bluuebunny/crossref_metadata_2025 huggingface dataset:
THE VITAMIN A CONTENT AND RETINOL ESTERIFYING ACTIVITY OF A KUPFFER CELL FRACTION OF RAT LIVER
Authors: HORI SAMUEL H. , KITAMURA TAKAKO
Published: April 2011

Kupffer cells were isolated from normal and A-hypervitaminotic rats, and their vitamin A content and retinol esterifying activity were assayed in order to examine whether the Kupffer cells actually participate in the storage of vitamin A. The intrahepatic distribution of vitamin A was also studied using frozen sections of fresh and formalin-fixed livers by means of the fluorescence microscope. Both quantitative and qualitative data indicate that Kupffer cells are not the main site of vitamin A storage. The fluorescence of vitamin A and its reactivity to SbCl3 decreased drastically after formalin treatment.
LOCALIZATION OF PEROXIDASE ACTIVITY IN THE DEVELOPING SUBMANDIBULAR GLAND OF NORMAL AND ISOPROTERENOL-TREATED RATS
Authors: YAMASHINA SHOHEI , BARKA TIBOR
Published: April 2011

The localization of peroxidase activity was studied in the developing submandibular gland of normal and isoproterenol-treated rats using a diaminobenzidine-H2O2 method. During postnatal development peroxidase activity was localized in the proacinar and acinar cells. The proacinar cells were characterized by the presence of polymorphic secretory granules that gave a strong peroxidase reaction, particularly in the glands of 1-day-old rats. In addition to the secretory granules, enzyme activity was demonstrated in the rough endoplasmic reticulum and nuclear envelope. The terminal tubule cells and duct cells were devoid of peroxidase activity. The secretory granules in the fully developed acinar cells revealed little or no enzyme activity. Isoproterenol stimulated the secretion of the peroxidase-positive granules from the proacinar cells. The stimulation was followed by a reaccumulation of peroxidase-positive secretory material. During this process enzyme activity was demonstrable in the Golgi complex. Isoproterenol had no effect on the terminal tubule cells. A less effective depletion of the secretory granules from the proacinar cells was seen after pilocarpine administration. Chronic administration of isoproterenol to 5-day-old rats led to an acceleration of the differentiation of acinar cells and to a hypertrophy of the gland, without significant change in the localization of peroxidase activity.
DETERMINATION OF l-HISTIDINE IN NANOGRAM AMOUNTS AND THE QUESTION OF ITS OCCURRENCE IN MAST CELLS
Authors: WILKINSON DAVID I. , GLICK DAVID
Published: April 2011

In an attempt to clarify the question of whether histidine is stored in the mast cell for coversion to histamine or whether the rate of conversion is rapid enough to prevent accumulation of histidine so that the rate-limiting step is the histidine uptake, it was found that no histidine was demonstrable in rat peritoneal mast cells by either quantitative analysis or paper chromatographic detection. Microadaptation of Hassall's method, based on conversion of l-histidine by histidase to urocanic acid and measurement of the latter by its absorbance at 277 nm, was made to permit determination of histidine in nanogram amounts in the presence of histamine. This adaptation was found reliable when compared with the o-phthalaldehyde method in estimation of l-histidine in serum and in insulin hydrolysate, and then it was applied to analysis of mast cells before and after l-histidine uptake in vitro. The adaptation should be generally useful in microanalysis of l-histidine in histologically and cytologically defined samples.
HISTOCHEMICAL AZO COUPLING OF PROTEIN HISTIDINE BRUNSWIK'S NITRATION METHOD
Authors: LILLIE R. D. , DONALDSON P. T.
Published: April 2011

The xanthoproteic reaction is accomplished with only 20% HNO3 with glacial acetic acid (HAc) as the solvent; in water 40% HNO3 is required. Tyrosine and 3-mononitrotyrosin readily azo-couple in alkaline solution with fresh p-nitrodiazobenzene in vitro; 3,5-dinitrotyrosine does not. In vitro p-nitrodiazobenzene at pH 8.5 does not couple with histidine, tryptophan or tyrosine after overnight nitration in tetranitromethane (TNM)-pyridine-0.1 N HCl 1:20:40. Histochemical nitration of tissue adequate to prevent the p-diazobenzenesulfonic acid, pH 1-0.02% azure A sequence reaction of hair medulla and arterial elastin of man, dog and rodents can be achieved by 40% HNO3 in glacial acetic acid-acetic anhydride (Ac2O) mixtures. Acetic anhydride should be 10% only, to restrict evolution of brown oxides of nitrogen and prevent undue section losses. Exposures of 4 hr at 3°C to 4:5:1 HNO3:HAc:Ac2O are effective and well tolerated. Nitration is also effectively accomplished by 6-hr 25°C exposures to mixtures of 1% tetranitromethane-pyridine and two volumes of water (pH 8.2) or 0.1 N HCl (pH 6.6). Even 10% TNM in dry pyridine and 2.5% in 16.3% pyridine alcohol (6 hr), 3°C exposures gave only partial tyrosine blockades. The pyridine-water TNM mixtures also prevented the Morel-Sisley tyrosine reaction and, with greater exposures, the postcoupled benzylidene indole reaction of tryptophan as well. Sites reactive to diazobenzenesulfonic acid-azure A after the best nitrations are probably assignable largely to histidine, though the presence of some unblocked tryptophan, purines and other reactive substances must be considered.
VITAL STAINING OF MAST CELLS WITH RUTHENIUM RED
Authors: LAGUNOFF DAVID
Published: April 2011

Ruthenium red, at a concentration of 0.005%, stains extracellular but not intracellular mast cell granules. This vital stain can be used to demonstrate that after degranulation with polymyxin B sulfate many granules retained within the domain of the cell have been extruded into the extracellular space. This interpretation is confirmed by electron microscopy. Indirect evidence indicates that some extruded granules may be recovered by the cell and sequestered in an intracellular space not accessible to ruthenium red.
ULTRASTRUCTURAL VARIATIONS IN N,N'-BIS(4-AMINOPHENYL)-N,N'-DIMETHYLETHYLENEDIAMINE (BED) OXIDASE LOCALIZATION IN RAT LIVER AND PAROTID GLAND WITH VARIATION IN LENGTH OF FIXATION
Authors: SHANNON W. ALLEN , SELIGMAN ARNOLD M.
Published: April 2011

The localization and reactivity of a terminal oxidase which oxidizes N,N'-bis(4-amino-phenyl)-N,N'-dimethylethylenediamine (BED) were studied in rat liver and parotid gland after varying the concentration of formaldehyde fixative and the length of fixation. Reaction product was observed in mitochondrial outer compartments, smooth elements of rough endoplasmic reticulum, some Golgi lamellae, perinuclear membranes and cytoplasmic membranous structures often associated with mitochondria. A reaction also occurred in the limiting membrane and, to some degree, in the material comprising the secretory granules of the parotid. The reaction in the mitochondrial outer compartment was extremely formaldehyde-sensitive. Controls in which diaminobenzidine (DAB) was substituted for BED showed reaction only in mitochondrial cristae and outer compartments, whereas controls without either reagent showed no reactivity.
FINE STRUCTURAL LOCALIZATION OF ACYLTRANSFERASE ACTIVITY IN RAT HEPATOCYTES
Authors: BENES FRANCINE M. , HIGGINS JOAN A. , BARRNETT RUSSELL J.
Published: April 2011

A method for the fine structural localization of acyltransferases catalyzing the transfer of acyl groups from palmityl-coenzyme A (CoA) to α-glycerophosphate based on the formation of a heavy metal precipitate at the site of CoA release is described. In this method CoA released through the action of the enzyme is oxidized by potassium ferricyanide, which is reduced to potassium ferrocyanide and precipitates in the presence of cupric ions. Acyltransferase activity was found to survive both fixation in glutaraldehyde and incubation in the presence of the capture reagent system used for cytochemistry. Reaction product marking the enzyme activity was located within the cisternae of the rough endoplasmic reticulum and to lesser, but significant extent, within the membranous envelope of the smooth endoplasmic reticulum. Reaction product was not associated to any significant extent with other membranous organelles. The significance of these observations in terms of the role of acyltransferases in liver function is discussed.
QUANTITATIVE ASSAY OF ESTERASES IN END PLATES OF MOUSE DIAPHRAGM BY ELECTRON MICROSCOPE AUTORADIOGRAPHY
Authors: SALPETER MIRIAM M. , PLATTNER HELMUT , ROGERS ANDREW W.
Published: April 2011

The inhibitor diisopropyl fluorophosphate (DFP) reacts covalently with a number of enzymes including acetylcholinesterase (AChE). The compound pyridine-2-aldoxime methiodide reactivates phosphorylated AChE faster than the other DFP-sensitive sites. Mouse diaphragm was incubated with 3H-DFP, and the radioactivity in the motor end plates was assessed by electron microscope autoradiography. Both the sites which are rapidly reactivated and those which are slowly reactivated by pyridine-2-aldoxime methiodide were evaluated. The distribution of silver grains was compatible with several alternative hypotheses, i.e., that both AChE and other DFP-sensitive enzymes are located on or around the postsynaptic membrane alone, are distributed uniformly throughout the synaptic cleft and are associated equally with both pre- and postsynaptic membranes. This distribution of developed grains was identical with that found in the larger end plates of mouse sternomastoid. The values for the number of molecules of 3H-DFP, whether expressed in terms of square micra of junctional membrane or of cubic micra of synaptic cleft, were also practically identical with those found for sternomastoid. These similarities between the diaphragm, a red muscle, and the sternomastoid, which is predominantly white, suggest that there may be a constant relationship between the sites that bind DFP and the unit dimensions of the subneural compartments concerned.
HISTOCHEMICAL AND CHEMICAL DEMONSTRATION OF REACTIVE ALDEHYDE GROUPS IN ELASTIN
Authors: ABATANGELO GIOVANNI , DAGA DANIELA , GIORDANO RENZO
Published: April 2011

To ascertain the presence of free aldehyde radicals in elastic fibers, a histochemical method using the Sawicki reagent (N-methyl-benzothiazolone hydrazone) was employed. Three separate studies were conducted on the elastic tissue of bovine ligamentum nuchae. The elastic fibers were tested (a) in native state, (b) after extraction using an autoclave method and (c) after hot NaOH treatment. Free aldehyde radicals were detected histochemically in native and treated fibers, indicating a close attachment of the carbonyl groups to the fibrous-protein component of the elastic material. Pretreatment of the sections with NaBH4 or with NaHSO3 abolished the staining, indicating that the aldehydes in elastin can be reduced or blocked by the addition of bisulfite compounds. The presence of free carbonyl compounds in elastin was confirmed by chemical data with a new chromatographic technique. Using QAE Sephadex column pretreated with sodium bisulfite, aldehyde-bearing peptide fractions could be isolated from the elastin fibers.
LIGHT AND ELECTRON MICROSCOPIC LOCALIZATION OF ANTIGENS IN TISSUES EMBEDDED IN POLYETHYLENE GLYCOL WITH A PEROXIDASE-LABELED ANTIBODY METHOD
Authors: MAZURKIEWICZ JOSEPH E. , NAKANE PAUL K.
Published: April 2011

Tissues embedded in polyethylene glycol (PEG) were used for the immunocytochemical localization of cellular antigens at the light and electron microscopic level. For demonstration of the method, growth hormone and luteinizing hormone were localized in the anterior pituitary gland of the rat embedded in PEG, using peroxidase-labeled antibodies. PEG is water-soluble, has a low melting temperature yet is firm enough to be sectioned at room temperature. Preservation of cellular ultrastructure is good. The use of PEG-embedded tissue permits precise correlation of light and electron microscopic observations of the same tissue section.
CHANGES IN THE SOLUBILITY OF IMMUNOGLOBULINS AFTER FLUORESCENT LABELING AND ITS INFLUENCE ON IMMUNOFLUORESCENT TECHNIQUES
Authors: ARNOLD W. , MAYERSBACH H. V.
Published: April 2011

The physical and chemical properties of fluorescein isothiocyanate- and dansylchloridelabeled immunoglobulins were investigated. A marked change in solubility after labeling was noted. Depending on the time and on the type of fluorescent labeling rabbit and bovine immunoglobulin G showed different tendencies for precipitate formation. Increasing the molar fluorescein-protein ratio as well as lowering the pH of the conjugate solutions caused pronounced insolubilization. An increase in the NaCl concentration up to 0.5 M had no significant effect on precipitate formation. A simple method for the elimination of nonspecific staining is given. The method is based on the fact that overlabeled components of fluorescein isothiocyanate conjugates tend to precipitate; this can be initiated and furthered by lowering the pH of the conjugate solution.
DEMONSTRATION OF INTRACELLULAR CARRAGEENAN BY A FLUORESCENT ANTIBODY TECHNIQUE IN GRANULOMA TISSUE
Authors: RICHER SUZANNE M. , MCCANDLESS ESTHER L.
Published: April 2011

Intracellular localization of λ-carrageenan in the carrageenan granuloma has been demonstrated by a specific fluorescent antibody to the polymer. In general, the fluorescent antibody staining correlated with the intracellular metachromatic staining. The fluorescent antibody technique did not confirm that the extracellular metachromasia was due to λ-carrageenan.
CHANGES IN DISTRIBUTION OF ENZYMES WITHIN THE LIVER LOBULE DURING ADAPTIVE INCREASES
Authors: WELSH FRANK A.
Published: April 2011

Quantitative determination of three enzymes in periportal and centrilobular cells of the liver lobule were made following experimental treatment to alter enzyme levels. Glutamic pyruvic transaminase was initially five times higher in periportal cells and increased 3-fold during a 3-day fast in periportal cells, and 4-fold in centrilobular cells. Tyrosine transaminase was initially distributed uniformly. Four hours after giving hydrocortisone it had increased somewhat more (6-fold) in portal cells than in central cells (4-fold). Preliminary results for glucose-6-phosphate dehydrogenase suggest that greater increases occur in central cells than portal cells during adaptation (recovery from fasting).
MECHANISMS OF IRON II AND IRON III SEQUENCE HEMATOXYLIN STAINS
Authors: LILLIE R. D. , PIZZOLATO P.
Published: April 2011

Undifferentiated Fe II and Fe III hematoxylins yield blue to black nuclei, keratin, keratohyalin, enterochromaffin, Paneth and eosinophil leukocyte granules, erythrocytes, myelin and bile casts, gray to blue smooth and striated muscle, variable collagen and elastin staining and lighter gray cytoplasms. Staining is more intense with formol-free fixations. Staining is not prevented by acid hydrolysis which abolishes the Feulgen reaction and cationic dye staining. Acetylation and sulfation have little effect. Methylation and nitrous acid deamination sufficient to prevent cationic and anionic dye staining, respectively, prevent staining of erythrocytes, muscle, cytoplasm and zymogen granules but not that of nuclei, keratin, keratohyalin, trichohyalin, Paneth and eosinophil granules. These blockade effects are more complete with Fe II than Fe III. Saponification after methylation restores staining. With sublimate, Carnoy, alcohol and methanol-chloroform fixations, 2 N NaNO2/HAc deaminates in 1 hr; with formol 18-24 hr or more may be required. The sites which stain with Fe II hematoxylin after adequate deamination have all given positive Sakaguchi reactions by α-naphthol, 8-hydroxyquinoline and 2,4-dichloro-l-naphthol technics . The new β-naphthoquinone-4-SO3Na method also shows the same sites. The combined deamination, Fe II, hematoxylin technic merits further investigation as a possibly specific method not entailing hypochlorite or strong alkali and yielding stable blue to black stains of protein-bound arginine. Without deamination it appears that lysine, hydroxylysine and histidine and histamine should also react. Mast cells are often readily identified in blue or violet-black but are not found after deamination. Alkaline benzil, 9,10-phenanthrenequinone, 1,2-cyclohexanedione and glyoxal treatments irregularly weaken the deamination-resistant Fe II hematoxylin staining, though they do not prevent it. Low reactivity of hair cortex and coarse collagen bundles (requiring higher iron concentrations) is assigned to lower penetrability. Goblet cell, salivary gland, gastric and other mucins, pepsinogen granules, lipofuscins and cutaneous melanins do not stain with either Fe II or III sequence hematoxylins, either directly, after periodic acid oxidation, or after aminoisophthalic acid condensation at periodic acid-oxidized gastric mucin. Thus carboxylic, sulfuric and sulfonic acid and aldehyde sites are excluded. With the acylation effects, hydroxyl, tyrosyl and histidyl sites also appear to be excluded, and iron binding appears to be largely restricted to lysyl and arginyl sites. Impairment of the β-naphthoquinone-4-SO3Na-arginine reaction by prior iron binding supports the arginyl participation.
IMMUNOFLUORESCENT LOCALIZATION OF α2u-GLOBULIN IN THE HEPATIC AND RENAL TISSUES OF RAT
Authors: ROY ARUN K. , RABER DOUGLAS L.
Published: April 2011

α2u-Globulin is an androgen-inducible rat urinary protein of hepatic origin. Examination of adult male rat liver sections with rhodamine-labeled anti-α2u-antiserum showed that the liver parenchymal cells are the site of α2u-globulin synthesis and that all of the parenchymal cells contain this protein. Immunofluorescent localization of α2u-globulin in male kidney sections revealed the existence of the protein in the cells of proximal convoluted tubules, in the loop of Henle as well as in the distal convoluted tubule. The collecting ducts did not bind the fluorescent labeled antibody. Intraperitoneally administered rhodamine-labeled α2u-globulin was found to be localized only in the kidney tubules in particulate form. It is concluded that α2u-globulin after its synthesis and release from the hepatic parenchymal cells is filtered into the renal tubular lumen and that both kinds of convoluted tubules as well as the loop of Henle are actively involved in α2u reabsorption.
IMMUNOFLUORESCENT ANTIBODIES TO BOVINE KERATOHYALIN AND IMMUNOLOGIC CONFIRMATION OF HOMOLOGY
Authors: GUSS STEPHEN B. , UGEL ARTHUR R.
Published: April 2011

Extraction of bovine hoof epidermis with 1.0 M potassium phosphate buffer (pH 7.0) and dialysis of the extract against distilled water results in the formation of macroaggregates. Fractionation of solubilized macroaggregates on polyacrylamide gels reveals a 13-member oligomeric series. Indirect immunofluorescence with antisera from rabbits immunized with either macroaggregates or gel slices with individual oligomers demonstrates specific fluorescence over keratohyaline granules. The reaction is tissue-specific, but antibodies cross-react with goat, sheep, wildebeest and rabbit keratohyaline granules, although not against human, rat or guinea pig keratohyalin. Immunofluorescent methods show that the macroaggregates are a component of keratohyalin and that the 13 oligomers are antigenically identical.
A HISTOCHEMICAL METHOD FOR THE DEMONSTRATION OF AMINOPEPTIDASE B ACTIVITY
Authors: MÄKINEN KAUKO K. , PAUNIO KEIJO U.
Published: April 2011

A histochemical method for the demonstration of aminopeptidase B activity is presented. The method is based on the specific activation of the enzyme by chloride ions present in physiologic concentrations (0.9% or 0.154 M) in the incubating medium, on the inhibition by phosphate ions and on the use of N-l-arginyl-2-naphthylamine as substrate. The liberated 2-naphthylamine was coupled with fast blue B.
INFERRED POLYMER SIZE OF AMYLOSE DURING EARLY SYNTHESIS BY PHOSPHORYLASE IN ZEBRINA
Authors: WHEELER GEORGE E.
Published: April 2011

Using an indirect method, it proved possible to follow certain aspects of phosphorylase-catalyzed new starch (amylose) synthesis during its early course in ground parenchyma cells of Zebrina internodes. The new starch stained purple to bluish purple (not red) with I2-KI from time of earliest detection, indicating long rather than short polymers. With increasingly longer incubation periods, there was an intensifying of the same colors but no appreciable change in hue. These results were interpreted as indicating "single chain" synthesis; i.e., new molecules, already started, are completed to full length before the synthesis of additional new ones is begun.
FLUORESCENCE HISTOCHEMICAL AND MICROSPECTROFLUOROMETRIC EVIDENCE OF TRYPTOPHYL PEPTIDES IN THYROID C CELLS OF CAT AND PIG
Authors: HÅKANSON R. , OWMAN C. , SUNDLER F.
Published: April 2011

In the thyroid gland of cat and pig, combined formaldehyde-ozone treatment induces a strong greenish yellow fluorescence (maximal excitation/emission, 370/510 mµ) in cells identified as C cells by argyrophil staining. With the formaldehyde-ozone treatment, authentic tryptamine as well as tryptophyl peptides in protein droplet models gave a similar, strong fluorescence. Combined formaldehyde-HCl treatment gave only moderate C cell fluorescence. With this treatment the fluorescence yield of tryptophyl peptides in protein droplet models was low, whereas that of tryptamine was high (of the same magnitude as after formaldehyde-ozone). Chemical analysis failed to reveal tryptamine in extracts of thyroid glands. Together, the microspectrofluorometric and chemical results indicate that the fluorogenic compound in the C cells of cat and pig is a tryptophyl peptide rather than tryptamine.
ESTERASE ISOZYMES FROM RABBIT SYNOVIAL FLUIDS NORMAL AND ARTIFICIAL JOINTS
Authors: ESTABROOKS LEWIS , SCHIFF ROBERT
Published: April 2011

Acetyl- and butrylesterase isozymes from knee and temporomandibular joint synovial fluid, serum and red cells of New Zealand White rabbits were compared by starch gel electrophoresis to the isozymes obtained from a false joint fluid produced in three animals by interposing a sheet of Silastic between segments of a fracture of the left subcondylar area. All fluids studied had two major regions of esterase activity that migrated at rates comparable to systems 1 and 2 of the red cell. However, inhibition tests differentiated the body fluid esterases from those of the red cell. Knee, temporomandibular joint and false joint fluids had similar zymograms. Analysis of the false joint fluid reinforced light microscopic findings that not only does a synovial membrane differentiate de novo but the membrane produces or maintains a fluid similar in esterase activity to the normal temporomandibular joint.
IN VIVO INCORPORATION OF 3H-GALACTOSE BY THYROID FOLLICULAR CELLS OF THE RAT, AS SHOWN BY ELECTRON MICROSCOPIC RADIOAUTOGRAPHY
Authors: HADDAD A.
Published: April 2011

Radioactive galactose was injected intravenously into rats and localized in thyroid follicular cells by electron microscopic radioautography at intervals ranging from 2.5 to 30 min after injection. The galactose label was mostly present in the Golgi apparatus at 2.5 min, with some of it in the adjacent rough endoplasmic reticulum. By 30 min, the label was found in apical vesicles and colloid. It was concluded that galactose is added to the carbohydrate side chains of incomplete thyroglobulin molecules during their travel through the cisternae of the endoplasmic reticulum into the Golgi apparatus; the uptake begins as this organelle is approached, but predominates within it. The thyroglobulin molecule which has thus been labeled is transported by the apical vesicles to the colloid.
OBJECTIVE CELL IMAGE ANALYSIS
Authors: BARTELS P. H. , BIBBO M. , WIED G. L. , BAHR G. F.
Published: April 2011

A system of computer programs processes digitized images of cells. The gray value arrays are considered as a two-dimensionally extended stochastic process. Supervised learning is employed to estimate the parameters of this process in different cell types, to derive classification algorithms and to compute a likelihood for correct classification for each cell. These were found to range from 10:1 to 106:1 for tumor cells from the female genital tract. A data bank for tumor cells has been established. Nonsupervised learning algorithms are used to examine sets of cells for homogeneity. Synthesized cell images of known stochastic properties can be generated to test the completeness of the derived classification rules. Under development are programs defining the changes observable in images of a given cell type during a disease process, by the deformation of the covariance matrix of image properties.
AUTOMATION OF THE FLUORESCENT TREPONEMAL ANTIBODY-ABSORPTION TEST
Authors: KAUFMAN GERALD I. , NESTER JAMES F.
Published: April 2011

The feasibility of automating the decision-making tasks of the human observer in the microscopic determination of Fluorescent Treponemal Antibody-Absorption test results was investigated. An argon-ion laser scanner-optical pattern recognition computer instrument was utilized. The results indicate that the use of area, perimeter and (perimeter)2/area parameters allows the identification of Treponema pallidum as distinguished from other, nonspecific fluorescing objects. It was concluded that complete automation of the Fluorescent Treponemal Antibody-Absorption test is feasible with existing technology.
THE EFFECT OF SOME FACTORS ON THE HISTOCHEMICAL DEMONSTRATION OF LIVER GLYCOGEN PHOSPHORYLASE ACTIVITY
Authors: LINDBERG L.-A. , PALKAMA A.
Published: April 2011

Existing methods for the histochemical demonstration of liver phosphorylase activity were investigated for possible application to a study of enzyme activity using the electron microscope as well as the light microscope. It was found that lead, in concentrations recommended in the literature, cannot be used as the precipitating agent because of its inhibitory effect on phosphorylase activity. The histochemical method based on the demonstration of enzymatically formed glycogen can be improved by adding ethylenediaminetetraacetic acid to the incubating solution. However, the addition of 3',5'-cyclic adenosine monophosphate is not as effective. It is not necessary to add glycogen to the incubating solution. In our opinion enzyme activity cannot yet be demonstrated electron microscopically by the precipitation of liberated phosphate. This is due to the inhibitory effect of high lead concentrations. Under the light microscope the method based on the demonstration of enzymatically formed glycogen is most reliable.
A VERTICAL MICROSYSTEM FOR DISCONTINUOUS ELECTROPHORESIS OF INSECT TISSUE PROTEINS USING THIN SHEETS OF POLYACRYLAMIDE GEL
Authors: BEEN ANITA C. , RASCH ELLEN M.
Published: April 2011

Proteins extracted from individual pairs of salivary glands or other larval tissues of Sciara coprophila (Diptera) were separated in a vertical microsystem for discontinuous electrophoresis using thin sheets of polyacrylamide gel cast in multiple layers of varying pore size. After electrophoresis at 150 volts for 40 min, gels were stained ( a) for total proteins with Coomassie brilliant blue, ( b) for glycoproteins with the periodic acid-Schiff reaction, ( c) for lipoproteins with Sudan black B or ( d) for nonspecific esterases with fast blue RR as coupler and α-naphthol acetate as substrate. Sequential application of these reactions to individual gel sectors permitted direct comparisons of protein profiles for 15-20 different samples of tissue extracts carried on a single gel sheet in adjacent lanes and thus subjected to identical conditions of electrophoresis. Representative photographs and densitometric scans are presented to show the suitability of thin gel sheets for autoradiography and for both qualitative and quantitative evaluation of tissue-specific differences in patterns of protein banding found for salivary gland cells, the gastric ceca, or the hemolymph of individual Sciara larvae sacrificed at particular stages of fourth instar development. Innovative details of methodology are presented, including the use of a microspectrophotometer to scan electropherograms of insect proteins and several types of human blood serum.
ELECTRON PROBE ANALYSIS OF THE CALCIUM DISTRIBUTION IN CELLS OF THE EMBRYONIC CHICK CHORIOALLANTOIC MEMBRANE II. DEMONSTRATION OF INTRACELLULAR LOCATION DURING ACTIVE TRANSCELLULAR TRANSPORT
Authors: COLEMAN JAMES R. , TEREPKA A. RAYMOND
Published: April 2011

Electron probe analysis of the embryonic chick chorioallantoic membrane reveals that calcium is located at discrete sites in the ectodermal cell layer of the membrane. The calcium loci are found at characteristic sites within distinctive "capillary-covering" cells. Evidence is presented that the calcium identified by electron probe analysis is that pool of calcium undergoing active transcellular transport. Mitochondria and/or endoplasmic reticulum do not appear to be responsible for the observed calcium sequestration. It is suggested that the calcium might be contained within endocytotic vesicles, and a hypothesis is advanced which could reconcile endocytotic transcellular transport by specialized cells with previously proposed models for intracellular calcium ion regulation.
THE FATE OF EXOGENOUS PEROXIDASE IN THE THYMUS OF NEWBORN AND YOUNG ADULT MICE
Authors: GERVIN SUSAN WINTER , HOLTZMAN ERIC
Published: April 2011

Horseradish peroxidase injected intraperitoneally into newborn and young adult mice is subsequently found in the thymus within the lumina of blood vessels and in perivascular areas. In the newborns, much peroxidase is also detectable throughout the thymic parenchyma; it is present in extracellular spaces and in pinocytotic vesicles and lysosomes in thymocytes and other parenchymal cells. In young adult mice, very little peroxidase escapes from the vicinity of blood vessels; cells resembling macrophages contain most of the tracer present outside of blood vessels and only those thymocytes located very near small venules or capillaries are exposed to peroxidase. The results suggest that the "blood-thymic" barrier is not simply a static set of structures that prevents penetration of potential antigens from the blood stream into the parenchyma; in young adult animals, active uptake of foreign molecules by macrophages and other cells is an important component of the barrier. In addition, the findings may shed light on aspects of the development of the immunologic system, since it appears, for example, that potential antigens can interact directly with far more cells in the newborn thymus than is true in the adult.
IMMUNOPEROXIDASE LOCALIZATION OF ANTIHAPTEN ANTIBODIES IN RATS RESPONDING UNEQUALLY TO ANTIGENIC STIMULATION
Authors: FRANÇOIS DOMINIQUE , ORIOL RAPHAËL , BINAGHI RUBEN A.
Published: April 2011

The present work reports on an electron microscopic study of the primary response of two strains of rats to the subcutaneous injection of the hapten 2,4-dinitrophenyl (DNP). When this substance is injected into the two strains used, there is either a weak or a strong production of circulating anti-DNP antibodies. Satellite lymph nodes were fixed in glutaraldehyde, incubated in a solution of horseradish peroxidase labeled with DNP and treated with diaminobenzidine + H2O2. A specific positive reaction was observed: ( a) in the perinuclear cisternae of numerous lymphocytes and lymphoblasts; ( b) in the perinuclear cisternae as well as in the peripheral cisternae of some plasmablasts; and ( c) in a recently described rare type of cell known as a "lymphoplasmacyte." In certain lymphocytes, there was an accumulation of the reaction product in the perinuclear cisternae, and sometimes in the peripheral cisternae, which, in some places, provoked a dilation of the cisternae. There was no significant qualitative difference between the two strains of rats.
QUARTZ FIBER ULTRAMICROBALANCE
Authors: MOSS M. L.
Published: April 2011

Design and operation of a quartz fiber cytoanalytical balance are described. The 2-µ fiber, 3-4 mm long, is mounted in a V-shaped notch in an aluminum slide for use on an ordinary microscope. The deflection is measured with the focusing adjustment by viewing through thin glass windows, which are flush with the surfaces of the slide. Since the opening to the balance faces away from the operator, weighings can be made without closing the balance and removing time manipulator needles employed for handling the specimens. Transverse sections of single muscle cells, 0.1 µg after freeze-drying, can be weighed to within 3% under ordinary atmospheric conditions.
MICROPHOTOMETRIC DETERMINATION OF ENZYME ACTIVITY IN SINGLE CELLS IN CRYOSTAT SECTIONS I. APPLICATION OF THE GEL FILM TECHNIQUE TO MICROPHOTOMETRY AND STUDIES ON THE INTRALOBULAR DISTRIBUTION OF SUCCINATE DEHYDROGENASE AND LACTATE DEHYDROGENASE ACTIVITIES IN RAT LIVER
Authors: NOLTE J. , PETTE D.
Published: April 2011

A gel film technique was adopted for kinetic enzyme activity determination in single cells using cryostat sections by means of a microscope photometer. The main principle of the method is a comparative activity determination, based on bipositional recording of initial reaction kinetics in two preselected measuring fields in the same tissue section. Systematic model experiments were performed to prove the fulfillment of the following conditions: ( a) validity of Beer's law; ( b) optimal concentrations of substrates, cosubstrates and dye within the reaction film, including evidence that the reaction rate is not limited by insufficient diffusion of these compounds; ( c) proportionality between local enzyme concentration or thickness of tissue sections and the recorded reaction rate; and ( d) specificity of the method as demonstrated by studying reaction rates in the absence of the substrate as well as in the presence of substrate and a specific inhibitor. The validity of the method was also examined by comparing the levels of succinate dehydrogenase activity in various rat tissues, as measured microphotometrically, with the enzyme levels as determined in homogenates. Microphotometric assays for nicotinamide adenine dinucleotide phosphate isocitrate dehydrogenase, lactate dehydrogenase and succinate dehydrogenase are described. Using these techniques it was found that: the periportal and central areas of the hepatic lobule in rat contain the same level of lactate dehydrogenase activity; succinate dehydrogenase activity is 1.6 times higher in the periportal area as compared to the centrilobular area. In experimental thyrotoxicosis, the ratio of enzyme activities in the periportal and central areas was 1.1.
LOCATION OF THE ANTIGEN, ANTIBODY AND ANTIGEN-ANTIBODY COMPLEXES IN DELAYED-TYPE HYPERSENSITIVITY SKIN REACTIONS TO HORSERADISH PEROXIDASE
Authors: STRAUS WERNER
Published: April 2011

The sites of intradermally injected horseradish peroxidase (HRP), of its antibody and of HRP-anti-HRP antibody complexes were investigated in the dermis of rabbits showing strong skin reactions of delayed hypersensitivity against HRP. Comparison was made with the location of intradermally injected HRP and its antibody in the dermis of immunized rabbits showing slight degrees of hypersensitivity of a nondelayed type and with the location of intradermally injected HRP in the dermis of nonimmunized animals. In all skin reactions of delayed hypersensitivity, a considerable proportion of the injected antigen was associated with collagen fibers, the reaction product appearing as "dots," "rodlets" and "strands" in linear array on individual collagen fibers in the interior of the bundles or as strands coating the outermost surface of the bundles. The reaction for the antibody was positive at the same sites. It was suggested that receptors for antigen-antibody complexes may be associated with collagen fibers. Free granular deposits containing antigen-antibody complexes seemed to originate from the collagen-associated granules after their enlargement and release. The extracellular granules often adhered to the antibody-positive surface membranes of lymphocytes. Other granular deposits (antigen-antibody complexes) seemed to be phagocytosed by monocytes (macrophages) and fibroblasts. In areas of tissue injury in the upper dermis, many mononuclear cells showed the reaction for the antibody on their cell surface. Aggregations of such cells were often located close to disintegrating bundles of collagen fibers. It was suggested that the encounter of the antigen and the antibody (antigen-antibody complexes) on the surfaces of cells and of collagen may trigger the release of cytotoxic factors. A few lymphocytes in the dermis of hypersensitive rabbits contained the specific antibody on their cell surface before skin lesions were elicited.
FLUOROMETRIC MEASUREMENT OF DEOXYRIBONUCLEIC ACID IN BONE MARROW CELLS THE MEASUREMENT OF MEGAKARYOCYTE DEOXYRIBONUCLEIC ACID
Authors: WESTE SANDRA M. , PENINGTON DAVID G.
Published: April 2011

The use of fluorochromes in the Feulgen reaction for the quantitative measurement of deoxyribonucleic acid (DNA) has been explored previously but is rarely applied. The occurrence of nonspecific fluorescence due to compounds other than DNA and the fading of the fluorochrome on excitation can cause variation in the measurements of nuclear fluorescence. The method of Ruch (in Introduction to Quantitative Cytochemistry, Wied, G. L., ed., Academic Press, 1966, p. 281) using auramine O as the fluorochrome has been modified to achieve improved nuclear fluorescence with a more linear rate of fading. A simple Zeiss microscope photometer has been used. Problems of instrumentation and optics, including lens changes during measurements on cells of widely varying sizes such as megakaryocytes, have been analyzed and correction factors have been calculated. Microfluorometric measurements of nuclear DNA are of the same order of accuracy as those made by integrating microdensitometry, without the need in the latter technique to compress large cells to a uniform thickness. Microfluorometry has been applied with the new staining method to analyze the ploidy distribution of bone marrow megakaryocytes.
ISOLATION OF PARIETAL CELLS FROM GLUTARALDEHYDE-FIXED RABBIT STOMACH
Authors: LEE SIN HANG
Published: April 2011

A method is introduced to isolate and purify parietal cells from the rabbit stomach which has been briefly perfused with 1% glutaraldehyde via the celiac artery. The fixed mucosa is initially digested with 1% trypsin and subsequently with 1% collagenase solution. The parietal cells are more resistant to the effects of these proteolytic enzymes than the other cell types in the gastric mucosa and can be recovered and purified by centrifugation in a discontinuous sucrose gradient. Glutaraldehyde fixation reduces the tendency of cell clumping and, therefore, facilitates isolation of the separated cells. The final yield from each adult rabbit stomach varies from 0.1 to 0.4 ml packed parietal cells of about 97% purity determined by nuclear counts. The nuclei and mitochondria of most of the isolated cells appear to be intact on electron micrographs, but the cell membrane is focally disrupted; the nuclear deoxyribonucleic acid (DNA) can be stained with Feulgen technique and extracted with perchloric acid for quantitative determination. Biochemical studies on incorporation of thymidine-3H into DNA of the parietal cells have been carried out in normal young adult rabbits and in rabbits under chronic histamine stimulation. The results show that there is no significant incorporation of thymidine-3H by mature parietal cells in any of the two groups of animals, although the whole mucosa DNA hydrolysates always exhibit a high specific activity.
CYTOFLUOROMETRIC DETERMINATION OF BASIC AND TOTAL PROTEINS WITH SULFAFLAVINE
Authors: LEEMANN U. , RUCH F.
Published: April 2011

The cytofluorometric determination of histones and total proteins with a single acid dye, Sulfaflavine, at the pHs 8.0 and 2.8, is described. Successive staining and measuring at the two pHs allow the determination of histones and total proteins in individual cells. Optimal staining conditions were determined; the specificity and the linearity of the staining were confirmed in model experiments (protein smears) and with different kinds of cells. The protein smears also served for calibration in absolute terms; the obtained protein values agreed with those calculated from dry weight measurements and with values from the literature. The influence of fixation (formalin and ethanol-acetic acid) and preparation, the reproducibility of the staining reaction and the inherent limitations of the method are discussed.
THE USE OF EXOGENOUS MYOGLOBIN AS AN ULTRASTRUCTURAL TRACER REABSORPTION AND TRANSLOCATION OF PROTEIN BY THE RENAL TUBULE
Authors: ANDERSON WINSTON A.
Published: April 2011

Exogenous myoglobin was used as an ultrastructural tracer to study its uptake, concentration and movement in successive segments of the rat nephron. The presence of myoglobin reaction product in intercellular gaps of the capillary endothelium and in fenestrae of peritubular capillaries was also shown. Myoglobin was visualized by a diaminobenzidine-H2O2 reaction, taking advantage of its peroxidatic activity. Myoglobin may be revealed in situ in formaldehyde-glutaraldehyde-fixed tissue and may be useful as a low molecular weight protein tracer.
NONOSMIOPHILIC TETRAZOLIUM SALTS THAT YIELD OSMIOPHILIC, LIPOPHOBIC FORMAZANS FOR ULTRASTRUCTURAL LOCALIZATION OF DEHYDROGENASE ACTIVITY
Authors: KALINA MOSHE , PLAPINGER ROBERT E. , HOSHINO YOSHINOBU , SELIGMAN ARNOLD M.
Published: April 2011

Monotetrazolium salts were designed and prepared which incorporate a phthalhydrazide moiety to make them lipophobic and a benzothiazole moiety to make them react with osmium tetroxide after they are reduced to the corresponding formazans. The tetrazolium salts themselves do not react with osmium tetroxide under the conditions used for the cytochemical demonstration of enzyme activities. Although all of the new formazans, when dissolved in dimethylformamide, were reoxidized to the tetrazolium salts by osmium tetroxide, they were not reoxidized by osmium tetroxide when they were dissolved in tetrahydrofuran or precipitated by reduction in tissue, but gave dark complexes. One of the tetrazolium salts, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT), was readily reduced by succinic dehydrogenase activity, gave a formazan which produced a dark osmium complex relatively rapidly and gave good localization of succinic dehydrogenase activity on the membranes of mitochondria of rat myocardial cells. Although this tetrazolium salt (BSPT) was not photoreduced by fresh cells of Elodea to stain chloroplasts as seen with nitroblue tetrazolium or distyryl nitroblue tetrazolium, the 5-p-nitrophenyl derivative of BSPT was photoreduced by chloroplasts. The preparation of BSPT and its use in demonstrating succinic dehydrogenase activity ultracytochemically is given here. The preparation and ultracytochemical use of the 5-p-nitrophenyl derivative in chloroplasts will be published later.
CHEMICAL CHARACTERIZATION OF UNCOATED ASBESTOS FIBERS FROM THE LUNGS OF ASBESTOS WORKERS BY ELECTRON MICROPROBE ANALYSIS
Authors: LANGER ARTHUR M. , RUBIN IVAN B. , SELIKOFF IRVING J. , POOLEY FRED D.
Published: April 2011

Lung tissues have been obtained from workmen with defined asbestos fiber exposure. These tissues have been prepared by the carbon extraction technique and examined with the electron microprobe analyzer. The uncoated fibers present in these specimens have been chemically characterized and compared with standard reference asbestos samples. The bulk chemistry of the fibers observed in lung tissues is similar to that of the reference fibers so that identification may be made. However, a statistical analysis of the measured emission characteristics from anthophyllite and amosite indicates that some magnesium loss has taken place. This loss appears to correlate with the magnesium content of the fibers. A slight iron increase was also noted in the same fibers, probably related to an incipient asbestos body formation.
NEW ASPECTS ON REACTION MECHANISMS IN THE FORMALDEHYDE HISTOFLUORESCENCE METHOD FOR MONOAMINES
Authors: BJÖRKLUND ANDERS , FALCK BENGT , LINDVALL OLLE , SVENSSON LEIF-ÅKE
Published: April 2011

The mechanisms of reactions underlying the fluorophore formation from indolylethylamines in the Falck-Hillarp histochemical formaldehyde method were investigated with the aid of thin layer chromatography and mass spectrometry of the fluorescent products formed in protein models and freeze-dried tissue. In the reaction of formaldehyde with tryptamine and 5-hydroxytryptamine, the main fluorophores formed were 3,4-dihydro-β-carboline and the 2-methyl-3,4-dihydro-β-carbolinium compound (from tryptamine), and 6-hydroxy-3,4-dihydro-β-carboline and the 2-methyl-6-hydroxy-3,4-dihydro-β-carbolinium compound (from 5-hydroxytryptamine). From these findings, it is concluded that the fluorophore formation in the Falck-Hillarp method proceeds as follows: In the first step of the reaction, the indolylethylamines react with formaldehyde to form low fluorescent 1,2,3,4-tetrahydro-β-carbolines. In a subsequent step, these products are converted to fluorophores in either of two ways: through an autoxidation to 3,4-dihydro-β-carbolines, or through a second, acid-catalyzed reaction with formaldehyde to yield 2-methyl-3,4-dihydro-β-carbolinium compounds. Experiments with radioactive tryptamine indicated that the two alternative fluorophore-forming pathways are of fairly equal importance. The latter of these fluorophore-forming reactions was not previously known, and the interesting properties and implications of this formaldehyde-induced and acid-catalyzed reaction are discussed.
PEROXISOMES IN DORSAL ROOT GANGLIA
Authors: CITKOWITZ ELENA , HOLTZMAN ERIC
Published: April 2011

Bodies with the morphologic and cytochemical characteristics of peroxisomes have been identified in the satellite and Schwann cells of rat dorsal root ganglia. They are membrane-delimited, round or oval structures which contain a moderately electron-dense matrix but lack a crystalline core. On incubation of the tissue in a cytochemical medium for demonstration of peroxisomes, these bodies show heavy deposits of reaction product. The reaction is inhibited by heating the tissue or by incubation in the presence of aminotriazole or dichlorophenolindophenol. In tissue incubated for acid phosphatase activity the bodies are not reactive, although lysosomes show reaction product.
DEVELOPMENT OF ENDOGENOUS PEROXIDASE IN FETAL RAT SUBMANDIBULAR GLAND
Authors: YAMASHINA SHOHEI , BARKA TIBOR
Published: April 2011

The prenatal development of endogenous peroxidase activity in the submandibular gland of rat was investigated by means of the diaminobenzidine-H2O2 histochemical method. The submandibular gland of a 16-day-old fetus was composed of cords of uniform, undifferentiated cells which contained no secretory granules and revealed no peroxidase activity. Peroxidase activity first appeared at the 17th day of gestation in the cisternae of the rough endoplasmic reticulum and nuclear envelope in a few cells. At the 18th day of gestation cells which exhibited reaction products in the rough endoplasmic reticulum and nuclear envelope also contained secretory granules with a strong peroxidase activity. During the last days of gestation the number of peroxidase positive cells, which contained numerous secretory granules, increased. The peroxidase-containing cells are the immediate precursors of the proacinar cells of early postnatal stages. During the same time period, when the peroxidase-containing cells differentiated, a second cell type also differentiated in the cellular cords. The development of this cell type was marked by the appearance of secretory granules stainable with toluidine blue. Through the prenatal development, this cell type revealed no peroxidase activity and was identified with the terminal tubule cell of the newborn. The morphologic and cytochemical findings indicate that terminal tubule cells and proacinar cells are committed cells; the former differentiate toward 2nd order intercalated duct cells and the latter transform to mature acinar cells.