An Improved Method for Identifying 8-O-Acetyl-N-Acetylneuraminic Acid

The periodic acid/thionin-Schiff/potassium hydroxide/periodic acid/fuchsin-Schiff sequence developed by Culling et al. frequently causes damage to sections and gives inconsistent results because of insufficient primary oxidation and difficulties in making the thionin-Schiff reagent. These disadvantages have been largely eliminated by more thorough primary oxidation and by replacing the original thionin-Schiff with a new cold thionin-Schiff. The effect of alkaline hydrolysis on thionin-aldehyde complexes was also studied and the reduction of color caused by this treatment was restored by a second thionin-Schiff reaction. The new sequence gives consistent results and imparts greater color to the thionin-Schiff reaction.     

Histochemical Comparison of the Casella, Bauer, and Periodic Acid Oxidation-Schiff Leucofuchsin Technics

Chromic acid, potassium permanganate and periodic acid apparently produce aldehyde from the same general group of substances. Chromic acid and potassium permanganate also destroy the aldehyde which they have produced, as well as that previously produced by periodic acid oxidation and by the Feulgen hydrochloric acid hydrolysis. Sulfite blockade, by occupying a considerable proportion of the aldehyde groups produced by periodic acid oxidation, creates a Schiff reaction similar to that produced by primary or secondary chromic acid or permanganate oxidation.

It is suggested that the periodic acid Schiff positive substances which fail to give distinct Bauer and Casella reactions, are those with relatively few reactive 1,2 glycol, 1,2 OH,NH₂, or 1,2 OH,NHR groupings per molecule.     

Histochemical Comparison of the Casella,Bauer, and Periodic Acid Oxidation-Schiff Leucofuchsin Technics

Chromic acid, potassium permanganate and periodic acid apparently produce aldehyde from the same general group of substances. Chromic acid and potassium permanganate also destroy the aldehyde which they have produced, as well as that previously produced by periodic acid oxidation and by the Feulgen hydrochloric acid hydrolysis. Sulfite blockade, by occupying a considerable proportion of the aldehyde groups produced by periodic acid oxidation, creates a Schiff reaction similar to that produced by primary or secondary chromic acid or permanganate oxidation.

It is suggested that the periodic acid Schiff positive substances which fail to give distinct Bauer and Casella reactions, are those with relatively few reactive 1,2 glycol, 1,2 OH,NH₂, or 1,2 OH,NHR groupings per molecule.     

The coating of mouse myocardial cells. A cytochemical electron microscopical study.

The coating of mouse myocardial cells has been investigated with a variety of cytochemical methods. The coating of the surface membrane gives a positive reaction with ruthenium red, colloidal thorium, phosphotungstic acid (PTA) at low pH, silver methenamine after periodic oxidation (PA-silver technique) and with silver proteinate after periodic oxidation and thiocarbohydrazide treatment (PA-TCH-silver technique). The coating of the T system gives almost similar results. The nexuses do not react with PTA nor with the PA-silver and PA-TCH-silver techniques, but they are strongly stained with ruthenium red which reveals periodic structures in their gaps. The specificities of the colloidal thorium technique and PAT staining have been tested by chemical treatments (methylation, acetylation, saponification), enzymatic digestions (pronase, trypsin, hyaluronidase, neuraminidase) and carbohydrate extractions (with 0.1 N NaOH and 0.05 M H2SO4). These cytochemical data indicate, considering the specificity of the reactions, that the coating of the membrane surface and the T system contains polyanionic groups. A part of them, at least, would belong to a carbohydrate-containing material (glycoproteins), whereas at the level of nexuses the sugar residues would probably be absent.     

Some comments on the PAS reaction of glycogen

Synopsis The periodic acid-Schiff (PAS) reaction has been studied in sections of mouse liver using a self-assembled microspectrophotometer. Increased colour intensity was obtained up to 4 hr of oxidation with periodic acid and 2 hr treatment with Schiff's reagent. The oxidation curve showed an initial, steep increase in colouration with a levelling off afterwards, that could not be attributed to loss of aldehyde groups. The results obtained from carrying out the PAS reaction on sections pretreated with -amylase suggest that the reaction takes place in two phases in which the outer glucosyl groups of the glycogen molecule are oxidized more rapidly than the inner ones.     

Black Periodic and Black Bauer Methods for Tissue Polysaccharides

m-Aminophenol gives a more rapid and more complete blockage of the Schiff reaction of aldehydes produced in tissue by oxidation with periodic or chromic acid than does aniline. Moreover, the phenol thus attached to aldehyde sites in tissue can then be azo coupled with alkaline solutions of fresh or stabilized diazonium salts. When so used at pH 8, fast black K, C.I. 37190, shows glycogen, mucins, basement membranes, muscle stroma, fungus cell walls, etc. stained black, on a somewhat grayish pink to red background.     

Histochemical Differentiation of Anterior Pituitary Cell Types by Contrasting Azocoupling and Mucoprotein Stains

Azo compounds couple with aromatic amino-acid nuclei in the cytoplasmic proteins of human anterior pituitary acidophile and basophile cells. In acidophile cells the reaction appears to be due primarily to tyrosine, but also in part to histidine. Procedures are given for the use of naphthanil diazo blue B (tetrazotized di-ortho-anisidine), with or without a second coupling agent (H acid; 8-amino-1-naphthol-3, 6-disulfonic acid), to demonstrate acidophile cells, in contrast to mucoprotein stains (periodic acid oxidation followed by leukofuchsin or leukothionin) for the cytoplasm of basophile cells. Evans blue can also be used to give a contrasting color to basophile cells.     

Histochemical Differentiation of Anterior Pituitary Cell Types by Contrasting Azocoupling and Mucoprotein Stains

Azo compounds couple with aromatic amino-acid nuclei in the cytoplasmic proteins of human anterior pituitary acidophile and basophile cells. In acidophile cells the reaction appears to be due primarily to tyrosine, but also in part to histidine. Procedures are given for the use of naphthanil diazo blue B (tetrazotized di-ortho-anisidine), with or without a second coupling agent (H acid; 8-amino-1-naphthol-3, 6-disulfonic acid), to demonstrate acidophile cells, in contrast to mucoprotein stains (periodic acid oxidation followed by leukofuchsin or leukothionin) for the cytoplasm of basophile cells. Evans blue can also be used to give a contrasting color to basophile cells.     

Fluorescence induction studies in isolated chloroplasts III. On the electron-transfer equilibria in the pool of electron acceptors of Photosystem II

The oxidation states of the primary and secondary electron acceptors of Photosystem II in isolated chloroplasts were measured simultaneously during their oxidation and reduction. The fluorescence yield and its variation were used as indicators for the oxidation states. An equilibrium treatment gives different contradicting results for different conditions: a low equilibrium constant (K ≈ 1) for the reaction between the (reduced) primary carrier to (oxidized) secondary carriers when the system is photoreduced, and a high equilibrium constant (K > 10, sometimes K > 100) when the system is oxidized in the dark. This discrepancy is discussed in terms of two alternatives: (a) influence of the light, by secondary interactions, on reaction parameters; (b) possibility of two photoreactions in Photosystem II.     

Alcianblue/PAS or PAS/alcianblue?

Summary The combined alcian blue (AB)/PAS technic is widely used for the detection and characterization of mucosubstances in tissue sections. Mostly the sequence AB/PAS is used, occasionally also the reserved sequence PAS/AB. The present study shows clearly that the sequence of the combined technic, i.e. AB/PAS or PAS/AB is substantially influencing the results. So it could be demonstrated that by using the combination PAS/AB originally PAS-positive and AB-negative reacting mucosubstances become AB-posltive. This could be caused by periodic acid oxidation followed by addition of hydrogen sulfite to aldehyde group thus providing secondary basophilic resp. AB positive material.     

Alcianblue/PAS or PAS/alcianblue? Remarks on a classical technic used in carbohydrate histochemistry

The combined alcian blue (AB)/PAS technic is widely used for the detection and characterization of mucosubstances in tissue sections. Mostly the sequence AB/PAS is used, occasionally also the reserved sequence PAS/AB. The present study shows clearly that the sequence of the combined technic, i.e. AB/PAS or PAS/AB is substantially influencing the results. So it could be demonstrated that by using the combination PAS/AB originally PAS-positive and AB-negative reacting mucosubstances become AB-positive. This could be caused by periodic acid oxidation followed by addition of hydrogen sulfite to aldehyde group thus providing secondary basophilic resp. AB positive material.     

Ethylenic Reaction of Ceroid with Performic Acid and Schiff Reagent

On oxidation with performic (or peracetic) acid ceroid becomes Schiff positive. This reaction is prevented by prior bromination, but not by acetylation. Interposition of bisulfite, semi-carbazide or phenylhydrazine blockades after the oxidation and before the leucofuchsin prevents the positive Schiff reaction on short exposure, but a long (3 hour) Schiff bath overcomes the blockade in all three instances. Prior acetylation blocks the periodic acid Schiff reaction of ceroid, but bromination does not.

These findings indicate the presence in ceroid of ethylenic groupings as well as 1,2 glycols.     

Spectroscopic study of permanganate oxidation reactions of oligonucleotides containing single base mismatches

The electrophoretic gel-based chemical cleavage of the mismatch method gives an incomplete view of the DNA conformational changes induced by a single base mismatch. This spectroscopic study investigates the permanganate oxidation reactions with matched and mismatched DNA under constant and variable temperature conditions. The results, which include the oxidation levels, reaction patterns with isosbestic points, color changes, thermal spectra, spectroscopy derivative, and gel separation and melting temperatures, provide a fundamental background for identification of oligonucleotides containing single base mismatches by chemical means.     

Quantitative PAS assay of some carbohydrate compounds and detergents.

A spectrophotometric method for determination of color development of glycocompounds subjected to PAS reaction was investigated with various carbohydrate compounds and related chemicals. The conditions of the oxidation with periodic acid was found to influence the amount of the colored Schiff dye produced. Mono- and di-saccharides (mannose, glucose and maltose) were PAS-negative. Glycogen was more reactive than dextran. When glycogen was hydrolyzed by amylase the intensity of the PAS product dropped until a certain limit probably reflecting the limit dextrin. The presence of proteins (albumin) or electrolytes (NaCl) did not influence the PAS reaction. Many non-ionic detergents commonly used in membrane biology such as alkyl glycosides and gluco-methyl alkanamides were strongly PAS-positive and so was the anionic detergent SDS while the zwitterionic detergents tested, such as CHAPS and CHAPSO, were PAS-negative. The color development of the spectrophotometric PAS reaction showed linearity with the concentration of a simple glycoprotein solution (peroxidase) and a complex solution (bovine serum). By the PAS reaction it was also possible to measure the content of soluble and membrane bound carbohydrate compounds in a pellet of liver cell membranes. We find that the PAS reaction is sensitive and reliable for quantitative estimations of complex carbohydrates as well as soluble and membrane-bound carbohydrate compounds. The latter should be treated with PAS-unreactive zwitterionic detergents.     

Studies on Synthetic Pyrethroid

Methyl αδ-dimethylsorbate, when submitted to the selective oxidation sequence by the actions of perbenzoic acid, mineral acid, lead tetraacetate and peracetic acid in this order, finally gives β-monomethyl mesaconate, which is converted after hydrolysis to mesaconic acid of the well-defined trans-configuration. The reaction sequence involves no process likely to invert the configuration, and thus another cogent chemical evidence for the trans-configuration of αδ-dimethylsorbic acid is obtained in agreement with the physico-chemical conclusion, previously reported.     

SILVER IMPREGNATION OF ULTRATHIN SECTIONS FOR ELECTRON MICROSCOPY

A new procedure is described for silver impregnation of thin sections for electron microscopy. Sections of various tissues, fixed in OsO₄ and embedded in methacrylate, were treated with an ammoniacal silver solution, directly or after oxidation with periodic acid or hydrogen peroxide. After OsO₄ fixation all cellular membranous systems exhibit a primary argentaffinity probably due to the reduction of ammoniacal silver solution by the reduced osmium bound to unsaturated lipids. Bleaching the sections with hydrogen peroxide removes the argentaffinity of protoplasmic structures. Treatment of the sections with periodic acid results in decreased argentaffinity of protoplasmic components while the argentaffinity of metaplasmic structures is greatly enhanced. The latter procedure appears particularly useful for enhancing the contrast of basement membranes.     

Black Periodic and Black Bauer Methods for Tissue Polysaccharides

m-Aminophenol gives a more rapid and more complete blockage of the Schiff reaction of aldehydes produced in tissue by oxidation with periodic or chromic acid than does aniline. Moreover, the phenol thus attached to aldehyde sites in tissue can then be azo coupled with alkaline solutions of fresh or stabilized diazonium salts. When so used at pH 8, fast black K, C.I. 37190, shows glycogen, mucins, basement membranes, muscle stroma, fungus cell walls, etc. stained black, on a somewhat grayish pink to red background.     

On the chemical basis of the Lowry protein determination

The copper-catalyzed oxidation of peptides and proteins by phosphomolybdic/phosphotungstic acid (Folin phenol reagent) was studied with respect to redox stoichiometry of color formation and nature of the oxidation products. From peptides without reducing side chains two reducing equivalents were transferred under ideal conditions to Mo6+/W6+ for each unit of tetradentate copper complex with concomitant formation of an imino peptide. Tyrosine and tryptophan side chains contributed four additional reducing equivalents. Oxidation of proline-containing peptides was greatly impaired as judged from color formation due to the interference of the imino acid with complex formation. Reaction of the oxidized peptides with 2,4-dinitrophenyl (DNP)-hydrazine gave a peptide amine and the DNP-hydrazone of a 2-oxoacyl peptide. The oxidation products from tetraalanine were identified as dialanine amide and pyruvoylalanine DNP-hydrazone. From the time course of the development of the blue color on reduction of Folin reagent with tetraalanine it was inferred that the reaction consisted of an initial (less than 5 s) oxidation to a Cu3+ peptide complex followed by slow changes in absorbance, especially above 0.2 mM. Due to these complications the two-electron stoichiometry has to be considered only as a limiting case for peptide concentrations below 0.02 mM.     

Electron histochemical study of 1:2-glycol groups in experimental hamster amyloid

Summary Leishmania-induced amyloid of hamster kidneys was studied by the periodic acid — thiocarbohydrazide — osmium tetroxide method which is analogous to the PAS reaction.The amyloid fibrils failed to give a reaction for 12-glycol groups. The ultrastructural distribution of mucosubstances containing 12-glycol groups was found to be localized in the ground substance between the fibrils.The reaction was unaffected by -amylase digestion, and chloroform-methanol extraction of lipids; it was dependant upon periodic acid oxidation.     

Polyvinyl pyridine metal complex as permanent antimicrobial finishing for viscose fabric

Viscose fabrics were treated with polyvinyl pyridine (PVP) using padding technique, followed by oxidation with hydrogen peroxide or peracetic acid, which was prepared by the reaction of tetra acetyl ethylene diamine (TAED) with hydrogen peroxide. Peracetic acid gives higher oxidation of PVP than hydrogen peroxide. FTIR study proved the formation of N-oxide as a result of oxidation. Incorporation of copper and silver ion onto oxidized PVP was also proved by FTIR. The antimicrobial study emphasise that Cu/oxidized PVP and Ag/oxidized PVP have retarded the growth of bacteria significantly, and Ag/oxidized PVP has a far better biocidal activity. The antibacterial activity of both metal ions survived after washing 10 times.