THE FUSION OF ERYTHROCYTES BY TREATMENT WITH PROTEOLYTIC ENZYMES AND POLYETHYLENE GLYCOL

1976 ◽  
Vol 18 (3) ◽  
pp. 503-512 ◽  
Author(s):  
James X. Hartmann ◽  
J. D. Galla ◽  
D. A. Emma ◽  
K. N. Kao ◽  
O. L. Gamborg
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Polyethylene Glycol ◽  
Proteolytic Enzymes ◽  
Fusion Process ◽  
Significant Extent ◽  
Multinucleated Cells ◽  
Peg Treatment ◽  
Previously Treated ◽  
High Degree

Polyethylene glycol (PEG) has been utilized to induce homokaryocyte formation in avian and mammalian erythrocytes previously treated with proteolytic enzymes. PEG of molecular weight 6,000–7,500 was found superior to 1,500 and 20,000 MW PEG. Cells exposed to protease alone, prior to PEG treatment, fused to a high degree (60–95% multinucleated cells), whereas trypsin or pepsin treatment alone allowed very little fusion (2.5%). Trypsin lowered the effectiveness of protease when used in combination. Cells which were not treated with proteolytic enzymes agglutinated in the presence of PEG but did not fuse to a significant extent (0.01%). Fusion was also markedly dependent upon the rate at which PEG was eluted during the fusion process. Electron microscopy indicated that fusion began during the elution of PEG from the agglutinated cells.

scholarly journals Changes in the Molecular Characteristics of Bovine and Marine Collagen in the Presence of Proteolytic Enzymes as a Stage Used in Scaffold Formation

Marine Drugs ◽  
2021 ◽  
Vol 19 (9) ◽  
pp. 502
Author(s):  
Marfa N. Egorikhina ◽  
Ludmila L. Semenycheva ◽  
Victoria O. Chasova ◽  
Irina I. Bronnikova ◽  
Yulia P. Rubtsova ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Scanning Electron Microscopy ◽  
Proteolytic Enzymes ◽  
Molecular Characteristics ◽  
Species Origin ◽  
Bovine Collagen ◽  
Hydrolyzed Collagen ◽  
Source Materials ◽  
Scanning Electron

Biopolymers, in particular collagen and fibrinogen, are the leading materials for use in tissue engineering. When developing technology for scaffold formation, it is important to understand the properties of the source materials as well as the mechanisms that determine the formation of the scaffold structures. Both factors influence the properties of scaffolds to a great extent. Our present work aimed to identify the features of the molecular characteristics of collagens of different species origin and the changes they undergo during the enzymatic hydrolysis used for the process of scaffold formation. For this study, we used the methods of gel-penetrating chromatography, dynamic light scattering, reading IR spectra, and scanning electron microscopy. It was found that cod collagen (CC) and bovine collagen (BC) have different initial molecular weight parameters, and that, during hydrolysis, the majority of either type of protein is hydrolyzed by the proteolytic enzymes within the first minute. The differently sourced collagen samples were also hydrolyzed with the formation of two low molecular fractions: Mw ~ 10 kDa and ~20 kDa. In the case of CC, the microstructure of the final scaffolds contained denser, closely spaced fibrillar areas, while the BC-sourced scaffolds had narrow, short fibrils composed of unbound fibers of hydrolyzed collagen in their structure.

scholarly journals The fusion of trout spermatozoa with Chinese hamster fibroblasts

1982 ◽  
Vol 53 (1) ◽  
pp. 307-321
Author(s):  
J.H. Yip ◽  
N.C. Bols
Keyword(s):  
Electron Microscopy ◽  
Polyethylene Glycol ◽  
Light Microscopy ◽  
Chinese Hamster ◽  
Cell Types ◽  
Culture Conditions ◽  
Peg Treatment ◽  
Sperm Nuclei

Polyethylene glycol (PEG) was used to fuse trout sperm with Chinese hamster fibroblasts (CHW-1102). As judged by light microscopy, PEG significantly increased the number of associations between the two cell types. Electron microscopy revealed that the sperm nuclei were in the cytoplasm of CHW-1102 in cultures that had been treated with PEG and in phagosomes of CHW-1102 in cultures not treated with PEG. In the cytoplasm many sperm nuclei had an extensive network of fibres whereas before fusion the sperm nuclei contained chromatin blocks which were packed together tightly. In phagosomes they consisted of dispersed blocks of chromatin. The chromatin of the few sperm that were found outside CHW-1100 cells but in the same culture that was treated with PEG also lacked fibres and consisted of loosely packed blocks. Most nuclei were unaltered by PEG treatment alone although in a few the packing of the chromatin blocks was loosened. Thus the decondensation of sperm nuclei within CHW-1102 cells appears to be brought about by the mammalian cytoplasm rather than by digestion in phagosomes, the culture conditions, or the treatment used to induce fusion.

scholarly journals Rapid low molecular weight polyethylene glycol embedding protocol for immunocytochemistry.

1989 ◽  
Vol 37 (10) ◽  
pp. 1549-1552 ◽  
Author(s):  
J Mowery ◽  
J Chesner ◽  
S Spangenberger ◽  
D C Hixson
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Polyethylene Glycol ◽  
Room Temperature ◽  
Low Molecular Weight ◽  
Fixed Tissue ◽  
Thin Sectioning ◽  
Membrane Antigens ◽  
Excellent Preservation ◽  
Peg 600

We describe an alternative polyethylene glycol (PEG) embedding procedure which utilizes PEG 200 for dehydration and PEG 600 for infiltration and embedding of perfusion-fixed rat liver. PEG 600 has a melting point of 22 degrees C, enabling infiltration of fixed tissue to be performed at room temperature. Sections (2 microM) cut in a cryostat at -20 degrees C and immobilized in agarose were readily labeled by immunoperoxidase protocols with monoclonal antibodies to hepatocyte membrane antigens. Subsequent examination by light microscopy or by electron microscopy after re-embedding in resin and ultra-thin sectioning showed excellent preservation of morphology, with minimal impairment of antigenicity.

scholarly journals Effect of Polyethylene Glycol Treatment on Acetic Acid Emissions from Wood

Forests ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1629
Author(s):  
Sarah Hunt ◽  
Josep Grau-Bove ◽  
Eleanor Schofield ◽  
Simon Gaisford
Keyword(s):  
Molecular Weight ◽  
Acetic Acid ◽  
Polyethylene Glycol ◽  
Solid Phase ◽  
Carbon Disulphide ◽  
Solid Phase Micro Extraction ◽  
Accelerated Corrosion ◽  
Archaeological Wood ◽  
Peg Treatment ◽  
Museum Display

Acetic acid is known to be emitted from sound wood and can accelerate damage to heritage materials, particularly metals. However, few studies have investigated the extent of acetic acid emissions from archaeological wood. This research utilised Solid-Phase-Micro-Extraction (SPME) GC–MS and lead coupon corrosion to identify volatile emissions from polyethylene glycol (PEG)-treated archaeological wood from the Mary Rose collection and assess if they could cause accelerated damage. In addition, the effect of PEG treatment on acetic acid emissions was investigated using sound wood samples. For sound wood, the PEG treatment acted as a barrier to acetic acid emissions, with higher-molecular-weight PEGs preventing more emissions. Archaeological wood, despite its age and high-molecular-weight PEG treatment, still emitted detectable concentrations of acetic acid. Moreover, they emitted a wider array of compounds compared to sound wood, including carbon disulphide. Like sound wood, when the archaeological wood samples were in a sealed environment with lead coupons, they caused accelerated corrosion to lead. This evidences that archaeological wood can emit high enough concentrations of volatile compounds to cause damage and further investigation should be performed to evaluate if this can occur inside museum display cases.

Evaluation of the dark field method for large association of spread DNA

1978 ◽  
Vol 36 (2) ◽  
pp. 190-191
Author(s):  
Douglas C. Barker
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Extraction Method ◽  
Field Method ◽  
Dark Field ◽  
Kinetoplast Dna ◽  
Field Electron ◽  
Field Electron Microscopy ◽  
Dark Field Electron

A number of satisfactory methods are available for the electron microscopy of nicleic acids. These methods concentrated on fragments of nuclear, viral and mitochondrial DNA less than 50 megadaltons, on denaturation and heteroduplex mapping (Davies et al 1971) or on the interaction between proteins and DNA (Brack and Delain 1975). Less attention has been paid to the experimental criteria necessary for spreading and visualisation by dark field electron microscopy of large intact issociations of DNA. This communication will report on those criteria in relation to the ultrastructure of the (approx. 1 x 10-14g) DNA component of the kinetoplast from Trypanosomes. An extraction method has been developed to eliminate native endonucleases and nuclear contamination and to isolate the kinetoplast DNA (KDNA) as a compact network of high molecular weight. In collaboration with Dr. Ch. Brack (Basel [nstitute of Immunology), we studied the conditions necessary to prepare this KDNA Tor dark field electron microscopy using the microdrop spreading technique.

Tissue section lifting for electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 86-87
Author(s):  
Adrian F. van Dellen
Keyword(s):  
Infectious Disease ◽  
Electron Microscopy ◽  
Tissue Culture ◽  
Organ System ◽  
Surrounding Tissue ◽  
Paraffin Sections ◽  
Surface Areas ◽  
Specific Determination ◽  
High Degree ◽  
Accuracy And Repeatability

The morphologic pathologist may require information on the ultrastructure of a non-specific lesion seen under the light microscope before he can make a specific determination. Such lesions, when caused by infectious disease agents, may be sparsely distributed in any organ system. Tissue culture systems, too, may only have widely dispersed foci suitable for ultrastructural study. In these situations, when only a few, small foci in large tissue areas are useful for electron microscopy, it is advantageous to employ a methodology which rapidly selects a single tissue focus that is expected to yield beneficial ultrastructural data from amongst the surrounding tissue. This is in essence what "LIFTING" accomplishes. We have developed LIFTING to a high degree of accuracy and repeatability utilizing the Microlift (Fig 1), and have successfully applied it to tissue culture monolayers, histologic paraffin sections, and tissue blocks with large surface areas that had been initially fixed for either light or electron microscopy.

Electron Microscopy in Molecular Biology

1967 ◽  
Vol 25 ◽  
pp. 2-3
Author(s):  
Cecil E. Hall
Keyword(s):  
Electron Microscopy ◽  
Molecular Biology ◽  
Noise Level ◽  
Molecular Structures ◽  
Material Structure ◽  
The Arts ◽  
Shadow Casting ◽  
Electron Image ◽  
High Degree ◽  
Very High

The visualization of organic macromolecules such as proteins, nucleic acids, viruses and virus components has reached its high degree of effectiveness owing to refinements and reliability of instruments and to the invention of methods for enhancing the structure of these materials within the electron image. The latter techniques have been most important because what can be seen depends upon the molecular and atomic character of the object as modified which is rarely evident in the pristine material. Structure may thus be displayed by the arts of positive and negative staining, shadow casting, replication and other techniques. Enhancement of contrast, which delineates bounds of isolated macromolecules has been effected progressively over the years as illustrated in Figs. 1, 2, 3 and 4 by these methods. We now look to the future wondering what other visions are waiting to be seen. The instrument designers will need to exact from the arts of fabrication the performance that theory has prescribed as well as methods for phase and interference contrast with explorations of the potentialities of very high and very low voltages. Chemistry must play an increasingly important part in future progress by providing specific stain molecules of high visibility, substrates of vanishing “noise” level and means for preservation of molecular structures that usually exist in a solvated condition.

Reconstruction of Two-Dimensional Projections of GP 32*I

1981 ◽  
Vol 39 ◽  
pp. 38-39
Author(s):  
H.A. Cohen ◽  
W. Chiu ◽  
J. Hosoda
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Uranyl Acetate ◽  
Distilled Water ◽  
Acetate Solution ◽  
Two Dimensional ◽  
Parent Molecule ◽  
T4 Bacteriophage ◽  
Electron Imaging ◽  
Better Than

GP 32 (molecular weight 35000) is a T4 bacteriophage protein that destabilizes the DNA helix. The fragment GP32*I (77% of the total weight), which destabilizes helices better than does the parent molecule, crystallizes as platelets thin enough for electron diffraction and electron imaging. In this paper we discuss the structure of this protein as revealed in images reconstructed from stained and unstained crystals.Crystals were prepared as previously described. Crystals for electron microscopy were pelleted from the buffer suspension, washed in distilled water, and resuspended in 1% glucose. Two lambda droplets were placed on grids over freshly evaporated carbon, allowed to sit for five minutes, and then were drained. Stained crystals were prepared the same way, except that prior to draining the droplet, two lambda of aqueous 1% uranyl acetate solution were applied for 20 seconds. Micrographs were produced using less than 2 e/Å2 for unstained crystals or less than 8 e/Å2 for stained crystals.

Structure of Myosin Subfragment-1 from Electron Microscopy and Crystallography

1988 ◽  
Vol 46 ◽  
pp. 156-157
Author(s):  
Donald A. Winkelmann
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Actin Filaments ◽  
Light Chains ◽  
Myosin Filament ◽  
Primary Role ◽  
Heavy Chains ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Globular Head

The primary role of the interaction of actin and myosin is the generation of force and motion as a direct consequence of the cyclic interaction of myosin crossbridges with actin filaments. Myosin is composed of six polypeptides: two heavy chains of molecular weight 220,000 daltons and two pairs of light chains of molecular weight 17,000-23,000. The C-terminal portions of the myosin heavy chains associate to form an α-helical coiled-coil rod which is responsible for myosin filament formation. The N-terminal portion of each heavy chain associates with two different light chains to form a globular head that binds actin and hydrolyses ATP. Myosin can be fragmented by limited proteolysis into several structural and functional domains. It has recently been demonstrated using an in vitro movement assay that the globular head domain, subfragment-1, is sufficient to cause sliding movement of actin filaments.The discovery of conditions for crystallization of the myosin subfragment-1 (S1) has led to a systematic analysis of S1 structure by x-ray crystallography and electron microscopy. Image analysis of electron micrographs of thin sections of small S1 crystals has been used to determine the structure of S1 in the crystal lattice.

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