bluuebunny/crossref_metadata_2025 huggingface dataset:
EFFECTS OF HORMONES ON HEPATIC GLUCOSE 6-PHOSPHATE DEHYDROGENASE OF RAT
Authors: HORI SAMUEL H. , MATSUI SEI-ICHI
Published: April 2011

The effects of hormones on the total activity and the isozyme pattern of glucose 6-phosphate dehydrogenase of livers of normal, castrated and adrenalectomized rats were studied. Sex difference in total enzyme activity and in the activity of one of the seven isozymes separated electrophoretically (band D enzyme) has been confirmed. Orchidectomy did not affect appreciably the enzyme activity; ovariectomy of young rats reduced the enzyme activity and abolished the sex difference. Adrenalectomy slightly reduced the enzyme activity in male and female rats, but did not eliminate the sex difference in isozyme pattern. Injection of dehydroepiandrosterone into normal and castrated female rats lowered the enzyme activity, whereas administration of estradiol benzoate to normal and castrated male rats strikingly increased the enzyme activity. In estradiol-treated males the isozyme pattern become female type. The effect of estradiol was inhibited by puromycin. Estradiol stimulated but dehydroepiandrosterone had little effect on hepatic 6-phosphogluconate dehydrogenase activity.
A TECHNIQUE FOR THE FIXATION AND DECALCIFICATION OF RAT INCISORS FOR ELECTRON MICROSCOPY
Authors: WARSHAWSKY H. , MOORE G.
Published: April 2011

A reproducible technique that provides sections of decalcified rat incisors for electron microscopy is described. Fixation is accomplished by perfusion with slightly hypertonic, neutral phosphate-buffered 2.5% glutaraldehyde for 30-60 min. Throughout the rest of the procedure, isotonic neutral solutions are used. For decalcification, 4.13% disodium ethylene diamino tetraacetic acid for 14-21 days is employed, followed by washing in phosphate buffer for 2 days, postfixation in 1% osmium tetroxide for 4 hr and embedding in Epon. Provided that the initial fixation by perfusion is good, the method gives satisfactory preservation of the fine structures of the cells and matrices of the incisor, in addition to maintaining the relationship of hard and soft tissues.
THE PRESERVATION OF THE FINE STRUCTURE OF CRYOSTAT-SECTIONED TISSUE WITH DIMETHYLSULFOXIDE FOR COMBINED LIGHT AND ELECTRON MICROSCOPY
Authors: ZAGURY DANIEL , MODEL PAT G. , PAPPAS GEORGE D.
Published: April 2011

A method which allows the observation of identical tissue components with both the light and electron microscopes is presented. The technique is based on the preparation of cryostat sections of organs with dimethylsulfoxide. This agent appears to prevent the damage to fine structure caused by freezing and thawing. The use of plastic sheets permits flat embedding of organ sections in Epon. Examination of the embedded sections, stained or unstained, in the light microscope allows the exact selection of material to be studied further in the electron microscope.
THE SITE OF FERRICYANIDE REDUCTION BY REDUCTASES WITHIN MITOCHONDRIA AS STUDIED BY ELECTRON MICROSCOPY
Authors: OGAWA KAZUO , SAITO TAKUMA , MAYAHARA HIROSHI
Published: April 2011

In the present investigation the ultrastructural site of ferricyanide reduction by reductases in mitochondria in tissues (heart and kidney) of the normal adult rat was studied by the copper ferrocyanide method developed recently in our laboratory. Substrates used were sodium succinate, cytochrome c, dihydronicotinamide-adenine dinucleotide (NADH2), sodium dl-β-hydroxybutyrate, phenazine methosulfate and ubiquinone. All reductases tested were positive in mitochondria and the activities of the succinate-ferricyanide reductase, the cytochrome c-ferricyanide reductase, the NADH2-ferricyanide reductase and β-hydroxybutyrate-ferricyanide reductase were most evident. An ambiguous trace of activity was observed when phenazine methosulfate and ubiquinone were used as substrate. It was also observed that not all of the mitochondria revealed the reductase activity and the existence of functional heterogeneity among mitochondria was postulated. In enzymatically positive mitochondria, the reaction product, copper ferrocyanide, was found both in the mitochondrial membranes and in spaces such as the intracristal space and the outer space. The functional significance of the positive reaction in spaces is briefly discussed.
ELECTRON MICROSCOPIC LOCALIZATION OF MITOCHONDRIAL ADENOSINE TRIPHOSPHATASE ACTIVITY
Authors: GROSSMAN I. WILLIAM , HEITKAMP DALE H.
Published: April 2011

Magnesium-dependent adenosine triphosphatase (ATPase) activity of unfixed, isolated, intact skeletal muscle mitochondria, frozen-thawed mitochondria and digitonin-disrupted submitochondrial particles was localized ultrastructurally after incubation in a Wachstein-Meisel reaction medium and correlated with chemical ATPase activity. The reaction precipitate was localized within the matrix of intact and partially disrupted mitochondria and between membranous fragments in the submitochondrial particles. These findings are consistent with an ATPase activity residing within the headpieces of the elementary particles of mitochondrial cristae with deposition of reaction precipitate within the adjacent matrix.
A COMBINED PROCEDURE FOR THE HISTOCHEMICAL FLUORESCENCE DEMONSTRATION OF MONOAMINES AND MICROAUTORADIOGRAPHY OF WATER-SOLUBLE DRUGS
Authors: MASUOKA DAVID , PLACIDI GIAN-FRANCO
Published: April 2011

A procedure for combining microautoradiography and the histochemical fluorescence method for aromatic monoamines on the same tissue section is described. The use of aqueous solutions is completely avoided, thus eliminating artifacts due to diffusion and dislocation of the labeled water-soluble drug and endogenous amines. The combination of these two techniques provides a means for obtaining data hitherto unavailable to correlate the site of binding and/or storage of water-soluble drugs with monoaminergic nerve cells and terminals.
HYALURONIDASE ACTIVITY OF ALVEOLAR MACROPHAGES
Authors: GOGGINS JOHN F. , LAZARUS GERALD S. , FULLMER HAROLD M.
Published: April 2011

Hyaluronidase activity was detected in preparations of isolated rabbit lung alveolar macrophages. The enzyme manifested a pH optimum of 3.9 with no activity detected above pH 5. Analysis of reaction products isolated from digests containing low enzyme concentrations revealed the production of large oligosaccharides from hyaluronic acid without the liberation of free N-acetylglucosamine or glucuronic acid. On the other hand, smaller oligosaccharides and free N-acetylglucosamine were obtained from digests with high concentrations of the crude enzyme. These data in conjunction with the findings of others indicate that macrophages contain hyaluronidase and other enzymes active against polymerized hyaluronic acid and its digestion products.
ACETYL DISULFIDE, (CH3COS)2, A MAJOR ACTIVE COMPONENT IN THE THIOLACETIC ACID HISTOCHEMICAL METHOD FOR ACETYLCHOLINESTERASE
Authors: KOELLE GEORGE B. , HORN ROBERT S.
Published: April 2011

Triple distilled thiolacetic acid (TA) was found to be much less active as a histochemical substrate for acetylcholinesterase (AChE) of mouse intercostal muscle than either the original, impure preparation, thiolacetic acid (practical, Eastman) or the residue from the first distillation. Iodimetric titration showed that the crude preparation had only 92-94% of its theoretical TA content, which fell to 70-72% with storage. Findings from column and thin layer chromatography and ether-water extraction indicated that the active material is relatively polar and acidic, and contains an S—S group. The addition of 0.0015-0.006 M acetyl disulfide (AcDiS) to 0.03 M triple distilled TA markedly enhanced the substrate activity of the latter so that it matched that of 0.03 M thiolacetic acid (practical); treatment with the reducing agents, sodium ascorbate and sodium borohydride, reduced the substrate activity of both the mixtures and thiolacetic acid (practical) but not that of triple distilled TA alone. Mass spectrometric examination of thiolacetic acid (practical), the distillation residue and a benzene extract of extemporaneously prepared AcDiS showed the presence in each of a peak at mass 150 (the molecular weight of AcDiS), which was not present in triple distilled TA. All except the AcDiS extract showed a peak also at mass 76 (the molecular weight of TA). It was concluded that AcDiS is the major component of thiolacetic acid (practical) that is responsible for its enhanced activity as a histochemical substrate for AChE.
CYTOCHEMISTRY OF 5-HYDROXYTRYPTAMINE AT THE ELECTRON MICROSCOPE LEVEL I. STUDY OF THE SPECIFICITY OF THE REACTION IN ISOLATED BLOOD PLATELETS
Authors: ETCHEVERRY GUILLERMO JAIM , ZIEHER LUIS MARIA
Published: April 2011

Blood platelets obtained from normal rabbits and those isolated from reserpine-treated animals and subsequently incubated in vitro with 5-hydroxytryptamine, norepinephrine and histamine were assayed for amine content or processed for examination under the electron microscope. With the glutaraldehyde-dichromate reaction for unsubstituted catechol- and indoleamines, reactive granules were observed in normal platelets. Formaldehyde fixation prior to the glutaraldehyde-dichromate reaction resulted in a similar image under the electron microscope. In platelets obtained from animals treated with reserpine a decrease of the amine content with a corresponding reduction in the number of dense granules was observed. Following incubation with 5-hydroxytryptamine the concentration of the amine increased markedly and the number of dense granules that reacted with both techniques became practically normal. In norepinephrine-incubated platelets dense granules were demonstrated with the glutaraldehyde-dichromate reaction, but no reactive products were observed using prefixation with formaldehyde. Histamine was also incorporated into depleted platelets but gave no reaction. It is concluded that prefixation with formaldehyde renders negative the reaction with catecholamines, leaving unaffected indoleamine-reactive sites. The previous assumption that the dense granules contain 5-hydroxytryptamine has been confirmed by such a cytochemical approach. The possibility that these organelles constitute a common storage site for different amines is discussed.
HISTOCHEMICAL DIFFERENTIATION BETWEEN TYROSINE AND CHLOROGENIC ACID IN PLANT TISSUES I. NITROUS ACID REACTIONS AND METAL CHELATION OF NITROSOTYROSINE
Authors: REEVE R. M.
Published: April 2011

Copper chelation of nitrosotyrosine has been found useful for histochemical localization for tyrosine in thick, fresh sections of large celled plant tissues. The nitrous acid reaction for ortho-dihydroxyphenolics also has been found useful for localization of chlorogenic acid, caffeic acid and dihydroxyphenylalanine in plant tissues. Application of these tests separately to serially adjacent sections demonstrated the distribution of tyrosine and chlorogenic acid in different plant tissues. Tests tube reactions on known substances verified specificity and also demonstrated that the presence of other amino acids and phenolics did not interfere with the positive test for tyrosine. The color reactions are sufficiently intense for stereoscopic microscopy and tested sections may be measured photometrically. Further adaptability of the nitrosotyrosine-metal chelate reaction to procedures for ultrastructural localization is suggested.
ALTERATIONS OF HEPATIC ENDOPLASMIC RETICULUM IN PORPHYRIC RATS
Authors: POSALAKI ZOLTAN , BARKA TIBOR
Published: April 2011

The alterations of hepatic microsomal fractions were studied in fasting rats given porphyrogenic doses of allylisopropylacetamide. A single dose of allylisopropylacetamide caused significant enlargement of the liver within 14 hr and in 24 hr the liver was about 49% heavier. Microsomal phospholipids of liver more than doubled in 48 hr after the administration of allylisopropylacetamide. This increase was accounted for principally by an increase in phospholipids of the smooth endoplasmic reticulum. Incorporation of 32P indi cated that the increase in microsomal phospholipids was caused mainly by a reduction of the rate of catabolism rather than by an augmentation of the rate of synthesis. Electron microscopic observations in agreement with the fractionation studies revealed a hypertrophy of the smooth membranes of the endoplasmic reticulum.
CYTOFLUOROMETRIC DETERMINATION OF LYSINE WITH DANSYLCHLORIDE
Authors: ROSSELET A. , RUCH F.
Published: April 2011

Dansylchloride (l-dimethylamino-naphthalene-5-sulfochloride) may be used for cytofluorometric determination of lysine. By means of model experiments on protein smears it is shown that the reaction must be carried out in ethanol if it is to be specific for amino groups. The fluorescence given by isolated rat liver nuclei treated with dansylchloride corresponds to the three classes of 2n, 4n and 8n nuclei. The dansylfluorescence of several kinds of sperms is proportional to their lysine content. In rat liver nuclei, 95% of the lysine is dansylated and the lysine content may be determined in absolute values by comparison with polylysine. In spermatozoa only 50% of the lysine reacts.
THE STAINING MECHANISM OF ALDEHYDE-FUCHSIN, WITH REFERENCE TO THE OXYTALAN FIBER IN THE MOUSE
Authors: MANDER STEPHANIE T. K. , MANDER L. N. , CARMICHAEL G. G.
Published: April 2011

Oxytalan fibers of the periodontal membrane in the mouse may be demonstrated, after periodate oxidation, by a number of aldehyde-fuchsin dye complexes. The reactive species in the stain is shown by visible spectrophotometry to be Schiff's base. The loss of staining activity after several weeks aging is due to the formation of the hexa-N-alkyl fuchsin, for which the mechanism is proposed. Experiments suggest that oxytalan fibers contain α-hydroxy amine residues which are oxidized by periodate to imines, which tautomerise to enamines. These enamines then behave as nucleophilic species in the staining process.
SIMULTANEOUS LOCALIZATION OF MULTIPLE TISSUE ANTIGENS USING THE PEROXIDASE-LABELED ANTIBODY METHOD: A STUDY ON PITUITARY GLANDS OF THE RAT
Authors: NAKANE PAUL K.
Published: April 2011

The peroxidase-labeled antibody method was modified to localize multiple tissue antigens in a single histologic section. The first antigen was localized by the indirect method and the antisera were removed from the section by elution, leaving the colored reaction products identifying the antigenic sites. The second and subsequent antigens were localized similarly, using substrates that develop reaction products of different colors. For the demonstration of the method, anti-human growth hormone (GH), anti-human thyrotropic hormone (TSH) and anti-human chorionic gonadotropin, which cross-reacts with rat luteinizing hormone (LH), were used with sections of pituitary gland of the rat. Anti-GH reacted with small acidophilic cells. Anti-LH was localized in oval shaped cells, and anti-TSH was localized in angulated basophilic cells. Some cells were devoid of these three antigens and no cell contained more than one of them.
A STUDY OF ARYL SULFATASE AND ACID PHOSPHATASE IN THE DEVELOPING NERVOUS SYSTEM OF THE RAT
Authors: CHOY A. E. , CRAVIOTO H.
Published: April 2011

Aryl sulfatases A and B and acid phosphatase activities were studied in the nervous system of rats aged 5, 10, 15, 20 and greater than 45 days. The enzyme reaction products appeared as granules localized in neurons of the cerebral cortex, brain stem, choroid plexus and ependyma and in blood vessels. The distribution and intensity of enzyme reaction product varied with the age of the animals. Aryl sulfatase B activity appeared earlier and reached a maximum of intensity sooner than the other enzymes. Acid phosphatase and aryl sulfatase A activities appeared at the same time. However, acid phosphatase had a greater intensity of activity than aryl sulfatase A. The increased activity of aryl sulfatase B was parallel with the maturation of neurons. It is suggested that aryl sulfatase B may be an essential enzyme for myelination.
QUANTITATIVE HISTOCHEMISTRY OF HYPOTHALAMUS II. THIAMINE PYROPHOSPHATASE, NUCLEOSIDE DIPHOSPHATASE AND ACID PHOSPHATASE IN THE ACTIVATED SUPRAOPTIC NUCLEUS OF THE RAT
Authors: JONGKIND JOHAN F.
Published: April 2011

The activities of nucleoside diphosphatases and thiamine pyrophosphatase (TPPase) that are associated with the Golgi apparatus and acid phosphatase were measured by quantitative histochemical methods both in histologically pure nucleus supraopticus and in an adjacent area of the anterior hypothalamus of the rat. In the nucleus supraopticus UDP-phosphohydrolase (UDPase), GDP-phosphohydrolase (GDPase) and TPPase activities increased 40% after a thirsting period of 3 days, while IDP-phosphohydrolase activity increased 18% and acid phosphatase activity decreased 25% after the same osmostic stress. The adjacent, nonsupraoptic anterior hypothalamic area did not show significant changes in activity of any of the enzymes studied. The activities of the Golgi-associated TPPase, UDPase and GDPase are likely to be reliable parameters for neurosecretory activity.
LEAD AND PHOSPHATE AS SOURCES OF ARTIFACT IN NUCLEOSIDE PHOSPHATASE HISTOCHEMISTRY
Authors: GANOTE CHARLES E. , ROSENTII ALAN S. , MOSES HAROLD I. , TICE LOIS W.
Published: April 2011

The contribution of lead and lead phosphate binding to tissue as a possible source of artifact in the Wachstein-Meisel histochemical procedure was considered. In addition, the relevance of lead-catalyzed substrate hydrolysis in tissue water and in the reaction media, as well as the efficacy of the enzyme inhibitors, para-mercurobenzoate and potassium fluoride were examined. Lead-catalyzed hydrolysis of substrate in tissue water does not play a significant role in reaction product deposition. Lead-catalyzed hydrolysis in the reaction media does produce significant amounts of lead phosphate. Furthermore, the enzyme inhibitors in addition to inhibiting enzyme activity alter lead binding to tissue, precipitate lead from solution and decrease tissue staining. Loss of tissue staining may result from a decrease in phosphate-trapping efficiency of lead when the free lead ion concentration is lowered in the reaction medium. Lead phosphate and lead localize as deposits of electron-dense material at tissue sites parallel to those observed with the complete Wachstein reaction mixture. It is concluded that tissue and chemical events other than enzyme activity may contribute to reaction product localization.
THERMAL ANALYSIS OF POLYANION METACHROMASY: EFFECT OF TEMPERATURE ON METACHROMATIC SOLUTIONS OF NUCLEIC ACIDS AND ACID MUCOPOLYSACCHARIDES
Authors: KELLY JOHN W. , CHANG LOUIS , ROTHSTEIN FRED
Published: April 2011

Spectra of toluidine blue with acid mucopolysaccharides and nucleic acids were recorded over the temperature range 10-70°C. Metachromatic ratios were inverse, linear functions of temperature. Intercepts and slopes of the thermal plots distinguished acid mucopolysaccharides from nucleic acids. Acid mucopolysaccharide reactions were thermally reversible. Nucleic acid reactions were not strictly reversible; such behavior was not attributable to thermal denaturation. An explanation for the unusual γ-peak of ribonucleic acid-toluidine blue metachromasy is offered in terms of terminal phosphate groups of ribonucleic acid. Solution data in which the polyanion-dye ratio was approximately 1, representing typical "metachromasy," were subjected to thermodynamic analysis. These data did not fit a common equation deriving equilibrium constants spectrophotometrically by variation of reactant concentrations. This failure supported the view of metachromasy as a phenomenon primarily involving dye-dye interaction. Another equation based only on temperature variation yielded a value of –4 kcal/mole for the chondroitin sulfate-toluidine blue complex, in good agreement with similar published values obtained both in solutions and stained materials.
THERMAL ANALYSIS OF POLYANION METACHROMASY: TEMPERATURE EFFECTS ON STAINED CELLS, TISSUES AND MODELS
Authors: KELLY JOHN W. , CHANG LOUIS
Published: April 2011

Metachromasy of toluidine blue-stained materials was examined at 5-95°C visually and 30-70°C microspectrophotometrically. Cells and tissues provided sites of acid mucopolysaccharides and nucleic acids, which were also prepared as films and droplets. As in similar studies of aqueous solutions, metachromatic ratios were inverse, linear, reversible functions of temperature, with the possible exception of deoxyribonucleic acid. An aqueous mounting medium (gelatin) supported maximum excursions of metachromasy during heating and cooling, although reversible loss of metachromasy occurs to lesser degrees in conventional media. Removal or denaturation of cartilage matrix protein merely increased over-all metachromasy; slopes of thermal plots were unchanged. All evidence suggests that metachromasy is not a fundamentally different phenomenon in solutions and solid systems. Temperature studies emphasize the role of structured water in metachromasy, interaction of water and other solvents and particularly solvent dielectric constant in relation to dye-dye interaction. The limited literature on temperature and biologic staining is reviewed.
HISTOCHEMICAL DEMONSTRATION OF SULFATED MUCOSUBSTANCES AND CATIONIC PROTEINS IN HUMAN GRANULOCYTES AND PLATELETS
Authors: DUNN W. B. , SPICER S. S.
Published: April 2011

Histochemical staining methods visualize sulfated mucosubstance in numerous small granules of early neutrophil leukocytes in appropriately processed human bone marrow smears. These reactive granules presumably constitute the counterpart in human neutrophils of the mucosaccharide-rich primary granules in rabbit heterophils. Neutrophils at a late developmental stage in human marrow or buffy coat smears reveal few or no granules with such reactivity. Strong staining for sulfated mucosubstance is readily demonstrable in numerous large cytoplasmic granules in human eosinophils. This reactivity diminishes but does not disappear during maturation of the eosinophil and its single population of cytoplasmic granules. Granules of human basophils at all developmental stages stain for sulfated mucosubstance. Platelets in human buffy coat smears reveal evidence for sulfated mucosaccharide. The mucosaccharide-containing granules of the three myeloid series also disclose acidophilia at high pH indicative of the presence of strongly cationic proteins.
DISTRIBUTION OF ACID AND ALKALINE PHOSPHATASE ACTIVITY IN UNDEMINERALIZED SECTIONS OF THE RAT TIBIAL DIAPHYSIS
Authors: WERGEDAL JON E. , BAYLINK DAVID J.
Published: April 2011

The distribution of acid and alkaline phosphatase activity in the rat tibia has been studied in transverse sections sawed from fresh undemineralized diaphyses. The endosteal resorbing surfaces had the highest acid phosphatase activity as demonstrated with 50 mM α-naphthol phosphate as the substrate. Strong activity was present not only in osteoclasts and adjacent osteocytes but extracellularly lining Howship's lacunae. In areas of bone formation, moderate acid phosphatase activity was present in osteoblasts and young osteocytes. In addition, a zone of activity was found at the mineralizing front where mineralization is initiated. This active zone ( a) was separated from the active periosteal cell layer by a zone of inactive osteoid and ( b) in formol-calcium-fixed demineralized sections extended into young bone. The identity of the mineralizing front was confirmed by tetracycline labeling. The activity at the mineralizing front had the properties of an enzyme. Alkaline phosphatase activity demonstrated with naphthol ASTR phosphate was largely associated with the osteoblasts and other mesenchymal cells at forming surfaces.
ORANGE FLUORESCENCE IN THE ACOUSTIC NERVE
Authors: ROSS MURIEL D.
Published: April 2011

Orange fluorescent granules are numerous in the acoustic nerve in the rat and the mouse. The granules are often accumulated in masses distributed along, or within, the acoustic nerve neurons within the cochlear modiolus. They are not found more peripheralward. The orange fluorescing granules are not autofluorescent; they are absent in sections mounted directly from the cryostat, and the fluorescence disappears in sections treated with water or dilute isopropanol. The fluorescence is sensitive to ultraviolet light and largely disappears after treatment with α-methyl- m-tyrosine or reserpine (24 hr). Heat alone induces some orange fluorescence, restricted to the distal ends of the axons of the neurons. Spectrofluorometric analyses of heat-induced and vapor-induced orange fluorescence have thus far failed to demonstrate significant spectral differences. It is concluded that orange fluorescence may indicate the presence of an amine in a distinctive storage form.
THE INTERMEDIATE MUSCLE FIBER OF RATS AND GUINEA PIGS
Authors: EDGERTON V. REGGIE , SIMPSON D. R.
Published: April 2011

The soleus, plantaris and gastrocnemius muscles of 60 Sprague-Dawley rats and 70 Hartley guinea pigs were studied histochemically. In the plantaris and gastrocnemius muscles, myosin adenosine triphosphatase activity differentiated intermediate fibers from red and white fibers as determined by malate dehydrogenase and succinate dehydrogenase and reduced nicotinamide adenine dinucleotide diaphorase activities. Contrary to what is commonly reported, red fibers could not be distinguished from white fibers on the basis of myosin adenosine triphosphatase activity as is commonly reported. The intermediate fiber was characterized by minimal menadione-mediated α-glycerophosphate dehydrogenase, phosphorylase and myosin adenosine triphosphatase activity and moderate malate dehydrogenase and succinate dehydrogenase and reduced nicotinamide adenine dinucleotide diaphorase activities. It is suggested that fibers with intermediate oxidative enzyme activity are physiologically slow, white fibers are fast and red fibers are moderately slow or even fast contracting fibers.
ON THE HISTORY AND MECHANISM OF ALIZARIN AND ALIZARIN RED S STAINS FOR CALCIUM
Authors: PUCHTLER HOLDE , MELOAN SUSAN N. , TERRY MARY S.
Published: April 2011

Alizarin (madder) has been used in textile dyeing since early antiquity. In histology calcium-alizarin or calcium-alizarin red S compounds are often referred to as "lake" or "complex." Chemical and infrared spectroscopic data showed that these compounds are salts, not chelates. In dye chemistry the term lake denotes a poorly soluble or insoluble salt of a water-soluble dye. Salt formation between calcium deposits in tissues and alizarin or alizarin red S is indicated by the sensitivity of these compounds toward dilute acetic acid. Acid dyes for lakes, which do not contain chelating groups, also stained calcium deposits selectively. Alizarin stained calcium deposits intensely only around pH 12. Alizarin red S colored calcium deposits selectively around pH 9; neutral and acid dye solutions produced severe diffusion artifacts. Chemical data indicate that alizarin red S can react with calcium via its sulfonic acid and/or its OH groups.
ELECTRON MICROSCOPIC DEMONSTRATION OF ZINC IN THE HIPPOCAMPAL FORMATION USING TIMM'S SULFIDE-SILVER TECHNIQUE
Authors: IBATA YASUHIKO , OTSUKA NAGAYASU
Published: April 2011

Ultrastructural localization of zinc in the hippocampus of rabbits and rats was investigated by the application of Timm's sulfide-silver and zinc-dithizonate methods. Best results were obtained by fixation of tissue in 3% glutaraldehyde saturated with H2S, followed by treatment of small blocks of tissue with Timm's solution for 1 hr. In sections cut from these blocks, silver particles were found on the synaptic vesicles in the terminal boutons of the mossy fibers. The nervous tissue of the hippocampal formation was devoid of silver particles, as were the perikarya of the granular cells of the fascia dentata and the pyramidal cells of the hippocampus. The cytochemical significance of zinc in nerve endings of the granular cells is discussed.
THE EFFECT OF CALCIUM CHLORIDE AND FORMALDEHYDE ON THE RELEASE AND COMPOSITION OF PHOSPHOLIPIDS FROM CRYOSTAT SECTIONS OF RAT HYPOTHALAMUS
Authors: ROOZEMOND R. C.
Published: April 2011

Calcium chloride and formaldehyde inhibit the release of phospholipids from cryostat sections of rat hypothalamus into water. The effect of calcium chloride is thought to be due to the formation of complex coacervates between calcium and phospholipids. The inhibitory effect of formaldehyde may be caused by denaturation of proteins which are associated with phospholipids. Both calcium chloride and formaldehyde influence the composition of phospholipids remaining in the tissue and of those that are released into the medium. Calcium chloride and formaldehyde decrease the amount of phospholipids that can be extracted from the whole tissue by chloroform-methanol, 2:1.
QUANTITATIVE AND HISTOCHEMICAL STUDIES ON THE DESORPTION AND READSORPTION OF ALDOLASE IN CROSS-STRIATED MUSCLE
Authors: ARNOLD H. , NOLTE J. , PETTE D.
Published: April 2011

Complete extraction of aldolase from minced rabbit psoas muscle was achieved by successive extraction steps in 0.1 M phosphate buffer. Aldolase was then readsorbed quantitatively to the depleted myofibrils. Extraction, readsorption and a final redsorption of the enzyme were followed quantitatively by enzyme activity determinations and qualitatively by histochemical staining of aldolase. The intracellular location of the readsorbed enzyme was found to be identical with that of aldolase in native muscle. In both cases, aldolase was localized within the isotropic bands. These results as well as the previously demonstrated binding of the enzyme to F-actin suggest that aldolase is located within the interfilamentary sarcoplasm of the isotropic bands and is probably also bound in vivo to the actin filaments.
PEROXIDASE-ASSOCIATED BINDING OF ESTRADIOL BY THE RAT UTERUS
Authors: BRÖKELMANN JOST
Published: April 2011

It has been shown that the addition of hydrogen peroxide to the incubation medium greatly increases the in vitro binding of tritiated estradiol or its degradation products to uterine tissue. The binding probably is covalent. It involves a hemoprotein which can be demonstrated histochemically by a peroxidase reaction. In light and electron microscopic radioautographs, the sites of radioactivity in uterine tissues after incubation with tritiated estradiol are mainly the specific granules of eosinophils and the cytoplasm of epithelial cells. The possible significance of an interaction between estradiol and a peroxidative hemoprotein is discussed.
BINDING OF LABELED RIBONUCLEIC ACID TO BASIC PROTEINS, A MAJOR DIFFICULTY IN RIBONUCLEIC ACID-DEOXYRIBONUCLEIC ACID HYBRIDIZATION IN FIXED CELLS IN SITU
Authors: DAS NIRMAL K. , ALFERT MAX
Published: April 2011

In an attempt to hybridize H3-ribonucleic acid (RNA) of onion roots to deoxyribonucleic acid of fixed cells in situ it has been found that such RNA binds only to cytoplasmic basic proteins (ribosomal?) as well as nuclear histones or protamines of diverse plant and animal materials. The bound RNA is relatively insensitive to mild RNase digestion. Most of these basic proteins cannot be removed from cells without distorting cell morphology, or without the prior removal of deoxyribonucleic acid. The binding characteristics of H3-RNA and basic proteins parallel alkaline fast green staining of histones. In the formalin-fixed cells, H3-RNA binds to the nucleus only after removal of deoxyribonucleic acid. The binding capacity is abolished after acetylation or deamination of lysine-rich histones; such treatments do not affect the binding ability of arginine-rich histones or protamines.
A QUANTITATIVE BIOCHEMICAL AND HISTOCHEMICAL STUDY OF THE LEAD METHOD FOR LOCALIZATION OF ADENOSINE TRIPHOSPHATE-HYDROLYZING ENZYMES
Authors: JACOBSEN N. O. , JORGENSEN P. LETH
Published: April 2011

The purpose was to study the nature of the adenosine triphosphatase (ATPase) localized to plasma membranes by the lead method and the significance of the lead-catalyzed hydrolysis of adenosine triphosphate (ATP) for the staining of fixed kidney. A modified Wachstein and Meisel's medium was used. The staining of sections of microsomal sediments from rat kidney was compared with the in vitro activity of (Na+ + K+)–, Mg++– and Ca++– ATPase in the presence of lead. The histochemical relevance of these observations was tested on sections of rat kidney. Localization of (Na+ + K+)–ATPase was not possible at concentrations of lead below the inactivating level (0.5 mM). Mg++–ATPase and Ca++– ATPase were only partially inhibited by 3.6 mM lead and were localized to plasma membranes in glutaraldehyde-fixed kidney. The staining followed a course of activation by Ca++ and Mg++ characteristic for enzymatic hydrolysis and different from the activation of nonenzymatic hydrolysis of ATP. Staining of fixed tissue was maximal at 2-3 mM lead and decreased at higher concentrations. In Mg++–deficient media membrane staining was weak or absent at 5.2-6.8 mM lead, where the rate of nonenzymatic hydrolysis of ATP was high. Thus, in the medium used the lead-catalyzed hydrolysis of ATP contributed little to the staining of fixed kidney.
ISOLATION OF THE PERIAXONAL SPACE OF THE CENTRAL MYELINATED NERVE FIBER WITH REGARD TO THE DIFFUSION OF PEROXIDASE
Authors: HIRANO ASAO , BECKER NORWIN H. , ZIMMERMAN H. M.
Published: April 2011

Horseradish peroxidase was implanted into rat forebrains and the distribution with respect to the periaxonal space was examined. The peroxidase filled the extracellular spaces. Usually, however, the flow stopped at the junction of the outer loop and the outermost lamella of the myelin sheath and at the outermost lateral loop at the node of Ranvier. Therefore, in most cases, the periaxonal space was devoid of peroxidase. Occasionally, however, peroxidase in small amounts evidently penetrated the junctions and could sometimes be clearly demonstrated within the periaxonal space.
HISTOCHEMICAL AND ULTRASTRUCTURAL DEMONSTRATION OF γ-GLUTAMYL TRANSPEPTIDASE ACTIVITY
Authors: RUTENBURG ALEXANDER M. , KIM HWAKYU , FISCHBEIN JEROME W. , HANKER JACOB S. , WASSERKRUG HANNAH L. , SELIGMAN ARNOLD M.
Published: April 2011

A simultaneous coupling azo dye method for the histochemical demonstration of γ-glutamyl transpeptidase activity using the new substrate γ-glutamyl-4-methoxy-2-naphthylamide has been described. The method appears superior to previously reported methods for γ-glutamyl transpeptidase activity and can easily be modified for the electron microscopic localization of the enzyme by bridging osmium to the copper chelate of the azo dye via thiocarbohydrazide. The optimum conditions for the histochemical reaction were developed and the distribution of enzymatic activity in the tissues of the rat is described for light microscopy and with rat pancreas for electron microscopy. The electron-opaque deposits were seen in the endoplasmic reticulum in the vicinity of the zymogen granules in the apical portion of the acinar cell.
AN IMMUNOGLOBULIN-ENZYME BRIDGE METHOD FOR LOCALIZING TISSUE ANTIGENS
Authors: MASON T. E. , PHIFER R. F. , SPICER S. S. , SWALLOW R. A. , DRESKIN R. B.
Published: April 2011

An immunohistochemical method not requiring conjugation of a label to antibody is described for visualizing tissue antigens. The method depends on binding of an enzyme label to the tissue antigen through the antigen-antibody reactions of an immunoglobulin-enzyme bridge. This bridge is composed of the following components in order: ( a) specific antiserum to the tissue antigen, ( b) antiserum against the Immune globulin of the species for component a, ( c) specific antiserum prepared against the enzyme label in the same species as component α and ( d) enzyme label. The bridge method employing horseradish peroxidase as the enzyme label has been applied to localization of cells forming growth hormone and chorionic gonadotrophin in the human adenohypophysis and placenta respectively.
CHANGING NUCLEAR HISTONE PATTERNS DURING DEVELOPMENT III. THE DEOXYRIBONUCLEIC ACID CONTENT OF SPERMATOGENIC CELLS IN THE CRAB EMERITA ANALOGA
Authors: VAUGHN J. C. , LOCY R. D.
Published: April 2011

The deoxyribonucleic acid (DNA) content of spermatogenic cells of the decapod crab, Emerita analoga, has been cytophotometrically determined at various stages of development, using Feulgen-stained nuclei. The haploid DNA value is found to be 2.9 x 10–12 g, regardless of the nuclear histone content, which drops to cytochemically indetectable levels by sperm maturity. In determining this value, a precise extinction coefficient for Feulgen-stained nuclei was first determined using chicken, frog and toad erythrocyte nuclei and also nuclei from various rat tissues. The effect of naturally occurring nuclear histone depletion on the quantitative Feulgen reaction has also been examined. Identical hydrolysis curves and Feulgen spectral absorption curves are found for somatic nuclei, which contain a full histone complement, and for mature sperm nuclei, which do not contain histones. Loss of nuclear histone does not lead to a change in total Feulgen dye bound per nucleus, as early, middle and late spermatids have equal DNA contents as reflected by Feulgen binding, and primary spermatocytes contain 4 times this value, as expected from the DNA constancy law. The effect of histones (and other proteins) on quantitative Feulgen microspectrophotometry is discussed.
LEAD ION AND PHOSPHATASE HISTOCHEMISTRY III. THE EFFECTS OF LEAD AND ADENOSINE TRIPHOSPHATE CONCENTRATION ON THE INCORPORATION OF PHOSPHATE INTO FIXED TISSUE
Authors: ROSENTHAL ALAN S. , MOSES HAROLD L. , TICE LOIS , GANOTE CHARLES E.
Published: April 2011

This communication attempts to separate and define the relationships between lead inhibition of tissue adenosine triphosphatase activity, lead. catalyzed adenosine triphosphate hydrolysis and reaction product localization when the Wachstein-Meisel reaction is applied to kidney. Using a radiochemical assay of adenosine triphosphatase activity and varying the concentration of lead nitrate or adenosine triphosphate, the quantity of phosphate bound to and released from tissue was determined. Depending on the relative concentrations of lead and adenosine triphosphate, two situations may exist. With low lead or high adenosine triphosphate concentrations, phosphate release by tissue exceeds phosphate trapped by tissue and substantial quantities of phosphate are lost to the medium. With low adenosine triphosphate or high lead concentrations more phosphate is bound in tissue than can be attributed to tissue enzyme activity. Possible explanations for these phenomenon are discussed.
CYTOCHEMICAL LOCALIZATION OF CHOLINESTERASE IN EMBRYONIC RABBIT CARDIAC MUSCLE
Authors: HAGOPIAN MARTIN , TENNYSON VIRGINIA M. , SPIRO DAVID
Published: April 2011

Cardiac muscle of rabbit embryos from day 9 through day 18 of gestation was studied by a modification of the Koelle-Friedenwald copper thiocholine technique for the localization of cholinesterase activity. In the earlier stages of development a cholinesterase, presumed to be acetylcholinesterase from its substrate and inhibitor specificity, is found in the abundant, randomly dispersed rough surfaced endoplasmic reticulum of the myoblast. Cytochemical end product is also occasionally found in the nuclear envelope, Golgi complex and subsarcolemmal cisternae. The localization of the enzyme first in the rough surfaced endoplasmic reticulum and Golgi complex and at later stages in the sarcoplasmic reticulum is significant with regard to the differentiation of the cardiac myocyte. Furthermore, the presence of this enzyme in contractile tissue before the appearance of nerves or nerve endings demonstrates an acetylcholine-cholinesterase system of myogenic origin.
STUDIES ON THE CHARACTERIZATION AND LOCALIZATION OF RAT PLACENTAL ESTERASES
Authors: BULMER D. , FISHER A. W. F.
Published: April 2011

The nonspecific carboxylic esterases of the rat placenta were studied by electrophoretic, biochemical and histochemical methods, with naphthyl and naphthol AS esters as substrates. The enzymes were characterized by the effects upon their activity of a group of organophosphate inhibitors and of eserine, p-chloromercuribenzoate and urea solution. After electrophoresis at pH 7.5, zymograms show an extensive pattern of anodal and cathodal bands similar for both maternal and foetal portions of the placenta. The B (carboxyl) esterases are sensitive to inhibition with E600 and diisopropyl phosphorofluoridate at very low concentrations. The A (aryl) and C (acetyl) esterases undergo progressive inhibition at concentrations greater than 0.01 mM, and only one A esterase resists a 1.0 mM concentration. Mipafox resembles the other organophosphates in its effect on esterases only when it is used fresh. Comparison of the zymograms with the histochemical preparations permits the localization of some of the individual enzyme species separated by electrophoresis.
ACID CATALYSIS OF THE FORMALDEHYDE CONDENSATION REACTION FOR SENSITIVE HISTOCHEMICAL DEMONSTRATION OF TRYPTAMINES AND 3-METHOXYLATED PHENYLETHYLAMINES
Authors: BJÖRKLUND ANDERS , STENEVI ULF
Published: April 2011

Hydrochloric acid catalyzes the formation of fluorophores in the histochemical condensation reaction between gaseous formaldehyde and certain phenylethylamines and indolylethylamines. Thus, when the formaldehyde reaction is carried out in the presence of minute amounts of HCl gas, 3-methoxy-4-hydroxyphenylethylamine, 3,4-dimethoxyphenylalanine and tryptamine displayed a fluorescence yield approximately 20-200 times higher than that obtained after standard formaldehyde treatment in normal air ( i.e., the conditions of the Falck-Hillarp method). This fluorescence intensity was nearly twice as high as that obtained from noradrenaline and dopamine under the standard conditions. The results indicate that the acid catalyzes the first step of the histochemical reaction, i.e., the Pictet-Spengler condensation reaction. In this step low fluorescent tetrahydroisoquinolines and tetrahydro-β-carbolines are formed, which are subsequently dehydrogenated to strongly fluorescent products. Low reactive aromatic amines and amino acids, such as phenylalanine, tyrosine, amphetamine and melatonin, gave no or only very low visible fluorescence after this treatment. Thus, the acid-catalyzed histochemical formaldehyde reaction described in this paper exhibits a good specificity for indolylethylamines and 3-hydroxylated or 3-methoxylated phenylethylamines and will also allow the distinction between these structurally related compounds on the basis of their reactivity in the condensation reaction.
METACHROMATIC STAINING OF SODIUM BISULFITE ADDITION DERIVATIVES OF GLYCOGEN
Authors: MALININ GEORGE I.
Published: April 2011

In fixed tissues and cell cultures glycogen readily formed sodium bisulfite addition compounds following oxidation with 0.5% aqueous periodic acid. Sodium bisulfite derivatives of oxidized glycogen developed intense metachromasia if stained with 0.5% toluidine blue at the 1.0-5.0 pH range. Induced metachromasia of tissue glycogen was dehydration-labile if the oxidation with periodic acid was carried out for less than 15 min. Oxidation in excess of 15 min resulted in formation of dehydration-resistant metachromasia. The induction of the two types of metachromasia was determined primarily by the duration of oxidation and only secondarily by the duration of addition reaction and sodium bisulfite concentration. The origin of the dehydration-labile metachromasia probably resides with the external glucose units of glycogen depositions, whereas the induction of dehydration-resistant metachromasia may be attributed to the sodium bisulfite derivatives of the internally located glucosyl groups of glycogen.
EFFECT OF HORSERADISH PEROXIDASE ON MICE LUNG CAPILLARIES' PERMEABILITY
Authors: CLEMENTI F. , BATTLES EMMA J.
Published: April 2011

Horseradish peroxidase has been reported to affect capillary permeability in several animal species, but not in mice. Two types of peroxidases, with different enzymatic activity, were used in mice at different doses, and the lung capillaries' permeability to the latter has been investigated. In animals injected with a low dose of a high reactive peroxidase, although a clear histochemical reaction was present in capillar lumina, the reaction product was never found outside capillaries' walls. In animals injected with the higher dose of peroxidase the reaction product was always found in pericapillary spaces and often in alveolar macrophages. It is suggested that peroxidase, in high dose, can also affect lung capillary permeability in mice.
LOCALIZATION OF THE ANTIGEN IN POPLITEAL LYMPH NODES OF RABBITS DURING THE FORMATION OF ANTIBODIES TO HORSERADISH PEROXIDASE
Authors: STRAUS WERNER
Published: April 2011

The localization of an antigen (horseradish peroxidase) in popliteal lymph nodes of rabbits was investigated in order to detect the possible interrelationship with the location of the specific antibody in the same tissue material. Staining procedures for peroxidase with benzidine, diaminobenzidine and 3-amino-9-ethyl-carbazole, as well as double staining procedures for the antigen and the antibody and for the antigen (or antibody) and acid phosphatase, were applied before and after adsorption of the antigen to sites of antibody in vitro. The appearance of the antigen in the cells lining the lymph sinuses, in reticular cells of medullary cords, in macrophages and in the "intercellular web" of lymphoid follicles was studied after a single and repeated injection of peroxidase, and the persistence of the antigen at these sites was observed. It was found that the localization of the antigen in the cortex and medulla of the lymph node was different depending on whether or not specific antibodies were present in the blood at the time of injection, and that at certain periods a considerable number of plasma cells and lymphoblasts contained the antigen together with the specific antibody.
THE QUESTION OF TRIAZENES IN THE TOTAL COLOR EFFECT OF THE PROTEIN AZO COUPLING REACTION IN HISTOCHEMISTRY
Authors: LILLIE R. D.
Published: April 2011

The suggestion that triazenes formed by union of alkalized diazonium salts with aliphatic amino groups contribute to the azo color reaction of tissue proteins is rejected. Deaminations of formaldehyde-fixed and other tissues were sufficient to render the previously oxyphil elements of these tissues basophilic to azure A-eosin B at the same pH level. After these deaminations there was no detectable difference in intensity or distribution of the azo coupling reaction. Freshly diazotized safranin O, dimethylphenosafranin (methylene violet) and the diazosulfanilic acid, azure A sequence technic were used as testing methods. Acid extraction even when prolonged to 24 hr and when acid concentration raised from 0.1 N to 0.24 N does not alter the intensity or distribution of the several azo coupling reactions as compared with unextracted preparations. Deliberate creation of triazenes by admixture of proline, diethylamine, hydroxylamine, hydrazine sulfate, glycine and uric acid, added to the diazo in stoichiometric excess, more or less completely inhibited azo coupling of all tissue elements. Uric acid and hydrazine were the most efiective, and then hydroxylamine, proline and diethylamine in that order. Reacidification of the uric acid compound to pH 2.2-2.5 for 10 min, filtration to remove liberated uric acid and realkalinization moderately restored the azo coupling capacity of diazosafranin. This illustrates acid destruction of a triazene of this diazotate. It is concluded that triazenes with tissue aliamino groups play no significant part in the final color effect of azo coupling with the diazonium salts used.