bluuebunny/crossref_metadata_2025 huggingface dataset:
THE KREBS CYCLE IN GLOMERULI OF NORMAL RAT KIDNEY AND IN COMPENSATORY HYPERTROPHY
Authors: NOWINSKI WIKTOR W. , PIGOŃ ANDRZEJ
Published: April 2011

The Krebs cycle in glomeruli of rat kidney was studied under normal conditions and in compensatory hypertrophy after unilateral nephrectomy. By using substrates of the cycle and measuring with the Cartesian diver technique the increase in oxygen uptake, it was established that the entire enzyme system is present, including succinic dehydrogenase. The latter could not be demonstrated by other authors who used older histochemical staining techniques. In compensatory hypertrophy it was thought sufficient to study activites of two enzymes only, isocitric dehydrogenase and succinic dehydrogenase. No change in the activity of either dehydrogenase was found. The conclusion was drawn that in hypertrophy, the glomeruli simply swell and serve as ultrafilters; the Krebs cycle is present, but produces ATP only for the maintenance of normal cell functions and thus does not undergo an increase in activity.
AN IMPROVED MICROFLUORIMETER FOR MEASURING BRIGHTNESS OF FLUORESCENT ANTIBODY REACTIONS
Authors: GOLDMAN MORRIS
Published: April 2011

A microfluorimeter suitable for measuring fluorescence of objects as small as bacteria is described. It consists of a fluorescence microscope equipped with one photomultiplier for intermittent measurements of observed objects, and another for continuous monitoring of the excitation beam. Output of the two photomultipliers is presented on a recorder as a ratio of the two currents. The light source is a high pressure mercury arc operated on de, and the instrument is aligned and standardized with the help of a standard fluorescent crystal mounted on a microscope slide. Measurements of intestinal amebae stained with a refined fraction of fluoresceinlabeled globulin showed high reproducibility on duplicate smears in spite of the presence of gross bacterial contamination. Standard errors of the means obtained from samples of 30 amebae averaged 5.6% of the means. The technique is capable of revealing extremely fine immunological distinctions in antigenic materials like intact microorganisms, virus particles and the like, even in the presence of tissue cells and other "contaminant" debris.
HISTOCHEMISTRY OF LYSOSOMES IN RAT HEART MUSCLE
Authors: ABRAHAM R. , MORRIS MARGARET , SMITH JENNIFER
Published: April 2011

Rat heart muscle was studied histochemically by the naphthol and lead methods for hydrolases (acid phosphatase, esterase, β-glucuronidase and aryl sulfatase). Activity of the enzymes studied is demonstrable in granules which by their staining properties are identified as lysosomes. These organelles are usually perinuclear in location and display variable amounts of enzyme activity. The heart lysosomes in chloroquine-treated rats are enlarged and aggregated together. The effects of the detergent Triton X-100 on the lysosomal hydrolases are discussed.
THEORETICAL AND EXPERIMENTAL ASPECTS OF ENZYME DETERMINATION IN A CYTOCHEMICAL MODEL SYSTEM OF POLYACRYLAMIDE FILMS CONTAINING ALKALINE PHOSPHATASE
Authors: VAN DUIJN P. , PASCOE E. , VAN DER PLOEG M.
Published: April 2011

A theoretical treatment of the influence of slow substrate diffusion on the kinetics of an enzyme reaction in a cytochemical system is given. Quantitative aspects of the effect of several parameters on the diffusion of substrate were studied experimentally on a model consisting of polyacrylamide films into which alkaline phosphatase was incorporated. Spectra and staining intensity of the films were measured in a special film colorimeter. Enzyme activity in the films was determined cytochemically (with the azo dye coupling method) and biochemically. An effectiveness factor for which diffusion is not rate-limiting proved to be attainable with both methods.
THE KINETICS OF THE PERIODATE-SCHIFF REACTION OF HUMAN LEUKOCYTE HOMOGENATES
Authors: OLSSON INGE , DAHLQVIST ARNE
Published: April 2011

The periodate-Schiff (PAS) reaction of homogenized human leukocytes has been studied with a quantitative test tube method. The leukocytes contain glycogen, which gives a slow formation of chromogen during the periodate oxidation, and one or several more rapidly reacting, nonglycogen substances. The latter substances are quantitatively more important for the PAS reaction than glycogen. Nonglycogenic, rapidly reacting PAS-positive substances are present in a high concentration in the specific leukocyte granules. A considerable fraction of the rapidly reacting PAS-positive material in leukocyte homogenates can be extracted with lipid solvents.
PURIFICATION AND QUANTITATION OF GLUTARALDEHYDE AND ITS EFFECT ON SEVERAL ENZYME ACTIVITIES IN SKELETAL MUSCLE
Authors: ANDERSON PAUL J.
Published: April 2011

Glutaraldehyde was quantitated by chemical titration, osmometry and ultraviolet spectrophotometry. Pure glutaraldehyde exhibited an absorption maximum at 280 mµ and absorbance at this wavelength conformed to Beer's law. A second maximum at 235 mµ indicated the presence of "impurities" (probably of polymeric origin), and effective purification was achieved by distillation or by repeated washing with charcoal of high surface area (Norit EX). Purified glutaraldehyde was stable when stored under nitrogen or freon at 4°C. Representative oxidoreductase, transferase and hydrolase activities were measured in supernatants and sediments of skeletal muscle homogenates that had been treated with glutaraldehyde samples of low, intermediate and high impurity content (based on relative absorbance at 235 mµ). Enzyme recovery was inversely related to impurity content, the highest levels being obtained from tissue treated with the purest glutaraldehyde.
A LIGHT AND ELECTRON HISTOCHEMICAL APPROACH TO THE NODE OF RANVIER AND MYELIN OF PERIPHERAL NERVE FIBERS
Authors: LANGLEY O. K. , LANDON D. N.
Published: April 2011

The Hale staining reaction has been used to study the nature and distribution of acid mucopolysaccharides associated with mammalian peripheral myelinated nerve fibers. Histochemical blocking reactions were employed to determine the specificity of the method. Electron micrographs of parallel preparations were examined to discover the fine structural localization of the optically visible Prussian blue pigment. The observations reported suggest that there is a specific localization of a sulfated mucopolysaccharide in the region immediately surrounding the axolemma at the node of Ranvier. Other parts of the fiber, in particular the myelin sheath, show a preponderance of carboxyl groups. Attention is drawn to the variation in the form and distribution of the Hale stain product after differing fixation procedures. The effect of osmium tetroxide on ferrocyanide-treated material is examined and discussed. Attention is directed to possible physiologic implications of the presence of material with the known ion exchange properties of a sulfated mucopolysaccharide in the immediate environment of the ionically active nodal axon membrane.
THE LOCATION OF ONUPHIC ACID IN HYALINOECIA TUBICOLA
Authors: PAUTARD F. G. E. , ZOLA H.
Published: April 2011

Onuphic acid, a sugar phosphate polymer extracted from the tube of Hyalinoecia tubicola, has been identified in the tube and in the body of the polychaete by histochemical methods based on the chemical properties of the macromolecule. Comparison of the results with probe microanalysis for selected elements suggests that onuphic acid is located in regions of high phosphorus density in the lateral-ventral glands, in proximity to sulfur but in the absence of calcium and any large local amounts of magnesium.
CORRELATED HISTOCHEMICAL STAINING AND S35O4= LABELING OF SALIVARY GLAND MUCOSUBSTANCES
Authors: LEPPI T. JOHN , SPICER SAMUEL S. , HENSON JACQUELINE G. , FIORAVANTI JOSEPHINE
Published: April 2011

Correlated histochemical and autoradiographic studies were conducted on rat, rabbit, cat and dog salivary glands. Mucous acini of rat minor sublingual (SL) rabbit SL and cat posterior SL and submandibular (SM) glands were stained by Alcian Blue at pH 1.0, aldehyde fuchsin or the high iron diamine technique. The distribution of cells with this type of basophilia corresponded with that of cells shown autoradiographically to incorporate S35O4= and accordingly to contain sulfomucin. The lack of periodic acid-Schiff and alcohol-resistant metachromasia at pH 0.5 of rabbit SL mucin resembles connective tissue mucopolysaccharide. Some mucous acini of the dog posterior SL gland incorporate S35-sulfate and the distribution of isotope-labeled acini correlates with that of acini showing histochemical staining indicative of sulfomucin. The remainder of mucous acini in this gland do not show uptake of radiosulfate, but have alcianophilia that is reduced by sialidase digestion. All of the mucous acini of dog SM gland show correspondence of staining for sulfomucin with S35O4–, incorporation. The seromucous demilunes of this gland manifest sialidase-labile alcianophilia. The staining of sulfomucin and S35 labeling in each of the salivary glands studied are eliminated by a 4-hr methylation at 60°C.
UBIQUINONE AND PHOSPHOLIPIDS AS LIMITING FACTORS IN THE HISTOCHEMICAL DEMONSTRATION OF SUCCINIC DEHYDROGENASE ACTIVITY
Authors: WOLMAN MOSHE , BUBIS JOSE JAIME
Published: April 2011

Deposition of ubiquinone and preferably of a crude phospholipid mixture and ubiquinone on cryostat sections of mouse kidney, liver and heart increased the intensity of staining for succinic dehydrogenase. A similar treatment partly restored the activity of this enzyme which was reduced by exposure to 60°C, long storage at 37°C, oxidation and treatment with lipid solvents. The restoration was present only in the case of mild noxious treatments. It is suggested that removal or structural alteration of ubiquinone and phospholipids may limit the rate of dehydrogenase activity; more drastic treatments affect the enzyme itself so that these factors cannot exert any restorative activity.
COMPARATIVE HISTOCHEMICAL LOCALIZATION OF LYSOSOMAL ENZYMES IN RAT TISSUES
Authors: HAYASHI MASANDO
Published: April 2011

Localization of acid phosphatase, β-glucuronidase and N-acetyl-β-glucosaminidase has been studied in various tissues of the rat with comparable simultaneous coupling azo dye techniques employing naphthol AS-BI compounds as substrates and hexazonium pararosanilin as a capture reagent. These three lysosomal hydrolases were widely distributed in tissues and were generally demonstrated as discrete granules in certain portions of the cytoplasm. Granules stained for each enzyme were localized at identical sites in most, but not all, tissues in which they were present. However, the intensity or number of granules stained for each of the enzymes was found to differ in varying degree among tissues. Acid phosphatase appeared to be more widespread in tissues than the two glycosidases. Distribution of the glycosidases was also found to differ considerably. Cells of the macrophage system, for example, displayed high β-glucuronidase activity, while N-acetyl-β-glucosaminidase was much less active in these cells. The latter enzyme was more active in the epithelium of the mucous membrane. Furthermore, in several tissues, the three enzymes exhibited qualitatively different localization. In these sites, one or two of the enzymes were not or were only faintly demonstrable while the other showed an intense reaction. These findings suggest that a functional differentiation among lysosomes in various tissues may exist.
ON THE HISTOCHEMISTRY OF AZO GROUP-FREE THIAZOLE DYES
Authors: KELIÉNYI GABRIEL
Published: April 2011

Analysis of primuline, thioflavine S and thioflavine T acid and basic azo group-free thiazole dyes showed that they were built up from a number of components which were characterized by physicochemical methods. It was found that the isolated components, as well as related substances of known chemical composition, have characteristic staining properties. Factors involved in the staining mechanism of the dyes and of components—dye concentration, pH, aggregation of the dye molecules—were investigated and their roles are discussed. Selectivity of these fluorescent staining methods was also studied.
ACID MUCOSUBSTANCE AND BASIC PROTEIN IN MOUSE PANETH CELLS
Authors: SPICER S. S. , STALEY M. W. , WETZEL M. G. , WETZEL B. K.
Published: April 2011

The histochemistry of mouse Paneth cell granules was investigated with alcian blue (AB, pH 1.0 or 2.5), periodic acid-Schiff reagent (PAS), high iron diamine (HID) and Biebrich scarlet (pH 9.5). After formalin fixation, an AB pH 2.5-PAS sequence demonstrates alcianophilic rims surrounding weakly PAS-reactive granule cores; the HID-AB and AB pH 1.0-PAS sequences show peripheral staining indicative of a sulfomucin in most granules and a carboxymucin in some granules, the latter predominantly in the distal small intestine. When stained with PAS alone, rims of Paneth granules stain intensely and cores weakly. After HgC12 fixation, HID staining indicative of sulfomucin occurs in the cores of most granules and in the rims of many granules. An AB-Biebrich scarlet sequence shows acid mucosubstance in the rims and strongly basic protein (presumably arginine rich) in the cores of the granules. The basic protein-rich cores are larger toward the bottom of the crypts and toward the distal small intestine. The acid mucosaccharide-rich rims appear thinner surrounding the larger granules at the bottom of the crypts. Fine structural studies have demonstrated dialyzed iron staining indicative of acid mucosubstance in the rims of mouse Paneth cell granules and in small profiles and some lamellae of the Golgi zone.
FREEZE-DRYING OF SMALL TISSUE SAMPLES AND THIN FROZEN SECTIONS BELOW –60°C A SIMPLE METHOD OF CRYOSORPTION PUMPING
Authors: STUMPF WALTER E. , ROTH LLOYD J.
Published: April 2011

Freeze-drying rates for three tissues have been determined by direct measurement of weight loss in a closed system, using an electrobalance. The half-drying times (T/2) at approximately 1 x 10–5 mm Hg with Dry Ice slush as coolant were determined for brain, kidney and liver and found to be 100, 50 and 30 hr, respectively, for 10-mg samples of tissue. A method for the freeze-drying of frozen sections in which the temperature of the tissue is maintained below –60°C throughout sectioning and drying is discussed. A simple portable, all glass, freeze-drying system utilizing cryosorption pumping is presented.
CORRELATION BETWEEN THE PRESENCE OF SULFATED POLYSACCHARIDES AND MINERALIZATION IN A MARINE COCCOLITHOPHORID PROTOZOON: HISTOCHEMICAL AND AUTORADIOGRAPHIC STUDIES
Authors: DOUGLAS STEVEN D. , ISENBERG HENRY D. , LAVINE LEROY S. , SPICER SAMUEL S.
Published: April 2011

The deposition of coccoliths, calcite-calcium carbonate-containing organic structures which align peripherally and are liberated into the environment by the primitive marine coccolithophorid chrysomonad protozoon Hymenomonas Mary Parke 156, follows the pathways of biphasic biologic mineral deposition without biochemical evidence of sulfur-containing polysaccharides. Tinctorial and autoradiographic evidence for the participation of sulfur-containing carbohydrate moieties is presented. Nutritional conditions and addition of inhibitors, which have been shown to interfere with the microscopic, electron microscopic and biochemical demonstration of mineral deposition, also inhibit the staining reactions associated in mammalian systems with acid mucopolysaccharides. These observations suggest that, even at the level of an evolving mechanism, sulfur-containing carbohydrates participate in some manner in the deposition of mineral onto a preformed organic matrix.
FINE STRUCTURAL LOCALIZATION OF ACID AND ALKALINE PHOSPHATASES IN CELLS OF RABBIT BLOOD AND BONE MARROW
Authors: WETZEL B. K. , SPICER S. S. , HORN R. G.
Published: April 2011

In rabbit heterophils, acid phosphatase activity occurs in primary (azurophil) granules which predominate in early cells and persist in mature cells and in tertiary granules which are seen only in mature cells. Alkaline phosphatase activity occurs in secondary granules which appear in intermediate heterophils and later predominate in mature cells. Acid phosphatase activity in heterophil Golgi zones coincides developmentally with the genesis of primary and, later, tertiary granules, whereas alkaline phosphatase in the Golgi complex coincides with secondary granulogenesis. In developing eosinophils, acid phosphatase reaction product occurs in Golgi elements, rims the spherical precursors of angular, mature granules and appears inconsistently within mature granules. Basophil myelocytes show acid phosphatase in Golgi elements but not in specific granules.

Additional acid phosphatase reactive structures include: granules of mononuclear cells; phagocytic vacuoles in macrophages; autophagic vacuoles in maturing erythroid cells; small dense granules of platelets; dense bodies in lipocytes; and Golgi elements of mononuclear cells, macrophages, nucleated red cells, megakaryocytes and lipocytes.

Localized deposits were absent in control specimens except for enzyme-independent nuclear staining in alkaline phosphatase preparations.
METHODS FOR THE STUDY OF SMALL PHAGOSOMES AND THEIR RELATIONSHIP TO LYSOSOMES WITH HORSERADISH PEROXIDASE AS A "MARKER PROTEIN"
Authors: STRAUS WERNER
Published: April 2011

Small phagosomes (micropinocytic vesicles and vacuoles) which had taken up injected horseradish peroxidase were identified by staining for peroxidase with benzidine and H2O2. Because of the small size of the granules and the possibility of artifacts, previously described procedures had to be modified in several respects. Prefixation of the tissue by perfusion at 37°C prevented artifacts of diffusion and adsorption of peroxidase. The blue product of the reaction of peroxidase with benzidine in the small phagosomes was preserved and fading to brown was prevented by cooling the tissue section to –10° to –15°C during its processing through polar media. The blue reaction product was stable as soon as the section was transferred to an apolar medium. Small phagosomes were visualized together with lysosomes and phago-lysosomes in the same cells by double staining for acid phosphatase and peroxidase in contrasting colors. The incubation for acid phosphatase was performed at 4°C since low temperature increased the stability of peroxidase in the acid medium. Factors which form the basis for other improvements of the procedure are discussed.
THE QUANTITATIVE HISTOCHEMISTRY OF HYPOTHALAMUS I. PENTOSE SHUNT ENZYMES IN THE ACTIVATED SUPRAOPTIC NUCLEUS OF THE RAT
Authors: JONGKIND JOHAN F.
Published: April 2011

The activities of two enzymes involved in the oxidative part of the pentose cycle and one glycolytic enzyme have been measured by quantitative histochemical methods both in histologically pure nucleus supraopticus and in an adjacent area of the anterior hypothalamus of rat. In the nucleus supraopticus, glucose 6-phosphate dehydrogenase activity increased 34% and 6-phosphogluconate dehydrogenase activity declined by 9%, while lactic dehydrogenase activity did not change significantly after a thirsting period of 6 days. The nonsupraoptic, adjacent anterior hypothalamic area did not show significant changes in activity of any of the enzymes studied.
DETERMINATION OF HISTAMINASE ACTIVITY IN HISTOLOGIC SAMPLES AND ITS QUANTITATIVE DISTRIBUTION IN INTACT HUMAN PLACENTA AND UTERUS
Authors: GUNTHER RONALD E. , GLICK DAVID
Published: April 2011

The spectrophotometric method for determination of histaminase, based on measurement of the hydrogen peroxide formed by the enzymatic oxidation of histamine, was modified and adapted to studies on microliter volumes of tissue (placenta) homogenates and microtome sections of tissue (human uterus and attached intact placenta at term). Optimal conditions for these assays were established. From analyses of tissue sections, peak enzyme activities were observed in the decidua, thus supporting the concept that this tissue is a significant source of the increased enzyme in blood plasma during pregnancy in the human. Inhibitor studies with aminoguanidine sulfate showed that the histaminase investigated in the human placenta was diamine oxidase. No histaminase activity was detected in isolated peritoneal mast cells from the rat or in homogenates of rat stomach mucosa.
SOME HISTOCHEMICAL OBSERVATIONS ON TRANSITIONAL EPITHELIUM OF MAN
Authors: MONIS BENITO , DORFMAN HOWARD D.
Published: April 2011

This report deals with a histochemical characterization of transitional epithelium of the urinary tract of man. The cytoplasm of cells of the innermost, luminal layer is distinctly stained by the periodic acid-Schiff procedure. Enhanced staining of the luminal border of these cells suggests the presence of a differentiation of the luminal surface ( surface mucous coat) which is selectively stained by Alcian Blue and colloidal iron. The alcianophilic surface mucous coat is seen in the innermost layer of transitional epithelial cells of the urinary tract extending from the area cribrosa down to the bladder. This indicates the presence of an acid mucinous component. Digestion with neuraminidase according to the procedure of Spicer and Warren demonstrates the presence of N-acetylneuraminic acid (sialic acid) in the free cell surface of the luminal layer of transitional epithelium of man. There is abundant glycogen in transitional epithelium of man. A very characteristic basement membrane underlies transitional epithelium. It reacts with periodic acid-Schiff and often with Alcian Blue. The neuraminidase digestion does not alter the staining of basement membrane with Alcian Blue. This suggests the presence of a sialidase-resistant form of sialomucin or a sulfomucin in basement membrane. The presence of periodic acid-Schiff-positive, nondigestible granules closely corresponds to acid phosphatase-reactive granules, presumably lysosomes. Within the transitional epithelium there were lacunae which contained abundant sialomucins. These structures have been described in old textbooks of histology but do not seem to be a normal component of transitional epithelium. The significance of these structures is unknown.
HISTOLOGIC LOCALIZATION OF FOLLICLE-STIMULATING AND LUTEINIZING HORMONES IN THE RAT TESTIS
Authors: MANCINI R. E. , CASTRO A. , SEIGUER A. C.
Published: April 2011

Follicle-stimulating and luteinizing hormones were labeled with fluorescein isothiocyanate, rhodamine B and ferritin. The histologic distribution of labeled hormones, injected intravenously into adult rats, was studied by means of fluorescence microscopy and Prussian blue reaction for ferritin-conjugated products. As controls, rats were injected with free labels, denatured labeled hormones and unlabeled hormones mixed with labels. As a result of labeling, a variable loss in biologic activity occurred. After injection of labeled hormones a fluorescent or blue colored material appeared in testicular and epididymal tissue, but was absent in other endocrine glands. Macrophages in the organs studied and kidney tubular cells showed accumulations of the same material. Five minutes to 3 hr after injection, labeled products were seen in vessels and extravascular connective tissue of the testis. Labeled follicle-stimulating hormone appeared in the tubule wall, in a few Leydig cells, in Sertoli cells and in some spermatogonial cells, while labeled luteinizing hormone was selectively localized in Leydig cells and fibroblast-like cells of the tubule wall. Control experiments revealed the presence of similar material in the vessels of testis and in Leydig cells and macrophages of other organs. Specificity based upon the heterologous origin of hormones, partial denaturation by tagging, the role of Sertoli cells in the accumulation of proteins and the phagocytic properties of Leydig cells is discussed.
HISTOCHEMICAL STUDIES OF OXIDATION AND REDUCTION REACTIONS OF THE BILE PIGMENTS IN OBSTRUCTIVE ICTERUS, WITH SOME NOTES ON HEMATOIDIN
Authors: LILLIE R. D. , PIZZOLATO P.
Published: April 2011

In parallel tests formol fixation preserves more hepatic bile pigment than chloroformmethanol. The latter extracts more yellow color into the solvent and a higher proportion of the remaining bile pigment is green. Titanous chloride-formol and bichromate-formol preserve about the same amount of pigment as formol; the color distribution between golden brown and green casts is altered only a little in the expected directions. Primary acetic silver and neutral silver nitrate fixations blacken bile casts in liver and kidney in a 4-hr fixation period; after formol fixation sections require ammoniacal or methenamine silver for a successful argentaffin reaction. Hepatic bile casts show a pK of 5 after formol and 6 after chloroform-methanol fixations, staining red-brown with azure eosin at lower pH levels and deep green blue at higher. A 4-hr methylation at 60°C produced strong oxyphilia in formol tissue at pH 6; saponification restored and enhanced the basophilia and also enhanced pH 6 basophilia in unmethylated sections. The last suggests liberation of the propionyl residues by hydrolysis of the glucuronide bonds. Staining with several red dyes effectively changed the color of green bile casts to brown; hence unstained controls are needed for evaluation of tests for oxidation of bilirubin to biliverdin. Green hepatic bile casts are reduced to yellow-brown by reducing agents: Na2S2O4, Na2S2O2, HC1 + Na2S2Q5, FeSO4. Use of reduced bile pigment may be needed to show convincingly the oxidative action of such agents as FeCl2, K2Cr2O7 and iodine. Bile casts in variously fixed livers show purplish to brownish red colors with the periodic acid-Schiff (glycol) reaction and also after saliva digestion. Saponification, to break the glucuronide bond with bilirubin propionyl residues, did not prevent the glycol reaction. If this reaction is due to glucuronic acid residues of conjugated bilirubin, they would appear to be otherwise bound to tissue, perhaps by an amide linkage to protein amino groups. Peptic digestion alone did not inhibit the glycol reaction; combination with saponification results in section loss. Previously sectioned paraffin blocks and cut paraffin sections on 2-3 months air oxidation present numbers of violet, purple and red bile casts. The same Gmelin colors are produced by 3-5-day oxidation at 60°C in 2.5% K2Cr2O7 by 1-2 hr at 24°C in 0.1% CrO3, and by short diammine silver baths followed by desilvering with iodine and thiosulfate, as well as by the dry Br,:CCl4 and aqueous OSO4 treatments previously reported (16). The Gmelin colors are not reduced by dithionite or thiosulfate.
HISTOCHEMICAL STAINING AND MICROCHEMICAL STUDIES OF FRUCTOSE 1,6-DIPHOSPHATE SPLITTING ENZYMES IN THE PANCREATIC ISLETS AND LIVER OF NZO MICE
Authors: BROLIN SVEN E. , BERNE CHRISTIAN , PETERSSON BIRGER , LARSSON ARNOLD
Published: April 2011

The ability to hydrolyze various fructose phosphates was studied by microscopic histochemical techniques. The liver cells and the tubular cells of the kidney split fructose 1,6-diphosphate, fructose 1-phosphate and fructose 6-phosphate, whereas only the vessels of the islets and exocrine pancreatic parenchyma showed a positive reaction with these substrates. Whether the splitting of fructose 1,6-diphosphate takes place at position 1 or 6 cannot be revealed by estimation of liberated phosphate. In samples prepared by Lowry's microtechniques, liberated fructose 6-phosphate was determined fluorometrically by measuring reduced nicotinamide adenine dinucleotide phosphate formation in coupled phosphoglucoisomerase and glucose 6-phosphate dehydrogenase reactions. The activity of hexose diphosphatase (EC 3.1.3.11) in the liver of the NZO mouse was in the same range (0.5-1 MKH) as that found in other species. Samples of both pancreatic islets and acini showed only insignificant activities which could be attributed to the nonspecific phosphatases present in the vessels. The absence of hexose diphosphatase in the endocrine cells of the islets suggests that these cells cannot reutilize triosephosphates in the pentose phosphate shunt or for gluconeogenesis.
LOCALIZATION OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN PRENEOPLASTIC AND CANCEROUS CELLS OF THE MOUSE SKIN INDUCED BY METHYLCHOLANTHRENE
Authors: SUGÁR JÁNOS , CSUKA ORSOLYA , TÓTH JÓZSEF
Published: April 2011

The localization of membrane-bound adenosine triphosphatase activity has been studied by both light and electron microscopy in the normal epidermis and in methylcholanthreneinduced preneoplastic alterations, i.e., hyperplasia, papilloma and also in similarly induced cancers of the skin of mice. In normal epidermis, the adenosine triphosphatase activity was seen to be located in contact with all of the cell membranes facing the intercellular spaces. In preneoplastic alterations, the enzyme activity was found along the cell membranes of the basal and suprabasal cells of the epithelial pegs and numerous dense deposits of lead phosphate could be seen on the surfaces of the cytoplasmic processes and in the enlarged intercellular spaces. The intensity of the reaction in preneoplastic conditions was greater than in the normal epidermis. No adenosine triphosphatase activity was observed in the cell membranes facing the basement membrane or in the desmosomes. In carcinoma a less intensive adenosine triphosphatase reaction was present and could be recognized on the surface of only few tumor cells.
CYTOPHOTOMETRIC DETERMINATION OF ALKALINE PHOSPHATASE ACTIVITY OF INDIVIDUAL NEUTROPHILIC LEUKOCYTES WITH A BIOCHEMICALLY CALIBRATED MODEL SYSTEM
Authors: VAN DER PLOEG M. , VAN DUIJN P.
Published: April 2011

A model system consisting of polyacrylamide films into which cell sonicate is incorporated was applied to investigate quantitative aspects of the cytochemical azo dye coupling method for alkaline phosphatase activity in neutrophilic leukocytes. The films were assayed in media containing naphthol AS-MX phosphate and 4-aminodiphenylamine diazonium sulfate. Optimal reaction conditions under which proportionality existed between the amount of reaction product and incubation time or enzyme concentration were determined. Since the enzyme activity in the films could also be measured chemically, using disodium phenyl phosphate as a substrate, a direct relation between the cytochemical and biochemical activity could be established. The azo dye coupling method was applied for the quantification of the enzyme activity. Air-dried microscopic preparations of exudate neutrophils were stained and the amount of dye formed in the cytoplasm of individual leukocytes was measured with a cytospectrophotometer based on the two-wavelength principle. By reference to the relation between biochemical and cytochemical activities in the model films, the cytochemically determined enzyme activity could be expressed in biochemical units. Independently, the average alkaline phosphatase activity per cell was determined by direct biochemical assay of the leukocyte suspension. The results showed that about 60% of the biochemically assayed activity in sonicate was demonstrated in the cell preparations by the cytochemical staining method. The discrepancy between the results of the two methods is discussed. The applicability of the model system for elucidation of the relationship between the results of cytochemical and biochemical enzyme determination in cells is stressed.
STRUCTURAL CHANGES OF ISOLATED RAT PERITONEAL MAST CELLS INDUCED BY ADENOSINE TRIPHOSPHATE AND ADENOSINE DIPHOSPHATE
Authors: DIAMANT BERTIL , KRÜGER PER GÖRAN
Published: April 2011

Procedures for the local application of chemical agents to individual rat peritoneal mast cells are described. The local application of adenosine triphosphate (ATP) to the mast cell surface did not induce degranulation of the cell, a process known to occur concomitantly with histamine release induced by compound 48/80 and antigen-antibody reactions. Instead, reversible configurational changes of the cell membrane gradually occurred without any striking changes in the over-all cell volume. Swelling was observed after incubation of mast cells with ADP as well as by intracellular electrical manipulations with pipettes filled with H3PO4. The reversal of swelling was also demonstrated. The results are discussed in relation to the known histamine-releasing effect of ATP on rat mast cells and the difference in the mechanisms of histamine release induced by ATP and degranulating agents are stressed.
ACETYL DISULFIDE, (CH3COS)2, AND BIS-(THIOACETOXY) AURATE (I) COMPLEX, Au(CH3COS)2–, HISTOCHEMICAL SUBSTRATES OF UNUSUAL PROPERTIES WITH ACETYLCHOLINESTERASE
Authors: KOELLE GEORGE B. , DAVIS RICHARD , DEVLIN MARTINA
Published: April 2011

The histochemical reaction for acetylcholinesterase (AChE) employing acetyl disulfide ([CH3COS]2, or AcDiS) as substrate and 0.006 M Pb(NO3)2 as capturing agent was characterized by (1) a sigmoid curve for velocity of hydrolysis versus substrate concentration and (2) a red precipitate (presumably [CH3COSS]2Pb) at sites of AChE at the motor endplates (MEP's) and (from spontaneous hydrolysis) in the supernatant solution. With the addition of 0.03 M thiolacetic acid (TA) to the incubation medium, which by itself produced little or no histochemical staining, the foregoing characteristics were changed to (1) a bell-shaped curve, with the peak at 0.003 M AcDiS, and (2) a black precipitate (presumably PbS) at the MEP's and in the supernatant solution; in addition, the velocity of hydrolysis was increased approximately 100-fold. Comparable differences were obtained with AcDiS as substrate when using ionic Au+ or Au(S2O3)2–3 as the capturing agent. Results are explained on the basis that in the absence of free heavy metal ion ( i.e., with the Pb[TA]2 or Au[S2O3]2–3 complex), AcDiS combines with AChE at both the anionic and esteratic sites, whereas, in the presence of Pb2+ or Au+, attachment of the substrate occurs only at the esteratic site of the enzyme. Results similar to the former type were obtained when the bis-(thioacetoxy) aurate (I) complex (Au[CH3COS]2– or Au[TA]2– served as both substrate and capturing agent. With both the AcDiS-Au(S2O3)2–3 and Au (TA)2– methods, extremely fine localization of AChE was obtained at the MEP's by electron microscopy.
NONSPECIFIC ESTERASES IN MOSQUITOS
Authors: FREYVOGEL THIERRY A. , HUNTER ROBERT L. , SMITH ELIZABETH M.
Published: April 2011

Esterase zymograms were prepared from 14 species and strains of Culicide mosquitoes by use of starch and polyacrylamide electrophoresis. The zymograms proved to be species- and strain-specific. Variations in the zymograms of individual mosquitos were demonstrated by use of the microgel technique. This technique is described. Sexual dimorphism was shown to occur in the zymograms of one species, Anopheles stephensi. The isozymes were characterized by their reactivity toward different substrates and inhibitors. Their distribution in the mosquito's organism was studied with the whole mount technique and with organ zymograms. The stomach is particularly rich in esterase isozymes. There is a close correlation of these esterases and the lipids known to occur in the stomach epithelium and the esterases may play a role in the digestion of the blood meal. Observations on the effect of storage of samples, at low temperature and while dried on the activity of some isozymes, are reported. It is also shown that certain isozymes apparently are activated by inhibitors.
FLUCTUATIONS IN THE ACID PHOSPHATASE ACTIVITY OF SPHEROSOMES IN GUARD CELLS OF CAMPANULA PERSICIFOLIA
Authors: SOROKIN HELEN P. , SOROKIN SERGEI
Published: April 2011

Acid phosphatase activity fluctuates in mature epidermal guard cells of Campanula persicifolia in response to changing light conditions. As demonstrated both by Gomori's method and by Burstone's simultaneous coupling azo dye technique in both unfixed and aldehyde-fixed epidermal strips, the enzyme is active in the spherosomes when the guard cells are flaccid and the stomata are either partially or fully closed. It is inactive when the cells are turgid and the stomata are fully open, regardless of whether opening had resulted from a photoactive or scotoactive process. In living preparations, such inactive spherosomes will gradually become active for acid phosphatase if turgid guarde clls are subjected to partial plasmolysis. Marked activity for acid phosphatase becomes manifest in the vacuoles as well as in the spherosomes of the guard cells if the plants are kept in the dark for many days. In senescent leaves the vacuoles of the guard cells are reactive even though the plants are grown in the light. The walls of the epidermal cells exhibit sporadic activity for the enzyme, but the walls of guard cells have not been observed to react. Nuclear staining sometimes is present after tissues are incubated in the Gomori medium, but it is considered to result from nonenzymic binding of lead on the nuclear surface. The spherosomes evidently are among the principal sites for acid phosphatase activity in plant cells. In guard cells the spherosomes are considered to be enzymically active when the cells are in a relatively catabolic phase of metabolism and to become inactive when the cells enter a relatively anabolic phase.
SELECTIVE STAINING OF NERVOUS TISSUE FOR LIGHT MICROSCOPY FOLLOWING PREPARATION FOR ELECTRON MICROSCOPY
Authors: BERKOWITZ LILLIAN R. , FIORELLO OLGA , KRUGER LAWRENCE , MAXWELL DAVID S.
Published: April 2011

The suitability and selectivity of several dyes have been tested on plastic-embedded sections of nervous tissue (0.5-1 µ) prepared for electron microscopy. Methods for plastic extraction and selective staining are described which provide satisfactory differentiation of principle cytologic features with good contrast and clarity of detail. The introduction of a hematoxylin "lake" has achieved the differential staining of oligodendroglia and astrocytes in cerebral cortex. The argyrophilia of cells and cell structure following 10-20% silver nitrate or silver nitrate in combination with ammoniacal silver permits identification of axon terminals.
HISTOCHEMISTRY OF MYELIN V. TRYPSIN-DIGESTIBLE AND TRYPSIN-RESISTANT PROTEINS
Authors: ADAMS C. W. M. , BAYLISS O. B.
Published: April 2011

Two protein classes can be identified histochemically in both central and peripheral myelin by means of their amino acid patterns, enzyme sensitivity and susceptibility to acid extraction. One protein class (proteolipid protein and neurokeratin) contains abundant tryptophan, some tyrosine and, possibly, some cystine-cysteine; it resists digestion with trypsin and extraction with acid. The other class is highly basic; it contains some tyrosine, but little tryptophan or cystine-cysteine; it is digested by trypsin and extracted with acid. The significance of these findings is discussed in relation to the myelin disintegration that is induced by proteolytic enzymes.
HISTOCHEMISTHY OF MYELIN VII. ANALYSIS OF LIPID-PROTEIN RELATIONSHIPS AND ABSENCE OF ACID MUCOPOLYSACCHARIDE
Authors: ADAMS C. W. M. , BAYLISS O. B.
Published: April 2011

Central nervous system and peripheral nerve myelin were studied with lipid and polysaccharide staining reactions, used in conjunction with enzyme digestions and solvent extractions. The colloidal iron (ferriphilic) and acridine orange reactions of myelin appeared to be due to an acidic sphingolipid (sphingomyelin and/or sulfatide). Acetone and trypsin unmasked the ferriphilic substance in fixed myelin, whereas phospholipase A increased the ferriphilia and acridine orange fluorescence of unfixed myelin. Both reactions were extinguished in fixed and unfixed myelin by preliminary extraction with choloroform-methanol. Trypsin removed considerably more lipids from unfixed than fixed myelin; it removed more cholesterol and less sphingomyelin than other lipids of fixed myelin. From these observations a model is presented of the intermolecular relationships between lipids and trypsin-resistant/trypsin-sensitive proteins in the myelin sheath.
RENSHAW ELEMENTS: LOCALIZATION AND ACETYLCHOLINESTERASE CONTENT
Authors: ERULKAR S. D. , NICHOLS C. W. , POPP M. B. , KOELLE G. B.
Published: April 2011

Fine microelectrodes filled with molar potassium acetate solution and saturated with fast green FCF dye have been used to record from and localize Renshaw elements in L7 of the cat spinal cord. Renshaw elements were identified electrophysiologically by their multiple spike responses to ventral root stimulation. Intracellularly recorded responses were obtained from 10 cells. After the recording procedures had been completed, dye was passed into the cell by applying a current through the electrode. Frozen sections were cut at 10 µ and stained for cholinesterases by the standard copper thiocholine method. Renshaw elements were found to be of extremely small size, apparently devoid of dendrites and in close proximity to motoneurons. Varying concentrations of acetylcholinesterase were found in the Renshaw elements. The distribution of these structures in the ventral horn of the spinal cord was more extensive than has been previously described. The question of their identification as interneurons or as the boutons of axon collaterals is discussed.
A HISTOCHEMICAL TEST FOR CITRULLINE ADAPTATION OF THE CARBAMIDO DIACETYL REACTION TO HISTOLOGIC SECTIONS WITH POSITIVE RESULTS IN PILOMATRIXOMAS (CALCIFYING EPITHELIOMAS)
Authors: HOLMES ELIZABETH J.
Published: April 2011

Slight quantitative differences exist in the chemical composition of various components and sheaths of the hair follicle. The one qualitative difference which has been found is the unique presence of the amino acid, citrulline, in the inner root sheath. Citrulline accounts for 6.1% of the dry weight of the sheath. In the past, Ehrlich's p-dimethyl aminobenzaldehyde reagent has been used to detect citrulline in the inner root sheath both chromatographically and histochemically. The histochemical color is a faint yellow and tryptophan interferes also, giving a positive reaction. A more specific test is given by Fearon's diacetyl monoxime reagent which reacts in hot acid solution with carbamido compounds. In the present investigation the carbamido diacetyl reaction was adapted for histochemical study by use of a silicone rubber adhesive for tissue sections that would withstand boiling hydrochloric acid. The reaction colors the inner root sheath a bright orange and when applied to pilomatrixomas is positive in some of the keratinous areas.
OSMIUM-CONTAINING COMPOUNDS WITH MULTIPLE BASIC OR ACIDIC GROUPS AS STAINS FOR ULTRASTRUCTURE
Authors: SELIGMAN ARNOLD M. , WASSERKRUG HANNAH L. , DEB CHANDICHARAN , HANKER JACOB S.
Published: April 2011

Osmium coordination compounds were prepared with a variety of acidic or basic ligands to produce water-soluble, brown-black monomers and occasional polymers bearing multiple basic or acidic groups. These colored osmium-containing substances served as basic or acidic stains in light microscopy that produced staining results similar to the familiar acidophilic or basophilic dyes. When ultrathin sections of glutaraldehyde-fixed, Araldite-embedded tissues were incubated in aqueous solutions of these osmium-containing stains for 3-18 hr, good contrast in the electron microscope was given to acidic or basic ultrastructures. The potentialities of the stains are illustrated and some results are correlated with chemical features of the stains.
CYTOCHEMICAL DEMONSTRATION OF ORNITHINE CARBAMOYLTRANSFERASE ACTIVITY IN LIVER MITOCHONDRIA OF RAT AND MOUSE
Authors: MIZUTANI AKIRA
Published: April 2011

In order to demonstrate ornithine carbamoyltransferase activity cytochemically, thin slices of liver and kidney of rat and mouse were fixed in cold acetone or formol-calcium, and incubated in a medium containing l-ornithine, carbamoyl phosphate, Tris-maleate buffer (pH 7.2), lead nitrate and sucrose. The specific reaction product occurred in the mitochondria of the hepatocytes only, and not in other cells of the liver or kidney. The specificity of the reaction was supported by the following observations. (1) The mitochondrial reaction was not obtained in sections incubated in a medium from which ornithine was omitted. (2) Other amino acids gave no reaction. (3) p-Chloromercuribenzoate suppressed the reaction. (4) The hepatocytes of chick (uricotelic) did not give the reaction. A nonspecific reaction in lysosomes and brush borders is caused probably by acid and alkaline phosphatase activities.
DETERMINATION OF GLUTATHIONE REDUCTASE ACTIVITY IN MICROGRAM SAMPLES OF TISSUE, QUANTITATIVE HISTOLOGIC DISTRIBUTION OF THE ENZYME IN THE RAT ADRENAL AND EFFECT OF ADRENOCORTICOTROPIN
Authors: SCHOR NORBERTO A. , GLICK DAVID
Published: April 2011

A fluorometric method for determination of glutathione reductase activity in microgram samples of tissue, i.e., microtome sections, based on measurement of the decrease of reduced nicotinamide adenine dinucleotide phosphate due to its oxidation on reaction with oxidized glutathione, was developed and applied to the quantitative histologic distribution of the enzyme in the adrenal gland of the rat. Single subcutaneous injections of adrenocorticotropin in saline solution (25 mg/kg) produced little change of enzyme activity in any of the histologic zones, although there was some tendency for the peak activity to shift from fasciculata to the fascicular-reticular border region. The possible interrelationship of glutathione reductase with ascorbic acid and reduced nicotinamide adenine dinucleotide phosphate in adrenal function was considered.
ABSORPTION OF HORSERADISH PEROXIDASE BY THE MUCOSAL CELLS OF THE DUODENUM OF MOUSE I. THE FASTING ANIMAL
Authors: HUGON J. S. , BORGERS M.
Published: April 2011

The uptake of horseradish peroxidase was studied in the duodenum of fasting mice. Five, 7 or 3 x 5 mg were injected in the tail vein. The protein rapidly diffused into the intercellular spaces of the villus and crypt epithelium. Subsequently, the marker was segregated in the dense bodies of the absorbing cells, particularly in the apical part of the cells. The tracer was never observed in the lumen of the gut but was blocked at the zonula occludens or degraded in the dense bodies. A diffuse staining of some cells was observed, and control experiments were made to test the possibility of artifacts.
HYDROLYTIC ENZYMES DURING SPERMATOGENESIS IN RAT AN ELECTRON MICROSCOPIC AND HISTOCHEMICAL STUDY
Authors: POSALAKI ZOLTAN , SZABÓ DEZSÖ , BÁCSI ERNÖ , ÖKRÖS ISTVÁN
Published: April 2011

The localization of lipids and the activities of nonspecific esterase, aryl sulfatase and acid phosphatase were studied in different stages of spermatogenesis in rats. In addition, the distribution of acid phosphatase activity was demonstrated electron histochemically. The spermatogenetic cycle was divided into two phases—corresponding to the first and the last four stages of Roosen-Runge-Giesel (RG) classification. Spermatids in the first phase contained abundant endoplasmic reticulum with rosette formation and well developed Golgi apparatus with numerous vesicles. They displayed high activity of hydrolytic enzymes but contained no appreciable amount of lipids. The Sertoli cells contained large lipid granules but showed minimal enzyme activity. During the second phase reduction of the cytoplasm of spermatids with fragmentation of the endoplasmic reticulum and Golgi lamellae, accumulation of lipids, aggregation of ribonucleo-protein particles, formation of residual bodies and marked decrease of enzyme activity were seen. The Sertoli cells contained large mitochondria, well developed endoplasmic reticulum and numerous dense bodies and revealed high activities of hydrolytic enzymes and rapid depletion of lipids. These ultrastructural and histochemical findings suggested an interaction between the Sertoli cells and the developing spermatids which probably contributed to the regulation of spermatogenesis.