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 demonstration of two acid groups in juxtaglomerular granules with basic triphenyl methane dyes,aldehyde-fuchsin and schiff's reagent

Summary Oxidation and bromination of mouse kidney JG cell-granules result in the production of cysteic acid from cystine; cysteic acid is capable of taking up rapidly and selectively certain basic triphenyl methane dyes including aldehyde fuchsin at lower pH levels.After treatment with periodic acid, bromine and hydrochloric acid, the JG granules or the nuclear chromatin also take up the basic triphenyl methane dyes (including aldehyde fuchsin) which contain amino groups, probable as a result of the production of aldehyde groups. Basic triphenyl methane lacking amino groups does not react with aldehydes.Some substance present in JG granules could be stained by aldehyde fuchsin after prior oxidation; HCl methyl violet 2B was taken up both with or without prior oxidation. Only strong methylation completely abolished these affinities which were restored after demethylation. These reactions are attributed to cystine.The staining of JG granules with dilute aldehyde fuchsin and dilute methyl violet 2B is not affected by oxidation, bromination, aldehyde blocking and hydrolysis; these reactions are abolished by mild methylation, but restored by subsequent saponification. These staining properties are due to the presence of carboxylic acid in JG granules.The positive PAS reaction of JG granules is due to the presence of 1.2-glycol in the same granules.     

The Use Of Lead Tetraacetate,Benzidine, O-Dianisidine and A “Film Test” In Investigating The Periodic-Acid-Schiff Technic

In order to obtain a better understanding of the “periodic-acid-Schiff” reaction, also known as the “periodic-acid fuchsin-sulfurous-acid” reaction, three types of investigations were carried out

1) The Schiff reagent was replaced by other aldehyde reagents: benzidine or o-dianisidine. There was no significant change in the histological distribution and intensity of the reactions occurring after periodic acid oxidation.

2) Periodic acid was replaced by another oxidizing agent: lead tetraacetate (dissolved in acetic acid). There was no significant change in the histological distribution of the reactions with the Schiff reagent, but some change in their intensity. It was concluded that 1,2-glycols and a-amino alcohols play the main role in the reactions with both oxidants. The presence of α-hydroxy acids in some types of mucous cells is suggested by the results with lead tetraacetate.

Incidently, glycogen and starch are not sufficiently oxidized by lead tetraacetate (in acetic acid) at room temperature to give positive reactions with the Schiff reagent, while cellulose and other periodic-acid-Schiff reactive substances are.

3) The staining of films of presumed reactive substances with the periodic-acid-Schiff technic C O the intense reactivity of many polysaccharides, mucopolysaccharides and mucoproteins, but not of ordinary proteins. (Hyaluronic and chondroitin sulfuric acid are, however, not reactive in vitro).

In conclusion, the periodic-acid-Schiff technic consists of an oxidation of 1,2-glycols and a-amino alcohols to produce aldehyde groups, which are then stained by the Schiff reagent. The “film test” reveals that these radicals are present in certain polysaccharides, mucopolysaccharides and mucoproteins.     

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.     

Characterization of vanadium bromoperoxidase from Macrocystis and Fucus: reactivity of vanadium bromoperoxidase toward acyl and alkyl peroxides and bromination of amines

Vanadium bromoperoxidase (V-BrPO) has been isolated and purified from the marine brown algae Fucus distichus and Macrocystis pyrifera. V-BrPO catalyzes the oxidation of bromide by hydrogen peroxide, resulting in the bromination of certain organic acceptors or the formation of dioxygen. V-BrPO from F. distichus and M. pyrifera have subunit molecular weights of 65,000 and 74,000, respectively, and specific activities of 1580 units/mg (pH 6.5) and 1730 units/mg (pH 6) for the bromination of monochlorodimedone, respectively. As isolated, the enzymes contain a substoichiometric vanadium/subunit ratio; the vanadium content and specific activity are increased by addition of vanadate. V-BrPO (F. distichus, M. pyrifera, and Ascophyllum nodosum) also catalyzes the oxidation of bromide using peracetic acid. In the absence of an organic acceptor, a mixture of oxidized bromine species (e.g., hypobromous acid, bromine, and tribromide) is formed. Bromamine derivatives are formed from the corresponding amines, while 5-bromocytosine is formed from cytosine. In all cases, the rate of the V-BrPO-catalyzed reaction is much faster than that of the uncatalyzed oxidation of bromide by peracetic acid, at pH 8.5, 1 mM bromide, and 2 mM peracetic acid. In contrast to hydrogen peroxide, V-BrPO does not catalyze formation of dioxygen from peracetic acid in either the presence or absence of bromide. V-BrPO also uses phenylperacetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid to catalyze the oxidation of bromide; dioxygen is not formed with these peracids. V-BrPO does not catalyze bromide oxidation or dioxygen formation with the alkyl peroxides ethyl hydroperoxide, tert-butyl hydroperoxide, and cuminyl hydroperoxide.     

Cytochemical reactions of human leprosy bacilli and mycobacteria: ultrastructural implications

Leprosy bacilli harvested from freshly biopsied tissue from cases of lepromatous, borderline and histoid leprosy were, in conjunction with Mycobacterium lepraemurium and representative mycobacteria, examined cytochemically with and without their pyridine-extractable acid-fastness. Unlike the mycobacteria, unextracted leprosy bacilli failed to give a positive response to the periodic acid Schiff test or to take up Sudan black B, toluidine blue O, alkaline methylene blue or safranin O. Once their acid-fastness was removed with pyridine, leprosy bacilli were stained by all of the foregoing dyes except Sudan black B, under this condition they remained gram positive. While permanent loss of acid-fastness from leprosy bacilli always resulted in a loss of acid hematein-fixing material (Smith-Dietrich-Baker tests), the reverse was not true. Mild aqueous saponification, bromination, or sequential treatment with lipase and phospholipase D resulted in a loss of acid hematein-positivity but not acid-fastness. After pyridine extraction, bromination, or aqueous saponification, true mycobacteria lost neither their acid hematein-positivity nor their acid-fastness. The acid hematein-positive material and the acid-fastness of both leprosy bacilli and mycobacteria were lost after treatment with alkaline ethanol. These cytochemical findings are discussed in the light of what is known of the ultrastructure of leprosy bacilli and mycobacteria, and of the occurrence of a dl-3, 4-dihydroxyphenylalanine oxidase in leprosy bacilli but not in mycobacteria. An effort is made to explain the rather unique cytochemical properties of leprosy bacilli. Since pyridine-extractable acid-fastness (and acid hematein-positivity) serve to distinguish human leprosy bacilli from M. lepraemurium, one or the other, or both, are suggested as bases for differentiating these two organisms in animal experiments designed to show the in vivo propagation of human leprosy bacilli.     

Basic Fuchsin in Acid Alcohol: A Simplified Alternative to Schiff Reagent

The use of Schiff reagent to demonstrate polysaccharides (after prior periodic oxidation) and nucleic acids (after prior acid hydrolysis) is unnecessary since the same results are obtained by substituting a 20 min staining in a 0.5% w/v solution of basic fuchsin in acid alcohol (ethanol-water-concentrated HC1, 80:20:1) followed by a rinse in alcohol. The shade of the basic fuchsin staining is a little yellower than that achieved with Schiff reagent but the selectivity, light fastness, response to different fixatives, and to prior histo-chemical blocking of the tissue section were much the same for the two methods. The need for prior oxidation or hydrolysis and the inhibitory effect of aldehyde blocking techniques indicate that basic fuchsin, like Schiff reagent, reacts with aldehyde groups. Infrared studies indicate that for cellulose the reaction product is an azomethine.     

Histochemical studies of the mechanism of the periodic acid-phenylhydrazine-Schiff (PAPS) procedure

Summary Histochemical investigations of the periodic acid-phenylhydrazine-Schiff (PAPS) procedure were carried out on tissues containing carbohydrate macromolecules known to produce on periodate oxidation, only sialic acid monoaldehydes or hexosedialdehydes or mixtures of the two. The results indicated that the PAPS reaction is a generalized phenomenon independent of the hydrazine or hydrazide used, the nature of the Schiff reagent or the presence of anionic groups. It is proposed that phenylhydrazine condenses with periodate-engendered sialic acid monoaldehydes to yield the corresponding phenylhydrazone and with periodate-engendered dialdehydes to yield the corresponding morpholine or azido derivatives. Subsequent Schiff treatment results in the reversal of the blockade of sialic acid monoaldehydes but not of the dialdehydes, thus leading to selective Schiff staining of sialic acid residues.     

Soybean peroxidase as an effective bromination catalyst*

Soybean peroxidase (SBP), an acidic peroxidase isolated from the seed coat, has been shown to be an effective catalyst for the oxidation of a variety of organic compounds. In the present study, we demonstrate that SBP can catalyze halogenation reactions. In the presence of H(2)O(2), SBP catalyzed the oxidation of bromide and iodide but not chloride. Veratryl alcohol (3,4-dimethoxybenzyl alcohol) served as a useful substrate for SBP-catalyzed halogenations yielding the 6-bromo derivative. Halogenation of veratryl alcohol was optimal at pHs below 2.5 with rates of 2.4 μm/min, achieving complete conversions of 150-μm veratryl alcohol in 24 h. The enzyme showed essentially no brominating activity at pHs above 5.5. SBP-catalyzed bromination of veratryl alcohol proceeded with a maximum reaction velocity, (V(max))(apparent), of 5.8 x 10(-1) μm/min, a K(m) of 78 μm and a catalytic efficiency (k(cat)/K(m) of 1.37 x 10(5) M/min at pH 4.0, assuming all of the enzyme's active sites participate in the reaction. SBP also catalyzed the bromination of several other organic substrates including pyrazole to produce a single product 1-bromopyrazole, indole to yield both 5-bromoindole and 5-hydroxyindole, and the decarboxylative bromination of 3,4 dimethoxy-trans-cinnamic acid to trans-2-bromo-1-(3,4 dimethoxyphenyl)ethylene. A catalytic mechanism for SBP-catalyzed bromination has been proposed based on experimental results in this and related studies.     

The Inhibition of Foam Cell Formation by Sodium Diethyldithiocarbamate

A prominent feature of human atherosclerosis is the lipid-laden foamy macrophage, which often also contains the insoluble pigment, ceroid. The culture of macrophage-like cells, P388Dis, with artificial lipoproteins composed of cholesteryl linoleate (CL) and bovine serum albumin (BSA) results in foam cell formation with lipoprotein uptake and the intracellular accumulation of ceroid. Ceroid accumulation is accompanied by the oxidation of the cholesterol ester as monitored by gas chromatography. The sodium salt of diethyldithio-carbamic acid (DDC) at 1-5 μM effectively inhibited lipoprotein uptake, cholesteryl linoleate oxidation and ceroid accumulation in cultures of P388D₁. Further studies showed that intracellular ceroid accumulation appeared to require the presence of cystine in the medium. Lipoprotein oxidation by this macrophage-like cell therefore appears to involve a mechanism dependent on cystine metabolism which is consistent with previous reports of macrophage-mediated lipoprotein oxidation. Studies on CL/BSA-induced ceroid accumulation in human monocytes also showed that DDC behaved in much the same manner. This inhibitory effect of DDC on foam cell formation, often considered a primary event of atherosclerosis, at concentrations as low as 1 μM, suggests the need for further, more comprehensive, studies on this compound's activities.     

Primary extracellular ceroid type lipopigment. A histochemical and ultrastructural study

Summary An extracellular ceroid-type (ECC) lipopigment which appears histologically as wavy hyaline membranes or, less frequently, as an amorphous solid or broadly reticulated mass is described. Its ultrastructure is either amorphous or membranous and consists either of simple linear membrane-like aggregates or elaborated trilaminar membranes. The histochemical profile is, as in histiocytic intracellular ceroid, dominated by autofluorescence, strong hydrophobicity, acid and extraction resistance. Staining for aromatic acid residues and periodic acid-Schiff positivity are strong but variable. Lectin receptors are either absent or sparse. The pigment is found solely within the lipid rich tissue debris, bound to processes marked by necrosis of adipose or steatosed tissues and interpreted as originatingde novo extracellularly from liquid unsaturated lipids under the influence of local enzymatic and nonenzymatic lipid oxidation catalysts. A hitherto unknown form of extracellular ceroid is the so-called membranocystic lesion found in Nasu-Hakola's disease, in several other conditions and in annular ceroid in human atheromas.     

Influence of some reactional parameters on the substitution degree of biopolymeric Schiff bases prepared from chitosan and salicylaldehyde

Some reactional parameters as mol ratio (salicylaldehyde:free amino groups), reaction time and temperature were investigated in order to improve the substitution degree (DS) in the preparation of biopolymeric Schiff bases from chitosan. In this case, the reaction of chitosan and salicylaldehyde was used as a probe system in order to produce the Schiff base. The use of 50% (mol/mol) salicylaldehyde excess, reaction time of 18 h and temperature of 55 °C permitted to obtain a DS of 60% without evidences of hydrolysis of the biopolymeric matrix or changes in its acetylation degree.     

Oxidation of tissue polysaccharides by periodic acid in dimethyl sulfoxide and its anhydrous and aqueous mixtures.

Periodic acid (1% w/v) solvated by anhydrous dimethyl sulfoxide (DMSO) readily induced a strong Schiff reaction in a variety of structures containing polysaccharides, but not glycogen. With the increasing amounts of water added to DMSO, glycogen was also oxidized, while the selective localization of other polysaccharides remained unimpaired. Periodate, solvated in the anhydrous acetic acid-DMSO mixture, rapidly induced concomitant oxidation of nucin and glycogen-containing structures. Sodium bisulfite addition derivatives of carbonyls, induced by periodate oxidation in DMSO, were stained meta- and orthochromatically with toluidine blue at controlled pH. Certain metachromatic tissue components were strongly birefringent in polarized light in contrast to the identical structures oxidized by aqueous periodate. Marked differences in staining reactions elicited in identical structures by periodate in DMSO as compared with aqueous periodate suggest that DMSO-periodate method considerably enhances the range of histochemical oxidations by periodate.     

Chitooligosaccharide-Induced Activation of o-Phenylenediamine Oxidation by Wheat Seedlings in the Presence of Oxalic Acid

A method for determination of oxidation of phenolic compounds by intact wheat seedlings using o-phenylenediamine (OPD) was developed. The reaction is initiated by the addition of oxalic acid to the incubation medium. It is suggested that an endogenous peroxidase and hydrogen peroxide formed during oxidation of oxalic acid by endogenous oxalate oxidase are involved in OPD oxidation. Treatment of plants with chitooligosaccharides (1-10 mg/liter) with acetylation degree of 65% and molecular masses of 5-10 kD significantly activated OPD oxidation by wheat seedlings.     

Steroids and related products. XLVI. The regiospecific alpha-hydroxymethylation of saturated carbonyl compounds via preformed enolate ions. An improved synthesis of 17-hdyroxymethyl 20-keto steroids

As demonstrated for pregnenolone, saturated ketones are conveniently alpha-hydroxymethylated by their transformation into a lithium enolate and by the reaction of the latter with formaldehydr. The 17-hydroxymethylpregnenolone prepared by this method in very good yield was readily converted to 17-hydroxymethylprogesterone; either by selective acetylation in position 17(1) and subsequent Jones oxidation, followed by hydrolysis, or by conversion to the 4,5-dibromo 3-ketone - by bis(tri-n-butyltin)oxide-bromine oxidation or by dibromination and oxidation with N-bromoacetamde - and debromination with zinc and acetic acid.     

Effect of UV-light on formation of fluorescent products of lipid peroxidation

The rate of fluorescent product formation during the peroxidation of polyunsaturated linolenic acid or egg phosphatidylethanolamine and also during the oxidation of linolenic acid together with a phenylalanine and synthetic phosphatidylethanolamine 1,5--3 times more intensive after previous UV-irradiation of the unsaturated fatty acid. Schiff bases are fluorescent products in amine containing systems which are produced in the reaction of the malonaldehyde with amines. It is possible that fluorochromes produced during the only unsaturated acid oxidation are the result of the radical recombination. Accumulation of the oxidated products determined by TBA-reactive substances does not inevitably correlate with the fluorescent intensity in explored systems.     

Polycations as prostaglandin synthesis inducers. II. Structure-activity relationships

Moderately high molecular weight polycations stimulate arachidonic acid release with concomitant synthesis and release of prostaglandins in cultured 3T3 mouse fibroblasts. We have examined a series of synthetic polycations for prostaglandin synthesis-inducing activity as an approach to defining the structural features required for activity. Extensive (greater than 80%) acetylation of poly(vinylamine) was tolerated without loss of activity, indicating that a uniform high density of charges is not required. However, complete acetylation of poly(vinylamine) abolished activity, indicating that some positive charges are required for activity. full activity was observed for charge densities in the range of one per two to one per six atoms of polymer backbone. Branched and linear polycations activated equally well. Location of the charge with respect to the polymer backbone did not affect activity in polymers bearing charges located up to seven atoms away from the backbone. Polycations lacking primary or secondary amino groups exhibited full activity, indicating that Schiff base formation is not required for activity. These results are consistent with a model in which activation involves electrostatic interactions with discrete anionic sites on the target cell.     

Polycations as prostaglandin sythesis inducers. II. structure-activity relationships

Moderately high molecular weight polycations stimulate arachidonic acid release with concomitant synthesis and release of prostaglandins in cultured 3T3 mouse fibroblasts. We have examined a series of synthetic polycations for prostaglandin synthesis-inducing activity as an approach to defining the structural features required for activity. Extensive (>80%) acetylation of poly(vinylamine) was tolerated without loss of activity, indicating that a uniform high density of charges is not required. However, complete acetylation of poly(vinylamine) abolished activity, indicating that some positive charges are required for activity. Full activity was observed for charge densities in the range of one per two to one per six atoms of polymer backbone. Branched and linear polycations activated equally well. Location of the charge with respect to the polymer backbone did not affect activity in polymers bearing charges located up to seven atoms away from the backbone. Polycations lacking primary or secondary amino groups exhibited full activity, indicating that Schiff base formation is not required for activity. These results are consistent with a model in which activation involves electrostatic interactions with discrete anionic sites on the target cell.     

Chemical characterization of the regularly arranged surface layer glycoprotein of Clostridium thermosaccharolyticum D120-70

Clostridium thermosaccharolyticum D120-70 possesses as its outermost cell envelope layer a square-arranged array of glycoprotein molecules. SDS/polyacrylamide gel electrophoresis of the purified surface layer showed a broadened band in the molecular mass range of about 115 kDa which, upon periodic acid/Schiff staining, gave a positive reaction. After proteolytic degradation of this material, two glycopeptide fractions were obtained. One- and two-dimensional nuclear magnetic resonance studies, together with methylation analysis and periodate oxidation, were used to determine the structures of the polysaccharide portions of these glycopeptides. The combined chemical and spectroscopic evidence suggests the following structures: (formula; see text).     

A new interpretation of the direct Schiff reaction of elastic connective tissue

A direct Schiff reaction of elastic tissues has been known for many years, but the nature of the native aldehyde-rich components has not been clear. In this study, chicken, quail, and rat embryos and adult rat lung, aorta, and kidney were fixed in methacarn or in a formalin solution, embedded in paraffin, and sections of 8-10 micron obtained. Rehydrated sections were incubated for various periods in solutions of the enzymes chondroitinase ABC, clostripain, collagenase, elastase, heparatinase, hyaluronidase, subtilisin Carlsberg ("protease"), or trypsin, and in solutions of phosphomolybdic acid or sodium borohydride. After incubation, sections were placed, without prior oxidation, in Schiff's reagent, and were ultimately observed and photographed in transmitted light or with blue or green epifluorescence. A Schiff-positive substance was found, always and exclusively, in elastic tissues of the vasculature and lungs, which was hydrolyzed by the proteolytic enzymes to an extent that ranged from complete loss of Schiff reaction in minutes (trypsin) to no loss of Schiff reaction in 22 hr (clostripain). The Schiff-reactive protein preceded the time of appearance of elastin in the early embryos. We conclude that the aldehyde-rich protein responsible for this reaction is a harbinger of elastogenesis in vivo and speculate that it may represent the elastic microfibril or a component thereof.