single Particle ◽  
Mass Density ◽  
Particle Surface ◽  
Uranyl Acetate ◽  
Electron Microscopic ◽  
Eukaryotic Ribosome ◽  
Negative Stain ◽  
Reconstruction Methods

We have studied the eukaryotic ribosome from two vertebrate species (rabbit reticulocyte and chick embryo ribosomes) in several different electron microscopic preparations (Fig. 1a-d), and we have applied image processing methods to two of the types of images. Reticulocyte ribosomes were examined in both negative stain (0.5% uranyl acetate, in a double-carbon preparation) and frozen hydrated preparation as single-particle specimens. In addition, chick embryo ribosomes in tetrameric and crystalline assemblies in frozen hydrated preparation have been examined. 2D averaging, multivariate statistical analysis, and classification methods have been applied to the negatively stained single-particle micrographs and the frozen hydrated tetramer micrographs to obtain statistically well defined projection images of the ribosome (Fig. 2a,c). 3D reconstruction methods, the random conical reconstruction scheme and weighted back projection, were applied to the negative-stain data, and several closely related reconstructions were obtained. The principal 3D reconstruction (Fig. 2b), which has a resolution of 3.7 nm according to the differential phase residual criterion, can be compared to the images of individual ribosomes in a 2D tetramer average (Fig. 2c) at a similar resolution, and a good agreement of the general morphology and of many of the characteristic features is seen.Both data sets show the ribosome in roughly the same ’view’ or orientation, with respect to the adsorptive surface in the electron microscopic preparation, as judged by the agreement in both the projected form and the distribution of characteristic density features. The negative-stain reconstruction reveals details of the ribosome morphology; the 2D frozen-hydrated average provides projection information on the native mass-density distribution within the structure. The 40S subunit appears to have an elongate core of higher density, while the 60S subunit shows a more complex pattern of dense features, comprising a rather globular core, locally extending close to the particle surface.

scholarly journals Proteolytic Processing of Pro-opiomelanocortin Occurs in Acidifying Secretory Granules of AtT-20 Cells

1997 ◽  
Vol 45 (3) ◽  
pp. 425-436 ◽  
Author(s):  
Shigeyasu Tanaka ◽  
Takao Yora ◽  
Kazuhisa Nakayama ◽  
Kinji Inoue ◽  
Kazumasa Kurosumi
Keyword(s):  
Secretory Granules ◽  
Specific Inhibitor ◽  
Tumor Cell Line ◽  
Microscopic Analysis ◽  
Proteolytic Processing ◽  
Acidic Ph ◽  
Electron Microscopic ◽  
Constitutive Secretion ◽  
Golgi Network ◽  
Acidic Organelles

Using antibodies specific for pro-opiomelanocortin (POMC), amidated joining peptide (JP), and the prohormone convertase PC1, we showed immunocytochemically that PC1 in a corticotrophic tumor cell line, AtT-20, was co-localized either with POMC or with amidated JP in secretory granules, and also confirmed that POMC was cleaved mainly in secretory granules. Analysis using DAMP (3- [2,4-dinitroanilino]-3'-amino- N-methyldipropylamine) as the pH probe suggested a correlation between POMC processing and acidic pH in the secretory granules. Bafilomycin A1, a specific inhibitor of vacuolar-type H+-AT-Pase, completely inhibited POMC processing and caused constitutive secretion of the unprocessed precursor. By contrast, chloroquine, a weak base that is known to neutralize acidic organelles, was unable to inhibit POMC processing. Electron microscopic analysis revealed that, in AtT-20 cells treated with bafilomycin A1, the trans-Golgi cisternae were dilated and few secretory granules were present in the cytoplasm. These observations suggest that acidic pH provides a favorable environment for proteolytic processing of POMC by PC1 but is not required, and that integrity of the trans-Golgi network and sorting of POMC into secretory granules are important for POMC processing. (J Histochem Cytochem 45:425–436, 1997)

Combined effects of aging and low temperature, long time heating on pork toughness

Meat Science ◽  
2019 ◽  
Vol 150 ◽  
pp. 33-39 ◽  
Author(s):  
Shengjie Li ◽  
Renchao Ma ◽  
Jinfeng Pan ◽  
Xinping Lin ◽  
Xiuping Dong ◽  
...  
Keyword(s):  
Low Temperature ◽  
Combined Effects ◽  
Long Time ◽  
Effects Of Aging

scholarly journals Structure and molecular analysis of RGR1, a gene required for glucose repression of Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (8) ◽  
pp. 4130-4138 ◽  
Author(s):  
A Sakai ◽  
Y Shimizu ◽  
S Kondou ◽  
T Chibazakura ◽  
F Hishinuma
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Electrophoresis ◽  
Daughter Cell ◽  
Glucose Repression ◽  
Pulsed Field Gel Electrophoresis ◽  
Null Mutation ◽  
Electron Microscopic ◽  
Transmission Electron ◽  
Transmission Electron Microscopic ◽  
Carboxy Terminal

An RGR1 gene product is required to repress expression of glucose-regulated genes in Saccharomyces cerevisiae. The abnormal morphology of rgr1 cells was studied. Scanning and transmission electron microscopic observations revealed that the cell wall of the daughter cell remained attached to that of mother cell. We cloned the RGR1 gene by complementation and showed that the cloned DNA was tightly linked to the chromosomal RGR1 locus. The cloned RGR1 gene suppressed all of the phenotypes caused by the mutation and encoded a 3.6-kilobase poly(A)+ RNA. The RGR1 gene is located on chromosome XII, as determined by pulsed-field gel electrophoresis, and we mapped rgr1 between gal2 and pep3 by genetic analysis. rgr1 was shown to be a new locus. We also determined the nucleotide sequence of RGR1, which was predicted to encode a 123-kilodalton protein. The null mutation resulted in lethality, indicating that the RGR1 gene is essential for growth. On the other hand, a carboxy-terminal deletion of the gene caused phenotypes similar to but more severe than those caused by the original mutation. The amount of reserve carbohydrates was reduced in rgr1 cells. Possible functions of the RGR1 product are discussed.

Sign in / Sign up

Export Citation Format

Share Document