Showing papers on "Glutaraldehyde published in 1973"

Polymerization of proteins with glutaraldehyde. Soluble molecular-weight markers.

Abstract: Glutaraldehyde is well known for its ability to react with proteins and to produce insoluble cross-linked aggregates. In contrast with this situation, conditions are described here which yield covalently linked soluble protein oligomers. The procedure is applicable to a wide range of proteins, and by slight variation in the reaction conditions, soluble polymers in the molecular weight range 3x10(4)-2x10(7) were produced. The products are valuable as molecular-weight markers, e.g. in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. The inherent similarities of these oligomers make them superior to commercial molecular-weight protein markers, which may have marked differences in composition and charge.

ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES : I. Effects of Trypsin, Phospholipase C, and pH on the Topography of Bound Positively Charged Colloidal Particles

Abstract: The effects of pH, trypsin, and phospholipase C on the topographic distribution of acidic anionic residues on human erythrocytes was investigated using colloidal iron hydroxide labeling of mounted, fixed ghost membranes. After glutaraldehyde fixation at pH 6.5-7.5, the positively charged colloidal particles were bound to the membranes in small randomly distributed clusters. The clusters of anionic sites were reversibly aggregated by incubation at pH 5.5 before fixation at the same pH. These results correlate with the distribution of intramembranous particles found by Pinto da Silva (J. Cell Biol.53:777), with the exception that the distribution of anionic sites on a majority of the fixed ghosts at pH 4.5 was aggregated instead of dispersed. The randomly distributed clusters could be nonreversibly aggregated by trypsin or phospholipase C treatment of intact ghosts before glutaraldehyde fixation. Previous glutaraldehyde fixation prevented trypsin and pH induced aggregation of the colloidal iron sites. Evidence that N-acetylneuraminic acid groups are the principal acidic residues binding colloidal iron was the elimination of greater than 85% of the colloidal iron labeling to neuraminidase-treated cell membranes. Colloidal iron binding N-acetylneuraminic acid residues may reside on membrane molecules such as glycophorin, a sialoglycoprotein which contains the majority of the N-acetylneuraminic acid found on the human erythrocyte membrane.

Glutaraldehyde fixation of isolated eucaryotic nuclei. Evidence for histone-histone proximity.

Abstract: Isolated chicken erythrocyte nuclei have been incubated with dilute concentrations of the bifunctional cross-linking agent glutaraldehyde (0–20 mM) in order to stabilize histone-histone interactions within the native nucleus. The kinetics of the disappearance of acid-soluble histones, free amino groups, and of individual histones have been observed to be pseudo first-order. Apparent first-order rate constants for the disappearance of individual histones correlate with the lysine mole percent of that fraction and follow the ranking, kapp: F1 > F2C > F2B ≥ F2A2, F2A1, F3. Histone polymers were observed to form very rapidly during the fixation reaction. Partial fractionation and amino acid analyses of these polymers support the view that they are composed principally of cross-linked (F2C)n molecules (where n = 2 to ∼8). The rate of glutaraldehyde reaction with free amino groups in histones is drastically reduced in solvents that promote chromatin decondensation (i.e., low ionic strengths in the absence of divalent cations) whereas the formation of cross-linked F2C polymers is less severely reduced. It is proposed that some F2C histones exist in close proximity within the isolated erythrocyte nucleus.

Antibody enzyme conjugates: the preparation of intermolecular conjugates of horseradish peroxidase and antibody and their use in immunohistology of renal cortex.

Abstract: The studies reported were done with the objectives of preparing several different peroxidase-labeled antibody conjugates and of testing their usefulness for the detection of soluble and insoluble tissue antigens. Glutaraldehyde, toluene diisocyanate and N,N'-dicyclohexyl carbodiimide were used to cross-link horseradish peroxidase to goat anti-rabbit immunoglobulin G; the resulting conjugates were characterized by molecular size and enzymatic and immunologic activity. They were then tested for their properties as immunohistologic reagents using 0.5-µ sections of freeze-substituted paraffin-embedded renal cortical tissue. Excellent results were obtained with a highly polymerized conjugate made with glutaraldehyde and with an unpolymerized conjugate made with toluene diisocyanate. With the use of these conjugates tissue localization of both soluble and insoluble antigens was achieved after subsequent fixation of tissue and counterstaining with periodic acid-Schiff-hematoxylin.

The preparation of intermolecular conjugates of horseradish peroxidase and antibody and their use in immunohistology of renal cortex

Abstract: The studies reported were done with the objectives of preparing several different peroxidase-labeled antibody conjugates and of testing their usefulness for the detection of soluble and insoluble tissue antigens. Glutaraldehyde, toluene diisocyanate and N, N’dicyclohexyl carbodiimide were used to cross-link horseradish peroxidase to goat antirabbit immunoglobulin G; the resulting conjugates were characterized by molecular size and enzymatic and immunologic activity. They were then tested for their properties as immunohistologic reagents using #{176}-5�Msections of freeze-substituted paraffin-embedded renal cortical tissue. Excellent results were obtained with a highly polymerized conjugate made with glutaraldehyde and with an unpolymerized conjugate made with toluene diisocyanate. With the use of these conjugates tissue localization of both soluble and insoluble antigens was achieved after subsequent fixation of tissue and counterstaining with periodic acid-Schiff-hematoxylin.

Preservation of Erythrocyte Ghost Ultrastructure Achieved by Various Fixatives

Abstract: We have evaluated the quality of ultrastructural preservation of erythrocyte ghosts achieved under various electron microscopic preparative conditions. Initially, negative staining was used to monitor gross morphology of the ghosts. Of several negative stains used (phosphotungstate, silicotungstate, ammonium molybdate, and uranyl acetate), all but uranyl acetate resulted in fragmentation of membranes and the appearance of small vesicular structures. Further, preservation of gross membrane ultrastructure was greatly enhanced when samples were fixed with either 4.0-5.0% glutaraldehyde or 1% osmium tetroxide (OsO4) before they were stained with uranyl acetate. We then examined the ability of these fixatives to preserve membrane fine structure, as monitored by thin-sectioning procedures. In these studies, fixation with 1% osmium tetroxide (alone or in conjunction with 5% glutaraldehyde) resulted in a trilamellar image about 95 A in width. Fixation with 5% glutaraldehyde alone provided a markedly different result. The membrane now appeared as a single line about 160 A wide with regions of varying electron density throughout. This result suggests that glutaraldehyde used alone may reveal the location of membrane proteins that are obscured or removed by OsO4 fixation. This point would seem to be supported by the results obtained when erythrocyte membranes were extracted with 5 mM EDTA after fixation in either 5% glutaraldehyde or 1% OsO4. While only 10% of the detectable protein was solubilized from glutaraldehyde-treated erythrocyte membranes, 85% was solubilized from OsO4-treated ghosts. Among these latter proteins are three that migrated on Ouchterlony double-diffusion agar plates at the same position as three known proteins with molecular weights of about 200,000. Additional studies indicated that, even during a routine pre-embedding procedure, OsO4 led to solubilization of as much as 8 times the amount of protein as glutaraldehyde alone. Although the erythrocyte membrane has a notoriously weak association with its proteins, we feel that our studies provide a cautionary note with regard to the use of OsO4 as a fixative in other membrane systems.

Studies on the Mode of Action of Glutaraldehyde on Escherichia coli

Abstract: Summary. Glutaraldehyde was readily taken up by Escherichia coli cells with an increase in solutions buffered to pH 7·9; it was paralleled by a corresponding increase in bactericidal activity. Attempts to desorb glutaraldehyde from the cells indicated that the drug molecules were firmly bound. Inhibition of synthesis of macromolecules was demonstrated. Cell walls of E. coli exhibited a much reduced rate of hydrolysis following treatment with glutaraldehyde.

Effects of Glutaraldehyde and Osmium Tetroxide on Hypotrichous Ciliates, and Determination of the Most Satisfactory Fixation Methods for Electron Microscopy

Abstract: SYNOPSIS. In electron microscope studies on hyppotrichous ciliates, cytolysis and/or body deformation–resulting from insufficient contact with glutaraldehyde and poor infiltration of Epon 812, particularly into the buccal cavity, usually were observed. Fixation experiments were carried out to examine the effects of some fixatives on Euplotes eurystomus, Oxytricha bifaria and Stylonychia mytilus to establish the best fixation technic applicable to all species of hypotrichous ciliates. Although the effects of fixation varied considerably with the species, 2 fully satisfactory fixation methods were developed by using OsO4 and glutaraldehyde. In one, a mixture of both fixatives was employed; in the other a very short application of OsO4 was followed by glutaraldehyde. The problem of infiltration was solved by using Spurr's low-viscosity embedding medium in place of Epon 812.

Effect of Glutaraldehyde on Cell Viability, Triphenyltetrazolium Reduction, Oxygen Uptake, and β-Galactosidase Activity in Escherichia coli

Abstract: Acid (pH 5) and alkaline (pH 8.5) glutaraldehyde solutions were compared for their effects on cell viability, oxygen uptake, and beta-galactosidase activities in Escherichia coli. The action of glutaraldehyde at pH 7 on dehydrogenase activity was also studied. Dehydrogenase activity was inhibited at aldehyde concentrations which had little effect on cell viability. In contrast, oxygen uptake and beta-galactosidase activity took place in cells killed by acid or alkaline glutaraldehyde. The effect of glutaraldehyde on dehydrogenase activity and beta-galactosidase activity of disrupted suspensions was also investigated. The dialdehyde was considerably less inhibitory to these enzyme systems than to those of whole cells, and it is thus feasible that the results with whole cells are a consequence of its interaction with, and strengthening of, the outer cell surface, thereby preventing ready access of substrate to enzyme.

Reaction of Glutaraldehyde with Foot-and-Mouth Disease Virus

Abstract: Summary Treatment of foot-and-mouth disease virus with 4% glutaraldehyde increases the diam. of the particles by 25% and makes them permeable to phosphotungstic acid so that they appear empty. The treated particles also resemble naturally-occurring empty particles in their low sedimentation coefficient (about 75S) but, in contrast to empty particles, they have a normal content of RNA and a higher than normal buoyant density in caesium chloride. The RNA can be removed from fixed particles by ribonuclease. Two models are suggested which account for these alterations in the structure of the virus particles. These results show that fixation with glutaraldehyde, far from maintaining the structural integrity of the virus particles, leads to considerable alterations in the arrangement of the RNA and protein subunits.

Interaction of glutaraldehyde with spheroplasts of Escherichia coli.

Abstract: . Glutaraldehyde, in the presence or absence of sodium bicarbonate, caused an increase in the optical density of spheroplasts of Escherichia coli. The aldehyde, particularly at alkaline pH, prevented lysis of spheroplasts when these were suddenly exposed to a hypotonic environment. It is proposed that the stabilization which occurs results from the crosslinking activity of glutaraldehyde on the cell envelope of E. coli.

Coupling of peroxidase to poliovirus antibody: characteristics of the conjugates and their use in virus detection.

Abstract: Antibody to poliovirus type 1 (Po-1) was coupled to peroxidase by use of glutaraldehyde or 4, 4' -difluoro,3, 3' -dinitro diphenyl sulfone. Glutaraldehyde was found to be the superior coupling agent, yielding conjugates that had up to 2.8 x 10(4) enzyme units/ml (75% of total enzyme input). Conjugates migrated as a single band when centrifuged in sucrose density gradients, demonstrating that the purification procedure used was effective in removing both noncoupled enzyme and heterogeneous antibody components. Conjugates were specific for Po-1 and did not adsorb to cells infected with unrelated enterovirus types. Adsorption of conjugates to Po-1-infected cells was demonstrable within 6 h postinfection.

An Electron-Microscope Study of the Reaction of Collagen with Some Monoaldehydes and Bifunctional Aldehydes

Abstract: Rat tail tendon collagen has been treated with the monoaldehydes, formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde and benzaldehyde, and with the dialdehydes, glyoxal and glutaraldehyde. The treated collagen was then examined in the electron microscope using a negative stain. Alterations to the native collagen pattern were seen with formaldehyde, acetaldehyde, propionaldehyde, glyoxal and glutaraldehyde. A correlation was noted between the changes in the band pattern and the degree of cross-linking estimated from chemical and physical changes in the treated collagen and from measurements of cross-linking made by other workers. The results indicate that the use of electron microscopy and negative staining may be a valuable method of assessing the degree of cross-linking in collagen treated with aldehydes and other reagent.

Water-insoluble penicillin acylase

Abstract: Water-insoluble enzyme preparations are formed from a penicillin acylase enzyme; a water-insoluble absorbent material, preferably a cross-linked acrylate polymer containing free carboxyl groups; a water-soluble dialdehyde, e.g., glutaraldehyde; and an aliphatic diamine, preferably a alpha , omega -diaminoalkane having 2 to 10 carbon atoms. The resulting enzyme preparations have improved activity in the production of 6-aminopenicillanic acid.

Perfusion fixation of rat molar pulp tissue for light microscopy.

Abstract: – Molar pulp tissue of 42 rats was subjected to cardiac perfusion fixation or to immersion fixation with glutaraldehyde and formaldehyde. The importance of variations in pressure and duration of cardiac perfusion was studied. The results indicated that the perfusion method was superior to immersion fixation. Best preservation of the tissues was obtained when perfusion was performed with 1.7% glutaraldehyde for 10–12 min under a pressure of 130 cmH2O, and leaving the animals for 4 h, without immersion fixation. Also perfusion with formaldehyde resulted in good preservation, provided the solutions were prepared from paraformaldehyde powder. In contrast, solutions made from commercial stock solutions of formaldehyde gave inferior results. For sections stained with hematoxylin-eosin, perfusion with glutaraldehyde was preferable, while perfusion with formaldehyde showed increased sensitivity in demonstration of dentinal tubules stained with alcian blue at pH 3.6. It is suggested that in future studies on rat pulp tissue, perfusion with glutaraldehyde and formaldehyde should replace immersion fixation.