bluuebunny/crossref_metadata_2025 huggingface dataset:
TRYPTAMINE OR TRYPTOPHYL PEPTIDES IN ENDOCRINE CELLS OF THE MAMMALIAN ADENOHYPOPHYSIS
Authors: HÅKANSON R. , LARSSON L.-I. , NOBIN A. , SUNDLER F.
Published: April 2011

In all species studied certain endocrine cells of the adenohypophysis exhibited intense fluorescence after treatment with formaldehyde-ozone but only weak fluorescence after treatment with formaldehyde alone. This is characteristic of both tryptamine and peptides with NH2-terminal tryptophan in protein droplet models. Excitation and emission maxima of the fluorophore in the pituitary were very similar to those of authentic tryptamine or tryptophyl peptide fluorophores. However, chemical analysis failed to demonstrate tryptamine in extracts of the pituitary, whereas large amounts of peptides with NH2-terminal tryptophan could be isolated and identified by fluorometry and thin layer chromatography. It is therefore suggested that the formaldehyde-ozone-induced fluorescence in the endocrine cells reflects the presence of tryptophyl peptides rather than tryptamine.
NUCLEOSIDE PHOSPHORYLASE ACTIVITY IN BLOOD VESSELS AND FORMED ELEMENTS OF THE BLOOD OF THE DOG
Authors: BORGERS M. , SCHAPER J. , SCHAPER W.
Published: April 2011

The distribution of nucleoside phosphorylase activity was investigated in dog coronary blood vessels and in buffy coat preparations of circulating blood cells of the dog using a histochemical method. The enzyme activity was found in the cytoplasmic sap of endothelial cells, adventitial fibroblasts and fibrocytes, and in the axonal cytoplasm of the nerves. Smooth muscle and macrophages revealed no enzyme activity. Of the circulating blood cells, neutrophils, basophils, monocytes and platelets showed a uniform enzyme reaction, whereas eosinophils and erythrocytes apparently lacked activity. A part of the lymphocyte population reacted strongly while some lymphocytes were completely devoid of activity.
HISTOCHEMICAL CHARACTERIZATION OF CELLULAR AND STRUCTURAL ELEMENTS OF THE HUMAN SPLEEN
Authors: LI C. Y. , YAM LUNG T. , CROSBY WILLIAM H.
Published: April 2011

Several histochemical methods were applied to identify the various cellular and structural elements of the human spleen. These included naphthol AS acetate esterase and reticulin stain for the sinuses, fluoride-resistant esterase for macrophages, alkaline phosphatase for vascular structures, chloroacetate esterase for neutrophils, cyanide-resistant peroxidase and chlorazol fast pink for eosinophils, toluidine blue for basophils and pseudoperoxidase for erythrocytes and erythroblasts. The methods are simple, sensitive and readily reproducible and are best used in combination, so that several types of cells can be identified simultaneously. The location of these cells in the spleen and the interaction between these cells are readily appreciated. These methods can be used to study the morphologic changes of spleen in various hematologic disorders.
DEMONSTRATION OF TRANSPORT ADENOSINE TRIPHOSPHATASE IN THE PLASMA MEMBRANES OF ERYTHROCYTE GHOSTS BY QUANTITATIVE ELECTRON MICROSCOPY
Authors: CHARNOCK J. S. , TREBILCOCK H. A. , CASLEY-SMITH J. R
Published: April 2011

A quantitative electron microscopic method for the demonstration of (Na+ + K+)-adenosine triphosphatase (ATPase) in human erythrocyte ghost membranes is described which differs from that employed previously by introducing several modifications which greatly reduce the possibility that variations in section thickness or contamination or sublimation in the electron beam could effect the result. This procedure makes use of the greater electron density of Cs+ rather than K+ ions as an activator of (Na+ + K+)-ATPase, thus avoiding the lack of specificity of other histochemical or cytochemical procedures. The transient formation of an ion-carrier state during the hydrolysis of ATP by erythrocyte ghost membrane (Na+ + K+)-ATPase was shown by a significant increase in membrane density when Cs+ ions accumulate at the membrane. This accumulation occurs only in the presence of ouabain. The increase in membrane specific gravity was a generalized phenomenon and was not localized at discrete areas of the membrane, suggesting that (Na+ + K+)-ATPase "pumping sites" are distributed uniformly throughout the total membrane structure.
LOW TEMPERATURE EMBEDDING IN GLYCOL METHACRYLATE FOR ENZYME HISTOCHEMISTRY IN PLANT AND ANIMAL TISSUES
Authors: ASHFORD ANNE E. , ALLAWAY WILLIAM G. , MCCULLY MARGARET E.
Published: April 2011

A method is described by which enzyme activity can be retained in plant and animal tissues embedded in glycol methacrylate at low temperature.
ALKALINE BISMUTH REAGENT FOR HIGH RESOLUTION ULTRASTRUCTURAL DEMONSTRATION OF PERIODATE-REACTIVE SITES
Authors: AINSWORTH STERLING K. , KARNOVSKY MORRIS J. , ITO SUSUMU
Published: April 2011

A simple technique is described for the ultrastructural localization of periodate-reactive mucosubstances and polysaccharides containing 1,2-glycols in thin sections of routinely fixed tissues. In this method the sugar residues are oxidized by periodic acid and the resulting aldehydes presumably reduce chelated bismuth subnitrate to metallic bismuth which then appears as a fine electron-opaque precipitate at the sites of the reducing sugars. The periodic acid-alkaline bismuth procedure provides a high resolution electron microscopic technique for demonstrating tissue sites of periodate-engendered groups very similar to the light microscopic periodic acid-Schiff reaction. The reaction can be prevented by the omission of periodate oxidation or alkaline bismuth subnitrate and by aldehyde blockage with the blocking agent, m-aminophenol. However, glycogen stains markedly without prior periodate oxidation, presumably through chelation of bismuth by hydroxyl groups. Other structures which stain without prior periodate oxidation are liver lysosomal dense bodies and, occasionally, ribosomes.
DISTRIBUTION OF GLYCOGEN WITHIN THE LIVER LOBULE
Authors: WELSH FRANK A.
Published: April 2011

Quantitative determinations of glycogen of periportal and centrilobular cells of the liver were made in normal, fasted, realimented and cortisone-treated rats. In most instances there was no significant portal-central difference in glycogen concentration. There were two exceptions. After 2 hr of refeeding following a 24-hr fast, the central to portal glycogen ratio was 1.3. During recovery from the glycogen overshoot, which occurs upon feeding after a prolonged fast, portal glycogen exceeded central glycogen by a factor of 1.8. It is suggested that some of the discordant staining findings on glycogen distribution arise from artifacts.
LOCALIZATION OF ACETYLCHOLINESTERASE IN THE TELEOST RETINA
Authors: NICHOLS CHARLES W. , HEWITT JAMES , LATIES ALAN M.
Published: April 2011

Acetylcholinesterase is the sole cholinesterase enzyme identifiable histochemically in the teleost retina. Acetylcholinesterase is present in both amacrine and ganglion cells in the retinas of all three species of fish studied. No sign of acetylcholinesterase activity was found in ganglion cell axons either in the nerve fiber layer of the retina or in the optic nerve. Evidence is presented for the presence of acetylcholinesterase activity in additional cell types within the nuclear layer. The distribution of acetylcholinesterase-containing cells in teleost retina is compared to that in other species.
COMPARISON OF ULTRASTRUCTURAL CHOLINESTERASE DEMONSTRATION IN THE MOTOR END PLATE WITH α-ACETYLTHIOL-m-TOLUENEDIAZONIUM ION AND 3-ACETOXY-5-INDOLEDIAZONIUM ION
Authors: DAVIS DONALD A. , WASSERKRUG HANNAH L. , HEYMAN IRWIN A. , PADMANABHAN K. C. , SELIGMAN GARRY A. , PLAPINGER ROBERT E. , SELIGMAN ARNOLD M.
Published: April 2011

The first cytochemical demonstration of a hydrolytic enzyme using a single agent as both substrate and capture reagent to form an osmiophilic polymer was reported earlier. The first results were obtained with substrates containing an enzyme-susceptible thiolester group and a diazonium group in the same molecule, which yielded osmiophilic polymers upon enzymatic hydrolysis via diazothioether formation linking the units. An enzyme-susceptible ester group has also been incorporated in the same indole molecule with a diazonium group to yield an osmiophilic polymer upon esterolysis via coupling to azo dye formation linking the units. The schemas of these two systems are given. The thiol substrate is α-acetylthiol-m-toluenediazonium ion (ATTD, compound II) and the indoxyl substrate is 3-acetoxy-5-indolediazonium ion (AID, compound VII). The preparation of AID is given here. Because of the strongly positive charges on the diazonium ion, neither ATTD nor AID is hydrolyzed by the aliesterases of rat kidney. The results with ATTD and AID in the ultrastructural demonstration of cholinesterases in the motor end plate of the rat are compared, as well as the influence of varying the method of fixation, substrate concentration, duration and temperature of incubation and the use of selected inhibitors with ATTD. Evidence was provided with ATTD that both acetylcholinesterase and pseudocholinesterase are present on the inner surface of the synaptic clefts of the motor end plate and that the deposits are discrete and not linear as has been reported with some other methods. On the other hand, deposits with AID are more finely granular and are also limited to the inner aspect of the synaptic clefts. The reasons for the differences in appearance of the deposits in ultrastructural preparations are discussed.
A COMPARATIVE CYTOCHEMICAL STUDY OF MICROBODIES (PEROXISOMES) IN GREAT ALVEOLAR CELLS OF RODENTS, RABBIT AND MONKEY
Authors: SCHNEEBERGER EVELINE E.
Published: April 2011

Lungs from rodents, lagomorphs and primates were briefly fixed in purified glutaraldehyde and incubated with diaminobenzidene and peroxide at pH 7.6 for the demonstration of peroxidase activity and at pH 9.0 for the demonstration of peroxidatic activity of catalase. Great alveolar cells of all animals except the rabbit contained round to elongated microbodies that stained at pH 9.0. In mice, rough and smooth endoplasmic reticulum and the perinuclear cisternae of these cells were also stained. At pH 7.6 there was no staining of great alveolar cells in any species, except in mice, where a light staining of the endoplasmic reticulum, perinuclear cisternae and microbodies persisted. In rodents, microbodies ranged in diameter from 0.13 µ in mice to 0.22 µ in guinea pigs. In monkeys they measured approximately 0.15 µ. Microbodies were not identified with certainty in rabbit great alveolar cells. In rodents the ratio of microbodies to mitochondria was roughly 1:1, whereas in primates it was roughly 1:2. Using appropriate inhibitors it was concluded that staining at pH 9.0 was due to peroxidatic activity of catalase within peroxisomes. Extraperoxisomal staining in mice was attributed to endogenous peroxidase.
IMMUNOFLUORESCENT LOCALIZATION OF TWO DEHYDROGENASES IN HONEYBEE FLIGHT MUSCLE
Authors: BROSEMER RONALD W.
Published: April 2011

Two enzymes involved in energy metabolism, glycerol-3-phosphate dehydrogenase and triosephosphate dehydrogenase, were localized in honeybee flight muscle with immunofluorescent techniques. Both enzymes were found at or near the Z-band cross-striations.
CYTOCHEMICAL LOCALIZATION OF PEROXIDASE ACTIVITY IN SACCHAROMYCES CEREVISIAE
Authors: TODD MICHAEL M. , VIGIL EUGENE L.
Published: April 2011

Cytochemical peroxidase activity in a haploid yeast ( Saccharomyces cerevisiae, strain D243) was studied in glutaraldehyde-fixed cells with a diaminobenzidine (DAB) procedure. In early stationary phase cells the predominant sites of peroxidatic activity were the mitochondrial cristae, with activity of lesser intensity on other mitochondrial membranes. The reaction required H2O2, was insensitive to aminotriazole but was blocked by cyanide and azide, by high peroxide concentration or by a decrease in medium pH from 8.5 to 6.0. The enzyme visualized was probably cytochrome c peroxidase, an enzyme known to be highly active in yeast mitochondria, although the possibility that H2O2 may somehow activate DAB oxidation via other mitochondrial heme proteins cannot be completely eliminated. The value of the DAB procedure as a means of specifically staining yeast mitochondria is discussed in relation to studies of mitochondrial biogenesis. No conclusive evidence of the presence of nonmitochondrial peroxisomal organelles (microbodies) was seen in cells grown on either glucose or galactose, but there was an indication of their presence in very low numbers in glycerol-grown yeast. Although catalase was clearly present, as evidenced by the rapid evolution of O2 from the H2O2-containing reaction media, it showed little apparent localized peroxidatic activity toward DAB. We were unable to demonstrate cytochrome oxidase activity in these cells with the usual DAB-cytochrome oxidase procedure.
SENSITIVITY IN ELECTRON MICROSCOPE AUTORADIOGRAPHY I. THE EFFECT OF RADIATION DOSE
Authors: SALPETER MIRIAM M. , SZABO MARIA
Published: April 2011

Sensitivity in electron microscope autoradiography using Ilford L4 emulsion was shown to be affected by radiation dose ( i.e., number of decays in test specimen per unit surface area). The sensitivity tended to be higher with lower doses. This dose dependence was most marked with Microdol X and least with gold latensification-Elon ascorbic acid development. Possible consequences for quantitation in electron microscope autoradiography are discussed.
MODIFICATION OF THE GELATIN SUBSTRATE PROCEDURE FOR DEMONSTRATION OF ACROSOMAL PROTEOLYTIC ACTIVITY
Authors: PENN ARTHUR , GLEDHILL BARTON L. , DARŻYNKIEWICZ ZBIGNIEW
Published: April 2011

In situ proteolytic activity in the heads of sperm of seven mammalian species has been demonstrated using autoradiographic film as a gelatin substrate. The film is first exposed and processed and then coated with the sperm sample. Proteolytic activity is monitored by following the appearance of "halos," which are areas of gelatin digestion and are found to surround the heads of reacting sperm. The proteolytic factor appears to be released from the acrosome and may be the protease with trypsin-like activity that has been found associated with the acrosome in biochemical studies. l-Chloro-3-tosylamido-7-amino-2-heptanone and diisopropyl fluorophosphate, both of which inhibit trypsin activity, apparently interact with a substance released from the acrosome but do not interfere with the formation of halos. Sperm treated with trichloroacetic acid, urea or formalin do not form halos.
THE NATURE OF NONSPECIFIC ESTERASES A SUBUNIT CONCEPT
Authors: CHOUDHURY S. ROY
Published: April 2011

To investigate the nature of esterase polymorphism, nonspecific esterases were investigated in a large number of organs obtained from rat. In starch gel electropherograms the capacity of ester hydrolysis was seen to decline predictably with successively longer chain carbon substrates. An increasing susceptibility to organophosphate inhibition was observed with progressive lengthening of the acyl chain in the substrate molecules. Attempts to hybridize an organophosphate-sensitive esterase with a resistant type yielded a few additional esterase species. Studies on an esterase fraction isolated by column chromatography revealed the highly relative nature of organophosphate inhibition. These suggested that nonspecific esterases all belong to one enzyme system that does not justify the present compartmentation into acetyl-, aryl- or carboxylesterases on grounds of substrate hydrolysis or organophosphate sensitivity. It is likely that esterase species are each built on a subunit structure and exhibit overlapping specificities through sharing of common subunits. The concept that a whole range of esterase subunits exist, each varying in its capacity to hydrolyze esters of up to an optimal chain length and arranged according to the dictates of specific tissue requirements, is in accord with the present findings.
MICROPHOTOMETRIC DETERMINATION OF ENZYME ACTIVITY IN SINGLE CELLS IN CRYOSTAT SECTIONS II. SUCCINATE DEHYDROGENASE, LACTATE DEHYDROGENASE AND TRIOSEPHOSPHATE DEHYDROGENASE ACTIVITIES IN RED, INTERMEDIATE AND WHITE FIBERS OF SOLEUS AND RECTUS FEMORIS MUSCLES OF RAT
Authors: NOLTE J. , PETTE D.
Published: April 2011

Comparative microphotometric determinations of succinate dehydrogenase, triosephosphate dehydrogenase and lactate dehydrogenase activities in single white, red and intermediate fibers in cross-sections of rat rectus femoris muscle and soleus muscle were performed. White fibers have the highest triosephosphate and lactate dehydrogenase activities and the lowest succinate dehydrogenase activity. Red fibers are highest in succinate dehydrogenase activity and relatively low in triosephosphate and lactate dehydrogenase activities. The intermediate fibers have medium succinate dehydrogenase activity and relatively low triosephosphate dehydrogenase activity. In the soleus muscle, the triosephosphate dehydrogenase activity of the red fibers exceeds that of the intermediate fibers. A parallel increase in succinate dehydrogenase is observed in the red fibers of musculus soleus and musculus rectus femoris after thyroid hormone administration. There is some evidence, however, that the red fibers of the two muscles investigated vary with regard to their absolute succinate dehydrogenase activities and also the triosephosphate dehydrogenase to succinate dehydrogenase activity ratio.
DEMONSTRATION OF CYTOCHROME OXIDASE ACTIVITY WITH DIAMINOBENZIDINE A BIOCHEMICAL AND ELECTRON MICROSCOPIC STUDY
Authors: REITH ALBRECHT , SCHÜLER BARBARA
Published: April 2011

The relationship between cytochrome oxidase activity assayed biochemically with 3,3'-diaminobenzidine (DAB) as substrate and the resulting electron microscopic staining reaction has been investigated. Mitochondria isolated from both fresh and perfusion-fixed (1% glutaraldehyde and phosphate buffer for 1-2 min) rat liver showed a significant increase in oxygen consumption, as measured by an oxygen electrode, after addition of DAB and were further stimulated by adding cytochrome c. 2,4-Dinitrophenol and antimycin had no effect on the oxidation of DAB; cyanide and azide completely inhibited DAB oxidation, resulting in the absence of any mitochondrial staining reaction. Already after incubation for only 2 min mitochondria exhibited electron-dense deposits on the surface of the inner mitochondrial membrane away from the matrix. The results indicate that DAB donates electrons at the cytochrome c level to the electron transfer system of the respiratory chain and demonstrate the direct relationship between the electron microscopic staining reaction and cytochrome oxidase activity.
ELECTRON MICROSCOPIC STUDY OF THE ADRENOCORTICOTROPIN-PRODUCING CELL WITH THE USE OF UNLABELED ANTIBODY AND THE SOLUBLE PEROXIDASE-ANTIPEROXIDASE COMPLEX
Authors: MORIARTY G. C. , HALMI N. S.
Published: April 2011

The technique involving use of unlabeled antibody and the peroxidase-antiperoxidase complex was used to identify the adrenocorticotropin (ACTH)-secreting cell in the anterior pituitary lobe of the rat and to localize ACTH in it electron microscopically in ultrathin sections. The ACTH cell is star-shaped, with processes extending around other cells, and contains secretory granules of a maximal diameter of 300 mµ arranged peripherally along the plasma membrane. Stain was observed on secretory granules, around them, in the Golgi complex and in rough endoplasmic reticulum. One day after adrenalectomy, the ACTH cell is degranulated and the staining intensity of its remaining granules and cytoplasm is decreased, suggesting release of ACTH stores. If cortisol is given 6 hr after adrenalectomy, 18 hr later the ACTH cells are well granulated and the granules stain more intensely than normal. In addition, staining around the granules and throughout the cytoplasm is more intense, suggesting that an early effect of cortisol is to block release of ACTH. Twenty-one days after adrenalectomy, the ACTH cells are greatly increased in numbers and have complex, tortuous processes filled with intensely stained secretory granules.
CARBONIC ANHYDRASE ACTIVITY IN RAT NEUROHYPOPHYSIS FOLLOWING OSMOTIC STRESS
Authors: BOER G. J.
Published: April 2011

Using the colorimetric micromethod of Mattenheimer and deBruin for the assay of carbonic anhydrase activity, a considerable loss of substrate (CO2 gas) from the incubation medium was measured during spontaneous hydration. Adding gaseous CO2 and coating the surface of the incubation medium with oil were employed in order to prevent this loss, which otherwise led to serious errors in the measurement of the enzyme activity. A mixture of 80% paraffin and 20% hexadecane as surface coating was satisfactory for reducing the CO2 exchange to negligible proportions. With this modified method, the carbonic anhydrase activity of frozen-dried samples of rat neurohypophysis was studied as a possible "marker" for pituicyte activity following osmotic stress. The enzyme activity increased about 130% after 6 days of water deprivation. Experiments with perfused tissue, however, indicated that this increase in enzyme activity was caused by an increase in the blood content of the neural lobe.
EVALUATION OF A CYTOCENTRIFUGE TECHNIQUE EFFECTS ON QUANTITATIVE DETERMINATION OF FEULGEN-DEOXYRIBONUCLEIC ACID AND TOTAL DRY MASS OF HUMAN LEUKEMIC CELLS
Authors: DE BAULT L. E.
Published: April 2011

The cytocentrifuge has been used as a tool to obtain preparations from suspension cultures suitable for quantitative cytochemical analysis. Three continuous suspension cultures of human lymphocytic cells (CCRF-CEM, acute lymphoblastic leukemia; CCRF-RKB, infectious mononucleosis; and EB-3, Burkitt's lymphoma) and a suspension of mouse inguinal lymph node lymphocytes were examined for quantitative differential effects on cellular dry mass and Feulgen-deoxyribonucleic acid (DNA) contents caused by cytocentrifugation. When using a cytocentrifuge technique which results in a dry preparation, a quantitative loss was observed for cellular dry mass but not for Feulgen-DNA. However, if a medium which prevented bursting, swelling or loss of cellular constituents during cytocentrifugation was used and if the preparation was maintained in the wet state (no air-drying), acceptable preparations for both dry mass and Feulgen-DNA quantitation can be obtained. These and other observations on centrifugal force, centrifuge time and suspending medium emphasize the importance of specimen preparation when quantitating cell parameters that may themselves be altered by the method of preparation.
ESTERASES IN HUMAN LEUKOCYTES
Authors: LI C. Y. , YAM L. T. , LAM K. W.
Published: April 2011

The esterases of human leukocytes were studied both by cytochemical methods and by polyacrylamide gel electrophoresis. The aim of the study was to clarify the specificity of various cytochemical methods and to establish specific methods for demonstrating the various types of esterases in the blood cells. Nine bands of isoenzymes were separable by polyacrylamide gel electrophoresis at pH 4.0. Bands 1, 2, 7, 8 and 9 were best demonstrated by naphthol AS-D chloroacetate and represented the activity of chloroacetate esterase as demonstrated by cytochemical methods in the granulocytes. Bands 3, 4, 5 and 6 were best demonstrated by naphthyl esters and represented "nonspecific esterases" as demonstrated by cytochemical methods in the monocytes, platelets and plasma cells. "Aminocaproate esterase" or "trypsin-like enzyme" was demonstrated only in the human mast cells. Its exact location in the gel system is unknown since pure human mast cell preparation was not available for study. These three groups of esterases had a substantial but not absolute substrate specificity. They can be specifically demonstrated in the various types of human blood cells only under properly controlled staining conditions.
CELLULAR LOCALIZATION OF GLUCAGON BY FLUORESCENCE MICROSCOPY REACTION OF NH2-TERMINAL HISTIDINE WITH o-PHTHALDIALDEHYDE
Authors: BRODY M. J. , HAKANSON R. , LUNDQUIST I. , OWMAN C. , SUNDLER F.
Published: April 2011

Exposure to o-phthaldialdehyde (OPT) causes intense fluorescence in A2-cells in sections from pancreas. Alloxan treatment of mice was followed by a marked increase in the number and fluorescence intensity of the OPT-reactive A2-cells, whereas cobalt chloride given to guinea pigs induced an almost total depletion of the cytoplasmic fluorescence with concomitant reduction of their argyrophilia. Microspectrofluorometric analysis of the fluorophore revealed excitation maxima at 370 and 420 mµ; the corresponding emission maxima were 430 and 490 mµ. This closely resembled the spectral characteristics of the OPT-induced fluorescence of authentic glucagon. It is concluded that OPT forms fluorphores wth glucagon in the A2-cells and that the NH2-terminal histidine residue of the polypeptide is responsible for the formation of the fluorophore. It is proposed that OPT is a useful reagent for the histochemical detection not only of histamine but also of histidyl peptides.
DIAMINOBENZIDINE REACTIVITY OF PEROXISOMES AND MITOCHONDRIA IN A PARASITIC AMEBA, HARTMANNELLA CULBERTSONI
Authors: CHILDS GEORGE E.
Published: April 2011

Peroxisomes in Hartmannella culbertsoni were identified by localization techniques for catalase employing diaminobenzidine and hydrogen peroxide at pH 9.0. The peroxisomal reaction required hydrogen peroxide as a substrate and was sensitive to aminotriazole and partially sensitive to cyanide and azide. A strong deposit of reaction product was observed in mitochondria under almost all conditions tested. There appeared to be two enzyme systems in the mitochondria which initiated the oxidation of diaminobenzidine. Peroxisomes were approximately 1 µ in length and were irregular in outline. These organelles possessed a finely granular matrix and were surrounded by a single membrane. Cores and vacuoles were seen in these bodies.
MAYER'S TANNIC ACID-FERRIC CHLORIDE STAIN FOR MUCINS
Authors: PIZZOLATO PHILIP , LILLIE R. D.
Published: April 2011

Tannic acid in aqueous solution is bound to mucins in formalin-fixed and formalin-free fixed tissues and its presence can be detected with ferric chloride as a dark gray, blue-black to black complex. This colored compound is readily extracted by acids and some chelating and bleaching agents and is changed to a reddish brown by alkalis. Hydrolysis in 1.2 N hydrochloric acid at 60°C for 4 hr or hot trichloroacetic acid prevents the tannin-iron reaction. Acetyl chloride or bromide is able to inhibit the binding of tannic acid to the mucosubstances and saponification restores the characteristic reaction. Several mechanisms for the attachment of tannic acid to the mucins appear possible.
DIFFUSION ARTIFACTS IN CYTOCHEMISTRY OF CATALASE
Authors: FAHIMI H. DARIUSH
Published: April 2011

The various causes of diffusion artifacts in cytochemical localization of catalase in rat hepatic peroxisomes have been investigated. It is noted that storage of tissue in buffer after the fixation with glutaraldehyde is mainly responsible for diffusion of catalase from peroxisomes. In the vicinity of such peroxisomes with diffusion, the product of oxidation of 3,3'-diaminobenzidine tetrahydrochloride (DAB) is localized on ribosomes and membranes of mitochondria and endoplasmic reticulum. In contrast, when the same tissue is reacted for catalase immediately after the fixation, no evidence of diffusion is observed. Prolonged incubation (up to 4 hr at 37°C) and storage of tissue after the reaction with DAB (up to 9 months) do not cause any diffusion of the reaction product. Similarly, there is no evidence of diffusion around the peroxisomes when the complete incubation medium is perfused into the liver immediately after the fixation with glutaraldehyde (perfusion-incubation technique). These findings indicate that the diffusion of heme protein catalase, rather than the diffusion of oxidation product of DAB, is responsible for focal cytoplasmic staining, which is seen occasionally in the vicinity of some peroxisomes.
UPTAKE OF TRITIUM-LABELED ADENOSINE TRIPHOSPHATE BY THE CYTOPLASM OF FIXED INTERSTITIAL CELLS FROM RAT TESTES
Authors: DARŻYNKIEWICZ Z. , GLEDHILL B. L.
Published: April 2011

The cytoplasm of histologically fixed interstitial cells of rat testes becomes heavily labeled after incubation in the presence of tritium-labeled adenosine triphosphate. The phenomenon shows a high degree of specificity; none of the other eight types of cells studied (five of nontesticular origin) exhibits significant labeling under the same conditions. The cell labeling seems to be nonenzymatic in character; specific macromolecules associated with retention of the label have not been identified. Label retained by the cytoplasm of interstitial cells withstands: (a) prolonged washings in cold 5% trichloroacetic acid; (b) washings in a solution of high ionic strength; (c) repeated washings with alcohol and with water; (d) heating to 100°C; and (e) treatment with enzymes hydrolyzing nucleic acids (deoxyribonuclease I, ribonuclease A, ribonuclease T2, micrococcal nuclease and phosphodiesterase).
HISTOCHEMICAL LOCALIZATION OF ADENYL CYCLASE IN THE FUNGUS PHYCOMYCES BLAKESLEEANUS
Authors: TU J. C. , MALHOTRA S. K.
Published: April 2011

The presence of adenyl cyclase in Phycomyces blakesleeanus has been investigated in situ histochemically by lead precipitation of pyrophosphate liberated by conversion of adenosine triphosphate to cyclic adenosine monophosphate. Dormant spores, heat-shocked spores, germinating spores and the growing zone of sporangiophore (stage IV) of the fungus were studied. Electron-dense lead precipitate was localized in association with plasma membrane and between the mitochondrial membranes and nuclear membranes in all stages investigated. This reaction product is not inhibited by sodium fluoride, and adenyl cyclase is the only known enzyme stimulated by sodium fluoride. Comparable studies on the hepatocytes of mouse liver showed the reaction product in association with plasma membrane only.
KINETICS OF HYDROLYSIS DURING THE FEULGEN REACTION FOR DEOXYRIBONUCLEIC ACID A REEVALUATION
Authors: RASCH ROBERT W. , RASCH ELLEN M.
Published: April 2011

Using thin films of Feulgen-stained chicken blood cells, we have found that the course of deoxyribonucleic acid (DNA) hydrolysis with 5 N HCl at room temperature over a period of 24 hr can be represented by equations based upon a simple kinetic model involving branched convergent reactions. In addition to its use for analysis of the Feulgen hydrolysis curves generated from avian erythrocytes and lymphocytes, the model has also been applied to previously published data on human lymphocytes. Use of the model to determine appropriate conditions for validating stoichiometry of the DNA-Feulgen reaction is discussed, with particular emphasis directed to possible implications for DNA-Feulgen cytophotometry of a two-compartment substrate moiety.
MORPHOLOGIC AND CYTOCHEMICAL IDENTIFICATION OF PEROXISOMES IN THE RAT PAROTID AND OTHER EXOCRINE GLANDS
Authors: HAND ARTHUR R.
Published: April 2011

Peroxisomes were identified in all three parenchymal cell types of the rat parotid gland. They averaged 0.33 µ in diameter in the acinar and intercalated duct cells, and 0.22 µ in the striated duct cells. They were closely related to the endoplasmic reticulum, occasionally in continuity with smooth surfaced cisternae and often embraced by ribosome-free portions of endoplasmic reticulum which paralleled their membrane. Glutaraldehyde fixation inhibited the endogenous peroxidase of the parotid gland and allowed visualization of the peroxisomes following incubation in alkaline diaminobenzidine medium. Peroxisomal staining was unaffected by varying H2O2 concentrations or low concentrations of KCN, but was prevented by aminotriazole and dichlorophenolindophenol. Examination of other exocrine glands after incubation in diaminobenzidine medium revealed the presence of peroxisomes in the pancreas, submandibular, lacrimal, nasal mucosal and von Ebner's glands. These studies indicate that peroxisomes are of widespread occurrence in exocrine tissues of the rat.
ON PHOSPHOTUNGSTIC ACID STAINING IV. SELECTIVITY OF THE STAINING REACTION
Authors: QUINTARELLI G. , BELLOCCI M. , GEREMIA R.
Published: April 2011

Phosphotungstic acid was used at different hydrogen ion concentrations and combined with glycogen and lysozyme, which were used as model systems for carbohydrates and proteins, respectively. Furthermore, in order to correlate the chemical analysis with the selectivity of the "staining" reaction, ultrastructural studies were carried out using mouse skin, aorta, esophagus and colon. When the heteropolyacid was used at a pH around 1.0, and not above 1.8, there was no interaction with glycogen whereas an extremely high lysozyme binding ensued. At these levels of hydrogen ion concentration the selectivity of the staining was quite apparent. The rise in pH of the phosphotungstic acid solutions determined a loss of specificity in the staining reaction accompanied by a consistent glycogen uptake. This anomalous reaction was interpreted as the result of a decomposition of the metal into two altogether different chemical compounds: phospho-11-tungstic acid and free tungstates. The latter, like the molybdates, are known to be excellent complexing agents with carbohydrates.
THE LOCALIZATION OF CATECHOLAMINE FLUORESCENCE TO DOG HYPOTHALAMIC NEUROMELANIN-BEARING NEURONS
Authors: BARDEN HERBERT , BARRETT ROBERT
Published: April 2011

By use of histochemical methods, a survey has been made of the localization of catecholamine to neuronal perikarya and processes in relation to neuromelanin accumulation in neuronal perikarya in the hypothalamus of dogs of various ages. Catecholamine was visualized in sections as a green fluorescence by means of the formaldehyde gasinduced fluorescence method of Falck and Hillarp. Neuromelanin, if also present in these sections, was identified on the basis of its characteristic optical properties as visualized through bright field, dark field and fluorescence microscopy. The study demonstrated a sequential replacement of temporally diminishing catecholamine fluorescence by accumulating neuromelanin within the same neuronal perikaryon wherein they were found to overlap during part of the 1st year of life. Catecholamine in processes in the perifornical region of the hypothalamus, where the neuromelanin-accumulating neurons were situated, was also diminished with age. If neuromelanin was extensively exposed to ultraviolet light, as required for viewing and for photography, the initially nonfluorescent pigment was converted to one which appeared fluorescent yellow. The larger and older accumulations of neuromelanin were more resistant to this induced alteration than the smaller and younger accumulations. These results support the concept that catecholamine contributes to the formation of neuromelanin although they do not exclude the possibility that the presence of both these substances in the same perikaryon is entirely coincidental.
ULTRASTRUCTURAL IDENTIFICATION OF CATECHOLAMINE-CONTAINING CENTRAL SYNAPTIC TERMINALS
Authors: BLOOM FLOYD E.
Published: April 2011

Cytochemical methods for the localization of central catecholamine-containing synaptic terminals have been developed from an extensive foundation of biochemical work and from extrapolation of results on the peripheral sympathetic nervous system. Direct localization of catecholamines in central nerve terminals in some parts of the brain can now be obtained by fixation with permanganates. More broadly applicable, but less direct localizing methods depend upon selective accumulation of tritiated catecholamines for autoradiography or the accumulation of reactive catecholamine congeners which act as markers with conventional fixation. The pattern of acute degenerative changes which result after treatment with 6-hydroxydopamine can also be used to provide an indirect localization of the terminals which had stored catecholamines. When the results of each of the methods are combined, the present techniques indicate that catecholamine-containing terminals in the brain can be identified more confidently than any other system of neurotransmitter substances. Nevertheless, there is considerable need for future cytochemical innovation.
NOTES ON SYNAPTIC VESICLES AND RELATED STRUCTURES, ENDOPLASMIC RETICULUM, LYSOSOMES AND PEROXISOMES IN NERVOUS TISSUE AND THE ADRENAL MEDULLA
Authors: HOLTZMAN ERIC , TEICHBERG SAUL , ABRAHAMS SUSAN J. , CITKOWITZ ELENA , CRAIN STANLEY M. , KAWAI NOBUFUMI , PETERSON EDITH R.
Published: April 2011

This paper reviews aspects of the origin and fate of synaptic vesicles and of the related catecholamine-containing secretion granules of the adrenal medulla. Most attention is given to evidence concerning the proposal that the membrane surrounding synaptic vesicles can originate from axonal agranular reticulum, participate in exocytosis and endocytosis and eventually undergo degradation in lysosomes of axons and perikarya. A number of relevant details of the roles of several organelles in neurons and other cells of the nervous system are discussed. The paper centers around microscopic and cytochemical work on a few experimental systems.
THE HISTOCHEMISTRY OF HYALURONIC ACID-CONTAINING MUCOSUBSTANCES
Authors: YAMADA KAZUYORI , HIRANO KENICHI
Published: April 2011

Digestion of histologic sections with Streptomyces hyaluronidase releases hyaluronic acid from hyaluronic acid-containing mucosubstances. Such treatment diminishes noticeably or eliminates the alcianophilia (pH 2.5) and metachromatic azurophilia (pH 4.5) of these mucosubstance-containing tissues. The present enzyme digestion procedure furnishes a useful method for the localization of hyaluronic acid in mucosaccharide histochemistry.
SAPONIFICATION-INDUCED INCREASE IN THE PERIODIC ACID-SCHIFF REACTION IN THE GASTROINTESTINAL TRACT MECHANISM AND DISTRIBUTION OF THE REACTIVE SUBSTANCE
Authors: REID P. E. , CULLING C. F. A. , DUNN W. L.
Published: April 2011

The mechanism and distribution of the KOH-periodic acid-Schiff (PAS) effect in rat large intestine, human sigmoid colon, rabbit rectum and rabbit Brunner's glands have been investigated following the abolition of normally occurring PAS-positive vicinal diols by means of a periodate oxidation-sodium borohydride reduction sequence. The KOH-PAS effect was shown to be independent of the initial oxidation time and could be abolished by pretreatment with either methanolic-HCl or alcoholic KOH. The evidence suggests that the KOH-PAS effect is due to the removal of an o-acyl-ester substituent on a potential vicinal diol. The alternative hypothesis, that the effect is due to an ester involving the carbonyl function of a macromolecule or a lactone, is less likely. The difficulty of interpretation of histochemical results following methylation is discussed.
SENSITIVITY IN ELECTRON MICROSCOPE AUTORADIOGRAPHY II. EFFECT OF HEAVY METAL STAINING
Authors: SALPETER MIRIAM M.
Published: April 2011

In quantitative electron microscopic autoradiography test specimens for sensitivity usually consist of plastic or protein films 500-1000 Å in thickness. For tritium, the applicability of sensitivity values derived from such specimens to biologic sections which had been stained with heavy metals was determined. It was found that, within the range of section thickness used for electron microscopic autoradiography, fixation with OsO4 followed by uranyl acetate staining increases self-absorption by less than 7% over that seen in Epon sections of the same thickness. A somewhat larger effect is seen in sections used for light microscopic autoradiography. The effect on electron microscopic autoradiographic resolution is estimated to be within 10%.
ACETYLCHOLINESTERASE ACTIVITY IN THE MYOTUBE AND MUSCLE SATELLITE CELL OF THE FETAL RABBIT AN ELECTRON MICROSCOPIC-CYTOCHEMICAL AND BIOCHEMICAL STUDY
Authors: TENNYSON VIRGINIA M. , KREMZNER LEON T. , BRZIN MIRO
Published: April 2011

Acetylcholinesterase (AChE) activity has been studied in the myotube and in extrajunctional skeletal muscle of the rabbit fetus. Some observations on the developing motor end plate are included. Both insoluble and soluble AChE's are present at all stages, but butyrylcholinesterase seems to be absent. Enzymic activity is high during myotube formation. The insoluble AChE probably corresponds to the cytochemical end product which is bound to the elements of the reticulum in the myotube, i.e., the nuclear envelope, the sarcoplasmic reticulum and some elements of the Golgi complex. The possibility that the enzyme-containing reticulum is involved in the spontaneous contraction of early myotubes is discussed. The number of active sites of reticulum-bound enzyme decreases markedly as the myotubes mature into early muscle. The soluble AChE is probably derived from the AChE-containing mononuclear cell and the Schwann cell which accompanies the spinal nerve to the motor end plate. These cells have a random distribution of end product and are probably the source of a similar end product found at the surfaces of these cells and at the sarcolemma of adjacent myotubes. The following possibilities that are discussed are that soluble AChE may (a) play a role in fusion of myotubes, (b) be involved in widespread sarcolemmal acetylcholine sensitivity and (c) contribute to the junctional enzyme. The randomly distributed end product in the AChE-containing mononuclear cell permits the tracing of its further differentiation. This cell appears to be a stem cell, which can fuse directly with the myotube; it can give rise to a more differentiated type of myoblast, as well as to the muscle satellite cell. AChE-containing mononuclear cells may also contribute to the cells associated with the neuromuscular junction. The soluble AChE in the muscle satellite cell seems to be associated with an "active" phase of this cell, since enzymic activity is not seen in the adult stage in which it is presumed to be dormant.
STRUCTURE AND STAINING CHARACTERISTICS OF MYOFIBER EXTERNAL LAMINA
Authors: ZACKS S. I. , SHEFF M. F. , SAITO A.
Published: April 2011

A variety of cytochemical methods were used to investigate the composition of mouse sarcolemma. We found that the sarcolemma is a complex composed of the myofiber cell membrane, a thin zone external to the cell membrane that contains groups that bind colloidal iron and thorium at low pH and a thicker amorphous layer that fails to stain with colloidal iron and thorium at low pH. The entire complex is periodic acid-Schiff-positive and stains with ruthenium red and strongly acid solutions of phosphotungstic acid. Although the specificity of these cytochemical stains is controversial, data obtained with them and from preliminary analyses of myofiber external lamina (EL) indicate that the myofiber cell coat is chiefly composed of glycoprotein containing a large number of carboxyl groups. The EL within the subneural apparatus of the neuromuscular junction differs from noninnervated areas in the fusion of Schwann cell and myofiber EL, the absence of collagen microfibrils, the more intense binding of divalent cations and the less intense stain with phosphotungstic acid in strongly acid solutions.
A NEW METHOD TO DISTINGUISH SEPARATE COMPONENTS IN CYTOCHEMICALLY STAINED MACROMOLECULES BY ANALYSIS OF THE KINETICS OF THE STAINING AND DESTAINING PROCESSES
Authors: DUIJNDAM W. A. L. , HERMANS J. , VAN DUIJN P.
Published: April 2011

A new method making it possible to analyze properties of the end products of cytochemical staining reactions in macromolecular substances not expressed in the absorption spectra is described. Mathematical analysis of the course of changes in the absorbance during staining or destaining of the macromolecular substance provides information about the reaction velocities of these processes for the individual components present as well as about the concentrations of the components thus detected. This analysis is performed by a procedure of computerized curve-fitting between experimentally determined and theoretically expected absorbance curves. The theoretical time course of absorbance is derived for a number of pseudo first-order reaction schemes, and a description is given of the way in which the most appropriate reaction scheme can be recognized and the parameters are estimated, both on the basis of the experimental absorbance curve. Possible complications due to dye impurities, depolymerization and diffusion processes are discussed.
SIZES OF END PLATE COMPARTMENTS, DENSITIES OF ACETYLCHOLINE RECEPTOR AND OTHER QUANTITATIVE ASPECTS OF NEUROMUSCULAR TRANSMISSION
Authors: SALPETER MIRIAM M. , ELDEFRAWI MOHYEE E.
Published: April 2011

The area of the postsynaptic membrane and the volume of the synaptic cleft were calculated for the end plates of the diaphragm and sternomastoid of mouse and rat. From these dimensions we were able to extrapolate, from data given by others on acetylcholine (ACh) released during neuromuscular transmission and on ACh receptor per whole end plate, to densities in the postneural compartments. The concentration of ACh in the cleft per nerve impulse was found to be ~ 10–5 M and the density of ACh receptor was between 5 and 10 x l03/µ2 of postsynaptic membrane. (This is approximately a factor of 2 to 3 higher than that for acetylcholinesterase at this site.) From these values we conclude that the concentration of ACh receptor in the plane of the postsynaptic membrane is considerably higher than that of ACh in the cleft during neuromuscular transmission. In addition the ACh itself is present at considerably higher concentrations than necessary to give optimal response. We calculated that the acytelcholinesterase plus ACh receptor together would occupy about 25% of the surface area of the postsynaptic membrane.
PROTON-INDUCED X-RAY ANALYSIS OF TRACE ELEMENTS IN TISSUE SECTIONS
Authors: JUNDT F. C. , PURSER K. H. , KUBO H. , SCHENK E. A.
Published: April 2011

A 3-meV proton beam from the MP Van de Graaff accelerator at the Nuclear Structure Research Laboratory of the University of Rochester has been used to induce characteristic x-rays of trace elements from a variety of biologic samples. The x-ray spectra have been measured with a high resolution Si(Li) detector which can detect and separate in energy x-rays from neighboring elements with Z > 13. Tissue homogenates, frozen sections and formalin-fixed, paraffin-embedded sections of various tissues obtained from surgical and autopsy specimens have been analyzed. Measurements have been made to check the sensitivity and reproducibility of the method. Data accumulated thus far indicate that this method is useful in evaluating absolute concentration of elements usually present in a given tissue and detecting environmental elements which may have accumulated in a given tissue.