scholarly journals Tissue fixation by osmium tetroxide. A possible role for proteins.

1979 ◽  
Vol 27 (5) ◽  
pp. 997-999 ◽  
Author(s):  
A J Nielson ◽  
W P Griffith
Keyword(s):  
Normal Tissue ◽  
Osmium Tetroxide ◽  
Chemical Nature ◽  
Side Chain ◽  
Cross Linking ◽  
Tissue Fixation ◽  
Reaction Products ◽  
Cholesteryl Acetate ◽  
Unsaturated Lipids

The osmiophilia, under the conditions of normal tissue fixation, of the histidine, lysine, tryptophan, cysteine and methionine side chain of proteins is suggested by in vitro studies on blocked amino acids representative of such protein side chains, and the chemical nature of the reaction products elucidated. The chemical feasibility of inter- or intramolecular cross-linking of protein by OsO4 at these and other sites is demonstrated, as in the cross-linking of protein with unsaturated lipids such as methyl oleate, methyl linoleate and linolenate, and cholesteryl acetate. The relevance of these results to the process of tissue fixation by OsO4 is discussed.

Tissue fixation and staining with osmium tetroxide: the role of phenolic compounds.

1978 ◽  
Vol 26 (2) ◽  
pp. 138-140 ◽  
Author(s):  
A J Nielson ◽  
W P Griffith
Keyword(s):  
Phenolic Compounds ◽  
Electron Density ◽  
Normal Tissue ◽  
Osmium Tetroxide ◽  
Direct Interaction ◽  
Plant Tissues ◽  
Tissue Fixation ◽  
Chelate Complexes ◽  
Animal Tissues

It has been postulated that phenol-containing areas of plant and animal tissues were osmiophilic, but proof of direct interaction between osmium tetroxide and phenolic materials, or the nature of such reactions, has been lacking. We find that, under conditions similar to those of normal tissue fixation, osmium tetroxide reacts rapidly with those phenols containing o-dihydroxy groups (including such species found in plant tissues) to give very stable chelate complexes. We conclude that these complexes are responsible for the observed electron-density in phenol-containing areas of tissue treated with osmium tetroxide, so that such phenols are indeed osmiophilic.

scholarly journals Generation of guanine–amino acid cross-links by a free radical combination mechanism

2014 ◽  
Vol 16 (23) ◽  
pp. 11729-11736 ◽  
Author(s):  
Yuriy Uvaydov ◽  
Nicholas E. Geacintov ◽  
Vladimir Shafirovich
Keyword(s):  
Free Radical ◽  
Amino Acid ◽  
Side Chain ◽  
Cross Linking ◽  
Two Photon ◽  
Ionization Method ◽  
Cross Links ◽  
Photon Ionization ◽  
Amino Acid Radicals

The key step of DNA–protein cross-linking in vitro is the combination of guanine neutral radicals with side-chain C-centered amino acid radicals produced by a two-photon ionization method.

scholarly journals A CHROMATOGRAPHIC AND SPECTROPHOTOMETRIC STUDY OF THE PRODUCTS OF THE REACTION OF OSMIUM TETROXIDE WITH UNSATURATED LIPIDS

1967 ◽  
Vol 34 (2) ◽  
pp. 627-638 ◽  
Author(s):  
Edward D. Korn
Keyword(s):  
Room Temperature ◽  
Osmium Tetroxide ◽  
Spectrophotometric Study ◽  
Dihydroxystearic Acid ◽  
Osmic Acid ◽  
Gas Liquid Chromatography ◽  
Reaction Products ◽  
Visible Spectroscopy ◽  
Unsaturated Lipids ◽  
Layer Chromatography

Thin films of methyl oleate, oleic acid, and di-octadecenoyl phosphatidylcholine were reacted with a 2% solution of OsO4 in water for 1 hr at 0°. As controls, methyl 9,10-dihydroxystearate and 9,10-dihydroxystearic acid were reacted with OsO4 in 0.25 N NaOH in methanol for 1 hr at room temperature. The reaction products were isolated, purified, and analyzed by thin-layer chromatography, gas-liquid chromatography, and infrared and visible spectroscopy. In all cases, the products were identified as diesters of osmic acid in which two molecules of fatty acids are linked through 1 molecule of osmic acid.

scholarly journals An Unusual Mechanism of Isopeptide Bond Formation Attaches the Collagenlike Glycoprotein BclA to the Exosporium of Bacillus anthracis

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Li Tan ◽  
Mei Li ◽  
Charles L. Turnbough
Keyword(s):  
Bacillus Anthracis ◽  
Bond Formation ◽  
Basal Layer ◽  
Side Chain ◽  
Cross Linking ◽  
Amino Group ◽  
Content Type ◽  
Isopeptide Bond ◽  
Isopeptide Bonds

ABSTRACTThe outermost exosporium layer of spores ofBacillus anthracis, the causative agent of anthrax, is comprised of a basal layer and an external hairlike nap. The nap includes filaments composed of trimers of the collagenlike glycoprotein BclA. Essentially all BclA trimers are tightly attached to the spore in a process requiring the basal layer protein BxpB (also called ExsFA). Both BclA and BxpB are incorporated into stable, high-molecular-mass complexes, suggesting that BclA is attached directly to BxpB. The 38-residue amino-terminal domain of BclA, which is normally proteolytically cleaved between residues 19 and 20, is necessary and sufficient for basal layer attachment. In this study, we demonstrate that BclA attachment occurs through the formation of isopeptide bonds between the free amino group of BclA residue A20 and a side chain carboxyl group of an acidic residue of BxpB. Ten of the 13 acidic residues of BxpB can participate in isopeptide bond formation, and at least three BclA polypeptide chains can be attached to a single molecule of BxpB. We also demonstrate that similar cross-linking occursin vitrobetween purified recombinant BclA and BxpB, indicating that the reaction is spontaneous. The mechanism of BclA attachment, specifically, the formation of a reactive amino group by proteolytic cleavage and the promiscuous selection of side chain carboxyl groups of internal acidic residues, appears to be different from other known mechanisms for protein cross-linking through isopeptide bonds. Analogous mechanisms appear to be involved in the cross-linking of other spore proteins and could be found in unrelated organisms.IMPORTANCEIsopeptide bonds are protein modifications found throughout nature in which amide linkages are formed between functional groups of two amino acids, with at least one of the functional groups provided by an amino acid side chain. Isopeptide bonds generate cross-links within and between proteins that are necessary for proper protein structure and function. In this study, we discovered that BclA, the dominant structural protein of the external nap ofBacillus anthracisspores, is attached to the underlying exosporium basal layer protein BxpB via isopeptide bonds formed through a mechanism fundamentally different from previously described mechanisms of isopeptide bond formation. The most unusual features of this mechanism are the generation of a reactive amino group by proteolytic cleavage and promiscuous selection of acidic side chains. This mechanism, which apparently relies only on short peptide sequences in protein substrates, could be a general mechanismin vivoand adapted for protein cross-linkingin vitro.

scholarly journals Evaluation and Implementation of Biocompatible Methods for the Cross-linking of Plasma Proteins

2021 ◽  
Vol 7 (2) ◽  
pp. 187-190
Author(s):  
Tom Bode ◽  
Kai Höltje ◽  
Sara Leal-Marin ◽  
Marc Müller ◽  
Birgit Glasmacher
Keyword(s):  
Plasma Proteins ◽  
Water Solubility ◽  
Patient Specific ◽  
Cross Linking ◽  
Reaction Products ◽  
Cross Link ◽  
Autologous Plasma ◽  
Amino Group Content ◽  
Primary Amino

Abstract Autologous plasma proteins can be used to fabricate patient specific cardiovascular implants but need to be cross-linked to increase their mechanical strength and reduce water solubility. Glutaraldehyde is the state-of-the-art solution but its reaction products have been shown to be cytotoxic and pro-inflammatory. In this work, it has been shown, that cross-linking of plasma proteins with biocompatible alternatives to glutaraldehyde is possible. This was achieved by identifying four candidate substances (thrombin, transglutaminase, genipin, EDC) from current literature and investigating their ability to cross-link porcine plasma proteins in vitro. The degree of crosslinking was examined using calorimetric (DSC) and spectroscopic (FTIR, Raman) methods, mapping the influence of cross-linking on the denaturation temperature and primary amino-group content of the proteins. It could be shown that thrombin, genipin and EDC are able to cross-link plasma proteins to a satisfactory degree and thus represent useful alternatives to glutaraldehyde. Transglutaminase, on the other hand, could not sufficiently cross-link the plasma proteins and was therefore ruled out as an alternative.

scholarly journals Tissue fixation and staining by osmium tetraoxide: a possible role for alkaloids.

1981 ◽  
Vol 29 (11) ◽  
pp. 1347-1348 ◽  
Author(s):  
M J Wright ◽  
M Schröder ◽  
A J Nielson
Keyword(s):  
Normal Tissue ◽  
In Vitro Studies ◽  
Plant Tissues ◽  
Tissue Fixation ◽  
Osmium Tetraoxide

In vitro studies on the reactions of osmium tetraoxide, OsO4, with isoquinoline, pyridine, quinuclidine, and a series of structurally related alkaloids suggest that these species, under the conditions of normal tissue staining and fixation, are potentially osmiophilic. The structure of the products and their reactions with unsaturated substrates are described. The relevance of these results to the process of tissue staining and fixation of plant tissues containing alkaloids by OsO4 is discussed.

Structure─activity relationships in semi-synthetic penicillins

1971 ◽  
Vol 179 (1057) ◽  
pp. 357-367 ◽  
Keyword(s):  
Antibacterial Activity ◽  
Chemical Nature ◽  
Side Chain ◽  
Structural Variations ◽  
Present Survey ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
Large Numbers ◽  
The Relationship

Investigation of the relationship between the antibacterial activity of penicillins and the chemical nature of the side chain began in earnest in 1957, when the isolation of 6-aminopenicillanic acid (6-APA) made possible the preparation of large numbers of N -substituted derivatives. Since that time some 1800 different penicillins have been prepared and studied in Beecham Research Laboratories, and the number examined throughout the world must amount to several thousands. The purpose of the present survey is to try to summarize the general patterns of structure-activity relationships which have emerged from these studies. 6-APA itself has only a low order of antibacterial activity, and the same applies to derivatives in which the amino group at position 6 is substituted by radicals other than acyl groups (figure 1). Hence it soon became clear that no advantage was to be gained from such considerable structural variations, and efforts were concentrated on preparing true penicillins containing acyl side-chains. Virtually all of these show considerable activity in vitro against at least some bacteria, but the spectrum of activity varies widely.

scholarly journals The proximal pathway of metabolism of the chlorinated signal molecule differentiation-inducing factor-1 (DIF-1) in the cellular slime mould Dictyostelium

1995 ◽  
Vol 306 (3) ◽  
pp. 735-743 ◽  
Author(s):  
P Morandini ◽  
J Offer ◽  
D Traynor ◽  
O Nayler ◽  
D Neuhaus ◽  
...  
Keyword(s):  
Side Chain ◽  
Stalk Cell ◽  
Reaction Products ◽  
Signal Molecule ◽  
Slime Mould ◽  
Phenolic Ring ◽  
Cellular Slime ◽  
Cytochrome P 450

Stalk cell differentiation during development of the slime mould Dictyostelium is induced by a chlorinated alkyl phenone called differentiation-inducing factor-1 (DIF-1). Inactivation of DIF-1 is likely to be a key element in the DIF-1 signalling system, and we have shown previously that this is accomplished by a dedicated metabolic pathway involving up to 12 unidentified metabolites. We report here the structure of the first four metabolites produced from DIF-1, as deduced by m.s., n.m.r. and chemical synthesis. The structures of these compounds show that the first step in metabolism is a dechlorination of the phenolic ring, producing DIF metabolite 1 (DM1). DM1 is identical with the previously known minor DIF activity, DIF-3. DIF-3 is then metabolized by three successive oxidations of its aliphatic side chain: a hydroxylation at omega-2 to produce DM2, oxidation of the hydroxy group to a ketone group to produce DM3 and a further hydroxylation at omega-1 to produce DM4, a hydroxyketone of DIF-3. We have investigated the enzymology of DIF-1 metabolism. It is already known that the first step, to produce DIF-3, is catalysed by a novel dechlorinase. The enzyme activity responsible for the first side-chain oxidation (DIF-3 hydroxylase) was detected by incubating [3H]DIF-3 with cell-free extracts and resolving the reaction products by t.l.c. DIF-3 hydroxylase has many of the properties of a cytochrome P-450. It is membrane-bound and uses NADPH as co-substrate. It is also inhibited by CO, the classic cytochrome P-450 inhibitor, and by several other cytochrome P-450 inhibitors, as well as by diphenyliodonium chloride, an inhibitor of cytochrome P-450 reductase. DIF-3 hydroxylase is highly specific for DIF-3: other closely related compounds do not compete for the activity at 100-fold molar excess, with the exception of the DIF-3 analogue lacking the chlorine atom. The Km for DIF-3 of 47 nM is consistent with this enzyme being responsible for DIF-3 metabolism in vivo. The two further oxidations necessary to produce DM4 are also performed in vitro by similar enzyme activities. One of the inhibitors of DIF-3 hydroxylase, ancymidol (IC50 67 nM) is likely to be particularly suitable for probing the function of DIF metabolism during development.

The Current Situation in Chemical Stabilization of Biological Materials

1972 ◽  
Vol 30 ◽  
pp. 132-133
Author(s):  
John H. Luft
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Molecular Level ◽  
Cross Linking ◽  
Chemical Stabilization ◽  
Specimen Preparation ◽  
Sufficient Condition ◽  
Radio Telescopes

With information processing devices such as radio telescopes, microscopes or hi-fi systems, the quality of the output often is limited by distortion or noise introduced at the input stage of the device. This analogy can be extended usefully to specimen preparation for the electron microscope; fixation, which initiates the processing sequence, is the single most important step and, unfortunately, is the least well understood. Although there is an abundance of fixation mixtures recommended in the light microscopy literature, osmium tetroxide and glutaraldehyde are favored for electron microscopy. These fixatives react vigorously with proteins at the molecular level. There is clear evidence for the cross-linking of proteins both by osmium tetroxide and glutaraldehyde and cross-linking may be a necessary if not sufficient condition to define fixatives as a class.

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