bluuebunny/crossref_metadata_2025 huggingface dataset:
CYTOCHEMICAL STUDIES OF LYSOSOMES, GOLGI APPARATUS AND ENDOPLASMIC RETICULUM IN SECRETION AND PROTEIN UPTAKE BY ADRENAL MEDULLA CELLS OF THE RAT
Authors: HOLTZMAN ERIC , DOMINITZ REGINA
Published: April 2011

The adrenalin-producing cells of the rat adrenal medulla have been studied by light and electron microscopy. Frozen sections of glutaraldehyde-perfused material were incubated for demonstration of "marker" enzymes for lysosomes (acid phosphatase, aryl sulfatase) and Golgi apparatus (thiamine pyrophosphatase). In addition, the uptake and fate of intravenously administered horseradish peroxidase was followed. Acid phosphatase activity is demonstrable in secretory granules, Golgi saccules, vesicles in the Golgi area and in the agranular tubules and cisternae (GERL) from which secretory granules appear to form at the inner surface of the Golgi apparatus. Endoplasmic reticulum with ribosomes on only one surface is closely apposed to both inner and outer aspects of the Golgi apparatus. Peroxidase is taken up in vesicles, tubules and "cup-like" bodies. The latter apparently transform into multivesicular bodies. A possible source of the acid phosphatase found in multivesicular bodies is the small vesicles from the Golgi apparatus or GERL.
THE FLUORESCENT MICROSCOPIC LOCALIZATION OF THIAMINE IN NERVOUS TISSUE
Authors: TANAKA C. , COOPER J. R.
Published: April 2011

A new fluorescent microscopic technique for visualizing thiamine in nervous tissue is described. Freeze dried sections are exposed to the vapors of cyanogen bromide and ammonia to convert thiamine to its fluorescent derivative thiochrome. The specificity of the procedure is ensured by utilizing monochromatic light for activation and a narrow band filter for emission and by scanning fluorescence after exposing the preparation to ultraviolet light at 365 mµ to destroy the thiochrome fluorescence. With this technique it has been shown that, with every preparation of nervous tissue that has been examined, thiamine is present only in membranes and not in axoplasm. This finding is discussed in relation to a suggested role of the vitamin in ion transport.
A NEW DIAZO REAGENT FOR SPECIFIC STAINING OF CONJUGATED BILIRUBIN IN TISSUE SECTIONS
Authors: DESMET V. J. , BULLENS A.-M. , DE GROOTE J. , HEIRWEGH K. P. M.
Published: April 2011

A new technique is presented for the specific histochemical demonstration of conjugated bilirubin in tissue sections, using the diazonium salt of ethylanthranilate. The specificity of this method was proved by using test materials and cholestatic tissue sections. There was no diffusion artifact under the prescribed working conditions. The method is applicable to fresh cryostat sections and frozen sections of cold, formol-calcium-fixed tissues. The method could not be satisfactorily applied for the staining of total bilirubin. The method using 2,4-dichloraniline remains the best available technique for the histochemical demonstration of total bilirubin, while the technique with ethylanthranilate proves to be the most specific method available for the demonstration of conjugated bile pigments.
MAST CELL STAIN FOR HISTAMINE IN FREEZE-DRIED EMBEDDED TISSUE
Authors: SHELLEY WALTER B. , ÖHMAN SVEN , PARNES HERBERT M.
Published: April 2011

A new method is described which permits staining of the histamine in mast cells of embedded tissue. Small specimens of fresh tissue, rapidly frozen in isopentane-liquid nitrogen, are dried in vacuo at –35°C, embedded in paraffin, sectioned and stained with o-phthalaldehyde in ethyl benzene. Mast cell histamine appears bright yellow as viewed under fluorescence microscopy. The procedure is standardized, suitable for a wide variety of tissues and remarkably free of diffusion artifacts. The uncut blocks may be stored for months with no changes in later histamine-staining potential. Alternatively, embedding may be done in polyethylene glycols with satisfactory results.
CHANGING NUCLEAR HISTONE PATTERNS DURING DEVELOPMENT I. FERTILIZATION AND EARLY CLEAVAGE IN THE CRAB, EMERITA ANALOGA
Authors: VAUGHN JACK C.
Published: April 2011

Application of cytochemical techniques to the early developmental stages of the decapod crab, Emerita analoga, shows the following. After fertilization, the sperm nucleus, which apparently contains no basic proteins prior to this stage, becomes associated with a class of weakly basic histones, which differ from adult type histones in their apparent inability to bind alkaline fast green and from protamines by their inability to bind bromphenol blue following acetylation. This class of histones only persists until the late pronucleus stage, by which time the chromosomes contain a class of histones that are indistinguishable from adult histones by qualitative cytochemical techniques. No further changes in the nuclear histones are detected in the zygote or early cleavage stages. These changing histone patterns during early crab development are discussed with reference to other similar studies in other organisms.
HYPERTROPHY OF LIVER CELL SMOOTH SURFACED RETICULUM FOLLOWING PROGESTERONE ADMINISTRATION
Authors: EMANS JOHN B. , JONES ALBERT L.
Published: April 2011

Progesterone given daily for several days to male golden hamsters was shown to promote an increase in liver weight and a striking increase in hepatic smooth endoplasmic reticulum. This increase in smooth reticulum observed by electron microscopy was confirmed biochemically through microsomal phospholipid measurements. The alterations in liver structure brought about by administration of progesterone are comparable to those induced by phenobarbital. Hypertrophy of the smooth reticulum is seen in the form of a delicate system of interwoven tubules which freely anastomose and often are seen in confluence with the lamellar profiles of rough reticulum. Progesterone-induced hypertrophy of the hepatic smooth endoplasmic reticulum demonstrates that this organelle is responsive to an endogenous compound normally present in the circulation, and suggests that stimulation by steroids may be responsible in part for the maintenance of microsomal hydroxylases and smooth reticulum in the normal hepatic cell.
THE FINE STRUCTURAL LOCALIZATION OF ALKALINE AND ACID PHOSPHATASE ACTIVITY IN THE RAT SUBMANDIBULAR GLAND
Authors: BOGART BRUCE I.
Published: April 2011

The lead capture method was employed to study the fine structural localization of nonspecific phosphatase activity in the rat submandibular gland. Alkaline phosphatase activity was observed in association with the plasma membrane and pinocytotic vesicles of the myoepithelial cell. A polarity in the distribution of alkaline phosphatase activity was described along the myoepithelial cell process. More reaction product was observed in association with the plasma membrane on the parenchymal surface than on the plamsa membrane on the stromal surface, where reaction product was confined mostly to the pinocytotic vesicles. Activity was evenly distributed over both surfaces of the portion of the myoepithelial cell characterized by the nucleus. Activity was also observed to be associated with pinocytotic vesicles of the endothelial cells and the plasma membrane of erythrocytes. No activity was observed in association with the ductal elements. Acid phosphatase activity was associated with membrane-bound structures in the acini and ducts. These structures took the form of lipofuscin granules in the acini and either multivesicular or dense bodies in the ducts.
CYTOCHEMICAL LOCALIZATION OF ACID MUCOSUBSTANCES WITHIN RABBIT, CHICKEN, FROG AND FISH SKELETAL MUSCLE FIBERS
Authors: DOUGHERTY WILLIAM J.
Published: April 2011

Skeletal muscles of rabbits, chickens, frogs and fishes were studied by bright field, phase contrast and polarization microscopy after treatment with: (1) Alcian Blue, pH 2.5, (2) Alcian Blue, pH 1.0, (3) low iron diamine, (4) high iron diamine, (5) dialyzed iron, pH 2.0, and (6) colloidal iron, pH 1.5. A prominent aspect of each of the muscles studied was transverse muscle bands which stained with Alcian Blue at pH 2.5, low iron diamine and dialyzed and colloidal iron reagents. Staining of transverse bands was not very intense with these reagents, suggesting that the materials demonstrated occurred in relatively low concentrations. Staining of transverse bands in chicken, frog and fish muscles was observed also after treatment with Alcian Blue at pH 1.0 or with high iron diamine. Staining of rabbit muscle with Alcian Blue at pH 1.0 or with high iron diamine was not detectable even after the most effective fixatives employed in this study, viz., Carony's fixative or buffered HgCl2. Phase contrast and polarization microscopy indicated that the transverse bands stained by the reagents employed corresponded to the I bands in agreement with previous electron cytochemical studies of skeletal muscles treated with dialyzed iron at controlled pH. Methylation for 4 hr at 60°C prevented I band staining with Alcian Blue and dialyzed iron in each of the muscles studied. Treatment of muscle sections with Vibrio cholerae neuraminidase, testicular hyaluronidase or ribonuclease under optimal conditions did not prevent Alcian Blue staining of I bands. Trypsin treatment of muscle sections did not prevent I band staining except when digestion was prolonged to the point that muscle striations were no longer recognizable. Saponification prior to hyaluronidase treatment reduced I band staining in each of the muscles studied. Saponification alone and saponification prior to neuraminidase digestion left I band alcianophilia intact. It was concluded that myofibrillar I bands of rabbit, chicken, frog and fish skeletal muscles contain acid mucosubstances. It was suggested that in each of these organisms an esterified, hyaluronic acid-like molecule is present within the I bands. Sulfated mucosaccharides occur as additional components in the I bands of fish, chicken and frog skeletal muscles; sulfated mucosaccharides apparently are not present in rabbit muscle I bands. Available evidence indicates that sialomucins are not present in the myofibrils of the organisms studied. The possible functional significance of I band acid mucosubstances in vertebrate skeletal muscles was discussed.
CYTOFLUOROMETRIC STUDY OF MAST CELL POLYANIONS II. ADULT RAT PERITONEAL MAST CELLS REGENERATING AFTER POLYMYXIN TREATMENT
Authors: MEYER SAM L. , SAUNDERS ALEX M.
Published: April 2011

Mast cells with metachromatic granules are not detectable in rats after polymyxin-B sulfate treatment. The morphologic and staining characteristics of the cells that repopulate the peritoneal cavity resemble those of mast cells of fetal rats in their maturation sequence, except that, in the adult, the sequence requires at least 56 days. During this time changes occur in the competitive staining of mast cells with acridine orange-sodium chloride, indicating that polyanion synthesis and storage in the granules is a multiphasic phenomenon.
FINE STRUCTURAL LOCALIZATION OF ACETYLCHOLINESTERASE USING ACETYL-β-METHYLTHIOCHOLINE AND ACETYLSELENOCHOLINE AS SUBSTRATES
Authors: KOKKO A. , MAUTNER H. G. , BARRNETT R. J.
Published: April 2011

Acetyl-β-methylthiocho1ine and acetylselenocholine were used as substrates for fine structural demonstration of acetylcholinesterase activity and were compared with acetylthiocholine. Essentially, the same localization of the enzyme activity was found with all of these substrates in the rat spinal cord. Acetyl-β-methylthiocoline proved to be the most specific for acetylcholinesterase in the cytochemical system used and the final product was deposited most rapidly with acetylselenocholine. When the method of Karnovsky and Roots was modified by substituting tartrate for citrate as a chelating agent, a fine crystalline end product was produced and a sharp localization with little evidence of diffusion was obtained. A fixative containing a mixture of formaldehyde and glutaraldehyde proved to be the best in preserving both enzyme activity and ultrastructure. The substrates were tested biochemically and the results substantiate the cytochemical data.
SEPARATION OF INTACT CORTICAL GRANULES FROM HOMOGENATE OF UNFERTILIZED SEA URCHIN EGGS BY ZONAL CENTRIFUGATION
Authors: SCHUEL HERBERT , WILSON WALTER L. , WILSON JEAN R. , SCHUEL REGINA
Published: April 2011

Cortical granules are localized in the peripheral cytoplasm of mature, unfertilized eggs. The rupture of these granules constitutes one of the initial morphologically observable responses of the egg to fertilization or parthenogenetic activation. These organelles were separated from homogenates of unfertilized sea urchin eggs ( Strongylocentrotus purpuratus) by fractionation in the A-XII zonal centrifuge using sucrose density gradients constructed in (0.5 M) KCl. Cortical granules were abundant in electron micrographs of the most rapidly sedimenting population of particles. This population was rich in acid nitrophenylphosphatase activity, contained particles which stained metachromatically with toluidine blue and also contained yolk platelets. Acid phosphatase activity was also associated with several populations of slower sedimenting particles. Yolk platelets were widely distributed in the gradient, and were found in all fractions examined. The mitochondria and cytochrome oxidase activity were sedimented a short distance from the starting boundary.
MICROFLUORIMETRIC STUDIES ON THE FORMALDEHYDE-INDUCED FLUORESCENCE OF NORADRENALINE IN ADRENERGIC NERVES OF RAT IRIS
Authors: JONSSON GÖSTA
Published: April 2011

The fluorescence concentration relationship of formaldehyde-induced fluorescence of noradrenaline in adrenergic nerves of rat iris was investigated by the use of isotope and microfluorimetric techniques. The irides first were depleted of their endogenous stores of noradrenaline either by reserpine or by the methylester of α-methyl- p-tyrosine (H 44/68) and then were incubated in vitro in a physiologic medium containing 3H-noradrenaline for partial replenishment of the nerves of their transmitter. The incubated irides were prepared as whole amounts and were exposed to gaseous formaldehyde for histochemical demonstration of noradrenaline. Microfluorimetric and isotope measurements were performed on the same preparations. The fluorescence intensity was found to be proportional to the noradrenaline concentration up to a value corresponding to 30-40% of the endogenous level, above which a concentration-dependent quenching of the fluorescence occurred. In the linear part of the relation, it was possible to perform fairly safe estimations of the fluorescence intensity by eye, but, if the nerves contained more than 40% of the endogenous content, usually no differentiation could be made, probably as a result of quenching effects. The quenching appears somewhat earlier and the relative fluorescence yield is lower when noradrenaline is stored in the intraneuronal amine storage granules, compared with the situation when the amine mainly is distributed extragranularly in the axoplasm. It can be concluded that, if changes in fluorescence intensity as compared with the control are observed, this reflects true changes in amine concentrations, but changes in amine concentration may escape detection when the concentration of amine is so high that quenching occurs.
SPECIFIC INHIBITION OF ACID PHOSPHATASE-3 IN POLLEN OF MAIZE BY THE DIAZONIUM SALT FAST GARNET GBC
Authors: EFRON YOEL
Published: April 2011

Fast garnet GBC, a diazonium salt commonly used for localization of acid phosphatase activity, was found to be a specific inhibitor for AP3, one of three acid phosphatase enzymes present in maize pollen. This enzyme was not inhibited by fast blue RR or fast blue BB, two other diazonium salts. A possible Inhibition by accompanying inorganic ions was ruled out.
ULTRASTRUCTURAL LOCALIZATION OF ACID MUCOSUBSTANCE AND ANTIMONATE-PRECIPITABLE CATION IN HUMAN AND RABBIT PLATELETS AND MEGAKARYOCYTES
Authors: SPICER S. S. , GREENE W. B. , HARDIN J. H.
Published: April 2011

For selective ultrastructural localization of acid mucosubstance in rabbit and human platelets and megakaryocytes, bone marrow and buffy coat specimens were fixed with formalin, glutaraldehyde or osmium tetroxide, sectioned at 40 µ and stained with the Rinehart-Abul-Haj solution of dialyzed iron. In specimens from both rabbit and man, dialyzed iron staining was observed within nucleoids of the cytoplasmic granules (α-granulomeres) of platelets and megakaryocytes, on the outer surface of the plasma membranes of platelets and megakaryocytes and on the luminal surface of demarcation membranes of megakaryocytes. These results were obtained following any of the three fixation procedures, except when nucleoids failed to stain after glutaraldehyde fixation. For ultrastructural localization of pyroantimonate-precipitable cation, bone marrow and buffy coat specimens were fixed in Komnick's solution of potassium pyroantimonate and osmium tetroxide. In specimens from both species, antimonate deposits were localized within the dense bodies (5-hydroxytryptamine organelles) of platelets and within nucleoids of cytoplasmic granules of platelets and megakaryocytes. The dense bodies were well preserved in platelets fixed in a pyrophosphate-osmium tetroxide solution but were poorly, if at all, preserved by osmium tetroxide solutions containing other buffers.
THE FATE OF WURSTER'S RED FREE RADICAL IN TISSUES WITH HIGH AND LOW OXYGEN UTILIZATIONS (HEART MUSCLE AND GINGIVA)
Authors: PERSON P. , FELTON J. H. , O'CONNELL D. J.
Published: April 2011

When N,N-dimethyl p-phenylenediamine is incubated with fresh frozen sections of rat heart muscle, there is a rapid, enzymatic, single electron oxidation of the diamine to form the pink Wurster's free radical with absorption maxima at 550 and 510 nm. This is followed by radical dimerization and other interactions to form a purple color accompanied by a new peak at 660-670 nm and finally a substantive blue dye. In contrast, with frozen sections of gingiva, instead of the pink color of the Wurster's free radical, one first sees a green color which slowly changes to blue. If heart muscle is kept at room temperature for 30-60 min or in a deep freeze for several days prior to use, it also exhibits formation of the green color. Spectrophotometric studies of the above tissue systems reveal that the new green color does not represent formation of a new compound with new absorption maxima. The same absorption maxima are present in each of the above systems; however, there are differences among the systems with respect to the rates of formation of the absorption maxima in different regions of the spectrum. The latter phenomena produce the color changes seen by the eye. It is suggested that the kinetics of Wurster's Red free radical interactions in tissue may be dependent upon the metabolic (redox) status of the tissue.
A NEW FLUORESCENT METHOD WITH PHENANTHRENEQUINONE FOR THE HISTOCHEMICAL DEMONSTRATION OF ARGININE RESIDUES IN TISSUES
Authors: MAGUN BRUCE E. , KELLY JOHN W.
Published: April 2011

Phenanthrenequinone, in an alkaline ethanolic solution, is shown to be specific for the guanidino group of arginine in tissues. The fluorescence measured in chick erythrocyte nuclei is highly reproducible among areas on one slide and among different slides. Benzil and cyclohexanedione, two agents known to be specific for the guanidino group, prevent 85% of the fluorescence developed in the phenanthrenequinone reaction. Glyoxal blocks phenanthrenequinone reactivity only partially, while alkaline formaldehyde has little effect. The effects of fixation and postfixation of chick erythrocyte nuclei indicate that fluorescence is most intense after formalin fixation, and that conformational factors may prevent ethanolacetic acid-fixed nuclei from reacting completely. By means of protein precipitation and prolonged incubation in ethanol, it was shown that the fluorescent chromophore is strongly bound to phenanthrenequinone-reacted gelatin and to tissue sections. Excitation and emission spectra are presented for phenanthrenequinone-reacted arginine, gelatin and a tissue section.
A TECHNIQUE FOR THE RADIOAUTOGRAPHY OF GERM CELLS IN WHOLE MOUNTS OF SEMINIFEROUS TUBULES
Authors: HUCKINS CLAIRE , KOPRIWA BEATRIX MARKUS
Published: April 2011

A technique has been described for preparing radioautographs of germ cells in intact segments of seminiferous tubules from rat testis. Pieces of tubules were dissected from testes, fixed in Bouin's fluid and stained in Harris hematoxylin. For radioautography, rectangular coverslips were first dipped in liquid Kodak NTB2 emulsion. The tubules were placed on the partially set emulsion, and thus became evenly coated on their viewing surface with a thin layer of emulsion. Following routine exposure and processing, the coverslips bearing these tubules were inverted onto microscope slides to give the final whole mount radioautographic preparation.
CYTOPHOTOMETRIC QUANTITATION OF GLUCOSE 6-PHOSPHATASE ACTIVITY IN RAT LIVER
Authors: ABRAHAMSON D. E. , RIGATUSO J. L. , LAZAROW A.
Published: April 2011

The Wachstein-Meisel lead sulfide precipitation method was used for the histochemical demonstration of glucose 6-phosphatase activity in fresh-frozen normal rat liver sections. Transmittances, microspectrophotometrically determined over cytoplasmic areas of stained sections, were used to calculate the optical density of the stain. In spite of the fact that no absorption maximum was found, the spectrum of glucose 6-phosphatase stain showed proportional increases with increased incubation time. The extinction at a fixed wavelength was shown to be a linear function of incubation time. Studies in which normal tissues were incubated in buffer-substrate and then removed and reincubated for an additional period of time in buffer-substrate containing a specific inhibitor of glucose 6-phosphatase indicate that the increase in optical density of the stain was a result of glucose 6-phosphatase activity.
A SIMPLE METHOD FOR DETERMINATION OF GLUTARALDEHYDE
Authors: FRIGERIO NORMAN A. , SHAW MICHAEL J.
Published: April 2011

A rapid method for determination of glutaraldehyde (GA) is described based on formation of the GA-bisulfite complex followed by iodometric titration of unreacted bisulfite. A 1-ml aliquot of 2-6% GA is added to 20 ml of 0.25 M Na bisulfite. A reaction time of 5 min is allowed, and the solution is titrated with 0.1 M triiodide. Blanks are run in parallel with 1 ml water or buffer. The method gave precisions of 0.2% or better and was not affected by phosphate, phosphate-sucrose or cacodylate buffers or by glutaric acid. Pure GA was prepared by vacuum distillation, analyzed by combustion and used as a primary standard. Deterioration of GA in water and buffer was studied by ultraviolet spectrometry and bisulfite titration. Ultraviolet extinction coefficients proved to be strong functions of time, temperature and initial concentration so that Beer's law was not obeyed. Deterioration led to the formation of polymeric materials, first water-soluble and then, with increasing polymerization, insoluble. Polymerization was most easily followed by observing the disappearance of aldehyde groups titrimetrically, since interpretation of the ultraviolet absorbances at 235: 280 mµ was subject to a multitude of conditions. Polymerization was independent of light, oxygen or transition metal ions, and nearly independent of temperature between 1 and 25°C. Under the proper conditions a partial, but slow, reversal of polymerization could be obtained. Because of the somewhat erratic deterioration shown by GA, periodic determinations of titer are advised if reproducible fixation is desired.
PREPARATION OF FAT CELL-"FREE" RAT MAMMARY GLAND
Authors: MOON R. C. , JANSS D. H. , YOUNG S.
Published: April 2011

The uptake and clearance of chemical carcinogens by mammary tissue are for the most part obscured by the large amount of fat surrounding the mammary parenchymal tissue. This is due primarily to the lipophilic nature of the carcinogenic polycyclic hydrocarbons. Therefore, it became necessary to develop a procedure whereby mammary gland adipose tissue cells may be separated from mammary parenchymal tissue without removal of intracellular lipid or the disruption of the tissue beyond histologic recognition. The procedure developed involves the incubation of the tissue with the enzyme collagenase in a tissue culture medium for 1-1½ hr. This is followed by several sodium chloride washes which effectively remove any adhering intact fat cells. Lipid content of the collagenasetreated preparation was decreased from approximately 60% to 2% when compared with the untreated control gland. The procedure described effectively separates intact fat cells from mammary parenchymal tissue but maintains a histologic state in which mammary ducts and alveoli may be readily identified.
INTRACELLULAR LOCALIZATION OF GLYCOGENOLYTIC AND GLYCOLYTIC ENZYMES IN WHITE AND RED RABBIT SKELETAL MUSCLE A GEL FILM METHOD FOR COUPLED ENZYME REACTIONS IN HISTOCHEMISTRY
Authors: SIGEL PETER , PETTE DIRK
Published: April 2011

A gel film technique has been developed for the histochemical localization of phosphorylase, phosphoglucomutase, hexokinase, glucosephosphate isomerase, phosphofructokinase, aldolase and triosephosphate isomerase. The staining reaction consists principally in coupling the specifically catalyzed reaction to a NADH2- or NADPH2-producing dehydrogenase reaction, which can be visualized by the phenazine methosulfate-mediated reduction of nitro-blue tetrazolium. The intracellular distribution of the respective enzymes was investigated in white and red rabbit skeletal muscles. Hexokinase was found to be associated with the mitochondria, and possibly also with the sarcolemma. A different type of location was observed for the other enzymes. These enzymes were found to be located at the site of the isotropic bands. Although it is suggested that these enzymes are located within the interfilamentary sarcoplasm of the actin filaments, no definite conclusion could be made on the basis of the presented histochemical findings. Especially, it could not be excluded that these enzymes might as well be associated with the transversal sarcotubular system. The very high concentration of glycogenolytic and glycolytic enzymes within the interfilamentary sarcoplasm or the transverse sarcotubular network suggests that these enzymes occur in the state of a labile multienzyme system.
DISTRIBUTION OF ACID PHOSPHATASE, β-GLUCURONIDASE AND N-ACETYL-β-GLUCOSAMINIDASE ACTIVITIES IN LYMPHOCYTES OF LYMPHATIC TISSUES OF MAN AND RODENTS
Authors: TAMAOKI NORIKAZU , ESSNER EDWARD
Published: April 2011

Acid phosphatase, β-glucuronidase and N-acetyl-β-glucosaminidase activities were demonstrated in lymphocytes of man and various rodents using formalin-fixed cryostat sections. Lymphocytes were heterogeneous in respect to the activity of these enzymes, depending on the location of cells in the lymphatic tissues. Acid phosphatase activity was demonstrable in lymphocytes located in the diffuse lymphatic tissue of the cortex of the lymph node and around the central arteriole of the splenic white pulp of all species studied. In man and in rat, the majority of lymphocytes in these areas also showed β-glucuronidase activity whereas, in the mouse, none had β-glucuronidase but many had N-acetyl-β-glucosaminidase activity. Most of the lymphocytes in the mantle of the germinal centers and in the lymphatic nodules of the splenic white pulp were negative for all three enzymes studied. The thymic lymphocytes were positive for acid phosphatase, but usually negative for β-glucuronidase and N-acetyl-β-glucosaminidase. The possible significance of these differences is discussed.
AN α-PARTICLE AUTORADIOGRAPHIC METHOD FOR QUANTIFYING THE INHIBITION OF ACETYLCHOLINESTERASE AT THE MYONEURAL JUNCTION
Authors: LANDOLT ROBERT R. , ZIEMER PAUL L. , CHRISTIAN JOHN E.
Published: April 2011

An existing histochemical method was adapted to α-particle autoradiography to provide a means of quantifying acetylcholinesterase inhibition at myoneural junctions. The α-particles were emitted from polonium 210 which was present in the precipitate after staining. A technique was designed for counting areas of dense α-particle tracks on the autoradiograms which corresponded to sites of acetylcholinesterase activity. An experiment was performed which showed that the track production rate was consistent for several diaphragm sections when incubated under identical conditions with no inhibitor. Tissue sections of this type, representing zero inhibition, were used as controls for inhibitor studies. It was also demonstrated that the track production rate was constant during the different exposure times used in the inhibition study. Diisopropylfluorophosphate was used to evaluate the autoradiographic method of quantifying acetylcholinesterase inhibition. A plot of log inhibitor concentration against percentage inhibition resulted in a smooth sigmoidal curve which is typical of irreversible inhibitors.
DEOXYRIBONUCLEIC ACID CYTOPHOTOMETRY OF STAINED HUMAN LEUKOCYTES I. DIFFERENCES AMONG CELL TYPES
Authors: MAYALL BRIAN H.
Published: April 2011

The deoxyribonucleic acid (DNA) content of individual human leukocytes was estimated cytophotometrically using visible light and spreads stained either with gallocyanin-chrome alum following ribonuclease digestion or with the Feulgen reaction. When the cells were measured on a scanning cytophotometer, significant differences in stain intensity were found among slides. Significant differences also were found among the leukocyte types. In gallocyanin-chrome alum preparations, monocytes measured 16% higher than small lymphocytes and 13% higher than neutrophilic granulocytes. In Feulgen preparations, monocytes measured 4% higher than small lymphocytes and 6% higher than neutrophils. These differences among cell types were independent of donor and stain intensity. Measurements of cells within types and within slides frequently showed close agreement, but it is only in this very limited context that the data are consistent with the hypothesis of DNA constancy. Measurements made on a two-wavelength cytophotometer showed a divergence of only 2.1% relative to similar measurements made on the scanning cytophotometer, which suggests that the differences observed among cells and types are unlikely to be artifacts of the instruments. Over-all, the data indicate either that there is variability in DNA content or that DNA is not being expressed correctly by the measured stain content.
CONTRIBUTION TO THE CYTOSPECTROFLUOROMETRIC MEASUREMENT OF 5-HYDROXYTRYPTAMINE IN ENTEROCHROMAFFIN CELLS
Authors: VIALLI MAFFO , PRENNA GIOVANNI
Published: April 2011

Cytofluorometry enables, under certain conditions, quantitative determination of the 5-hydroxytryptamine content of enterochromaffin cells. Preparations were obtained by: (1) fixation with 10% neutral formalin, (2) freeze-drying and exposure of the sections to formaldehyde released from aqueous formalin or paraformaldehyde and (3) Falck's method. For measurements the Leitz MPV microspectrophotometer and microspectrograph with a television oscilloscopic system were used. Absorption spectra, excitation spectra, emission spectra, photolysis and emission intensity recovery in cells were measured. The corrected data obtained included: absorption peak, 390 nm; excitation peak, 360 to 370 nm; and emission peak, 560 nm. Comparative observations were made on the fluorescence of the background.
A METHOD FOR THE FINE STRUCTURAL LOCALIZATION OF MONOAMINE OXIDASE
Authors: BOADLE MARGARET C. , BLOOM FLOYD E.
Published: April 2011

The light microscopic histochemical method for the demonstration of monoamine oxidase depends upon the reduction of a tetrazolium salt to a dark colored insoluble formazan by the aldehyde which is produced by oxidative deamination of tryptamine. This method has been adapted for electron microscopy by use of an osmiophilic ditetrazolium salt, thiocarbamyl nitroblue tetrazolium. Guinea pig kidney cortex, a rich source of monoamine oxidase, was fixed in 2% buffered paraformaldehyde, cut into 5O-8O-µ frozen sections, washed in buffered sucrose and incubated in the reaction medium at 37°. The reaction was terminated by rinsing in buffered sucrose. Tissues were exposed to 1% buffered OsO4 for 1 hr and then embedded in Araldite. Electron-dense deposits were observed between the inner and outer membranes of mitochondria in cells of the proximal tubules. These deposits were absent from controls from which substrate had been omitted and were not seen in experimental tissues treated with a monoamine oxidase inhibitor, parnate, in tissues incubated at 0° instead of 37°C or in tissues overfixed with 4% paraformaldehyde.
LOCALIZATION OF β-GALACTOSIDASES AND ACID PHOSPHATASE IN THE SMALL INTESTINAL WALL COMPARISON OF ADULT AND SUCKLING RAT
Authors: NORDSTRÖM CHRISTER , KOLDOVSKÝ OTAKAR , DAHLQVIST ARNE
Published: April 2011

By horizontal sectioning of fresh frozen pieces of rat intestinal wall different parts of the villi and crypts were isolated. The sections were collected in groups, homogenized and used for enzyme analyses. Single sections for histologic examination were taken before and after each collection. A quantitative comparison of the distribution of the two intestinal β-galactosidases, EC 3.2.1.23 (neutral and acid β-galactosidase), and the acid phosphatase, EC 3.1.3.2, in jejunum and ileum of adult and suckling (12 days old) rats was performed. The neutral β-galactosidase, which corresponds to the enzyme usually called lactase, was always present along the villi with the highest activities in the apical halves of the villi. This distribution profile is typical for digestion enzymes. The acid β-galactosidase, which is chiefly a heterogalactosidase, and acid phosphatase were found to have a rather flat distribution profile with about the same activities along the villi and in the crypts. The only exception was the ileum of suckling rat, in which especially the acid β-galactosidase showed higher activity in the villi and decreased toward the crypts. The results support the concept that the neutral β-galactosidase is responsible for the digestion of dietary lactose, while the acid β-galactosidase seems to have a different functional significance. That this enzyme is distributed in parallel with the acid phosphatase is consistent with the suggestion that the acid β-galactosidase may be a lysosomal enzyme.
FINE STRUCTURAL LOCALIZATION OF ACETYL COENZYME A CARBOXYLASE IN RAT HEPATOCYTES
Authors: YATES R. D. , HIGGINS JOAN A. , BARRNETT H. J.
Published: April 2011

Acetyl coenzyme A (CoA) carboxylase, the enzyme catalyzing the first step in fatty acid biosynthesis, has been localized in the hepatocytes of glutaraldehyde-fixed rat livers. This reaction in which the hydrolysis of adenosine triphosphate (ATP) is coupled to the synthesis of malonyl-CoA utilizes biotin, bicarbonate, acetyl-CoA, ATP and manganese or magnesium. ATP hydrolysis yields adenosine diphosphate and phosphate, the latter of which is subsequently converted to a lead phosphate precipitate. In tissues incubated in a complete medium, lead phosphate was seen to be closely associated with the outer surfaces of the membranes of the granular endoplasmic reticulum. When ATP was omitted from the medium no intracellular lead deposit was noted. A small amount of reaction product was seen when bicarbonate, biotin or acetyl-CoA was omitted from the medium. Reaction product was not seen when the biotin inhibitor avidin was added to the incubation medium or when tissue sections were boiled in buffer prior to incubation. The possibility of using a similar technique in studying other coupled enzyme systems is considered.
SEX DIFFERENCE AND GONADAL HORMONE INFLUENCE ON SYRIAN HAMSTER KIDNEY ESTERASE ISOZYMES
Authors: LI JONATHAN J. , KIRKMAN HADLEY , HUNTER ROBERT L.
Published: April 2011

Thirteen esterase isozymes were separated from kidney extracts of normal adult Syrian hamsters ( Mesocricetus auratus) using the zymogram technique with vertical starch gel electrophoresis and naphthyl esters as substrates. The activities of two esterases were altered by gonadectomy and by treatment with testosterone or estradiol-17β but not with progesterone. One of these esterases showed negligible activity in castrated males and in normal and spayed females. The activity of this esterase increased appreciably in castrated males treated with testosterone or estradiol and in testosterone-treated spayed females. This enhanced activity, however, was not consistent in spayed females treated with estradiol. No sex differences in renal esterases were observed in immature hamsters but a variation was detected in normal adults. The difference appears to be of normal enzyme levels rather than the absence of any esterase. Under the influence of estradiol and progesterone, separately or in combination, distinct alterations in kidney esterase profiles were observed. Certain other quantitative hormonal effects were found. These hormone-dependent esterases do not appear to be cholinesterases, as they were unaffected by eserine. The marked sensitivity of various hamster kidney esterases to estrogen appears to be a unique response and may be related to the estrogenic induction of renal adenocarcinoma, initiated under similar conditions, but not found with androgen-treated or other estrogenized animals. These findings indicate that gonadal effects on the hamster kidney may be of a more complex nature than has been considered heretofore.
THE HISTOCHEMICAL DEMONSTRATION OF PROTEASE ACTIVITY IN RAT AND GUINEA PIG ADIPOSE TISSUES
Authors: SHEAR M.
Published: April 2011

Protease activity is demonstrable histochemically in adipose tissues of the rat and guinea pig, using the gelatin-silver substrate film technique with incubation of fresh frozen cryostat sections at 37°C for 3 hr in a humid atmosphere at pH 5.2 and 7.7. Serial sections stained with hematoxylin and eosin are used for localization. Control sections are treated with absolute alcohol, 4% formalin, 1% sodium iodoacetate and heat as inhibitors. It is uncertain whether this protease activity is related to the pituitary adipokinetic peptide inactivators which have been demonstrated biochemically in rat, mouse and hamster adipose tissues.
QUANTITATIVE HISTOCHEMISTRY OF RACHITIC RAT CARTILAGE ENZYME ACTIVITY DURING HEALING INDUCED BY VITAMIN D AND STARVATION
Authors: DIXIT PADMAKAR K.
Published: April 2011

Quantitative histochemical methods were used for assaying several dehydrogenases in various cell zones of the rachitic rat epiphyseal cartilage during healing brought about by either vitamin D administration or fasting for 48 hr. 6-Phosphogluconic dehydrogenase activity was significantly greater in the cells of both the proliferating and hypertrophic zones of rachitic control rats as compared to those from vitamin D-treated or fasted rats. Activity of 6-phosphogluconic dehydrogenase in the cells from proliferating and hypertrophic zones was identical in the different groups. Lactic dehydrogenase activity of proliferating and hypertrophic cells obtained from rachitic controls was markedly higher than those from the fasted animals. Minor differences were noted in the malic and isocitric dehydrogenases and peptidase in the cartilage cells obtained from vitamin D-treated, untreated and fasted rats.
HISTOCHEMICAL METAL UPTAKE REACTIONS OF THE BILE PIGMENTS OF OBSTRUCTIVE ICTERUS AND OF HEMATOIDIN
Authors: LILLIE R. D. , PIZZOLATO P.
Published: April 2011

Mordanting with CrO3 and K2Cr2O7: but not with K2CrO4 induces acetic hematoxylin staining (pH 2.5) of bile pigment granules, bile casts and erythrocytes in icteric human livers. Mordanting with tri- or divalent salts of Cr, Fe, Cu, and Sn also induces hematoxylin staining of these objects at pH levels determined by the specific requirements of the metal ion concerned. Mordant concentrations of 1, 10 and 50 mM were used for Cr+++, Fe+++, Fe++, Cu++ and Sn++ salts. Higher concentrations yielded somewhat denser staining, but did not extend the pH range of hematoxylin staining downward. Cr+++ required a hematoxylin pH level of 6-7, Cu++ of 5-7, Fe+++ of 2.8-7, Fe+ of 5-7 and Sn++ of 2.8-7. Alkaline pH levels were not used. Fe+++ and Fe++ uptake were also shown by the positive Prussian and Turnbull's blue reactions obtained after both valency mordantings, indicating partial reduction of Fe+++ and partial oxidation of Fe++. Fe++ and Sn++ are bound equally well by bile cases in untreated sections and in sections previously reduced 24 hr in 0.1 M H2SO3 mixtures. Colors produced were blue-black for hexavalent and trivalent Cr, black for Fe+++ and Fe++, dark blue for Cu++ and dark red for Sn++. Hematoidin colors blue-black with acetic hematoxylin after 6-hr 0.2% CrO3 oxidation, with and without prior dithionite desiderization of the concurrently present hemosiderin in pulmonary, cerebral and splenic infarcts.
STUDIES ON DEOXYRIBONUCLEOPROTEIN IN LEUKOCYTES AND RELATED CELLS OF MAMMALS VII. THE FAST GREEN HISTONE CONTENT OF RABBIT LEUKOCYTES AFTER HYPOTONIC TREATMENT
Authors: GARCIA ALFREDO MARIANO
Published: April 2011

Peripheral leukocytes of rabbit were subjected to the following treatments: (1) 3 parts blood to 2 parts water; (2) 0.95% sodium citrate; (3) Ohnuki's medium. Treated and control leukocytes were then smeared, fixed in 9:1 methanol-commercial formalin and stained for histones according to Alfert and Geschwind. Cytophotometry was carried out with the Barr and Stroud integrating microdensitometer. The cells treated with water increase their dye content by 50%; this increase is less marked (30%) in the cells incubated in Ohnuki's mixture, while there is a 15% decrease in case of citrate. The spread of values "between cells" is larger than the values found with the Feulgen reaction because of factors other than chromatin compaction. (1) There are fluctuations in staining intensity in different areas of the same slide. (2) Nonhistone substrates take the dye, such as hemoglobin of erythrocytes, cytoplasm and specific granules of heterophil granulocytes. (3) Apparently, a certain amount of histones is lost when nuclei swell in hypotonic citrate. It is concluded that, although histone dye binding behaves in a manner similar to Feulgen with respect to changes in the physical state of the deoxyribonucleoprotein complex, the quantitation of fast green histone is a less reliable method for the study of these changes.
THE EFFECT OF FIXATION WITH FORMALDEHYDE AND GLUTARALDEHYDE ON THE COMPOSITION OF PHOSPHOLIPIDS EXTRACTABLE FROM RAT HYPOTHALAMUS
Authors: ROOZEMOND R. C.
Published: April 2011

Fixation of rat hypothalamus in 4% formaldehyde + 1% CaCl2 for 24 hr at 0°C reduced the amount of extractable ethanolamine phospholipids considerably. This decrease may be caused by hydrolytic cleavage of the vinyl ether bond in phosphatidalethanolamine and by reaction of formaldehyde with the free amino groups in ethanolamine phospholipids. Evidence is presented that the reaction with free amino groups may be the main cause for the decrease of extractable phospholipids when dealing with a fixative that contains glutaraldehyde and is buffered at pH 7. In this case no phosphatidylserine and hardly any phosphatidylethanolamine could be detected in the tissue extract. It is presumed that these phospholipids are fixed to proteins by the cross-linking action of glutaraldehyde involving free amino groups of proteins and phospholipids.
CYCLIC 3',5'-NUCLEOTIDE PHOSPHODIESTERASE IN BRAIN
Authors: BRECKENRIDGE BRUCE McL. , JOHNSTON ROBERT E.
Published: April 2011

The distribution of cyclic 3',5'-nucleotide phosphodiesterase activity in the rabbit central nervous system has been studied with a quantitative micromethod. A wide range of values between various structures was observed, but correlations with cellular density, monoamine content or other enzymes were not apparent. Phosphodiesterase activity is not uniquely associated with particular layers of the cerebral cortex or with specialized synaptic structures in the olfactory bulb. The enzyme level was unaltered in the rat superior cervical ganglion after section of preganglionic fibers, and in the cerebral cortex after lesion of the medial forebrain bundle. Major disparities in comparing these results with those of a Gomori type procedure may be explained in part by the inhibition of the enzyme by lead ion.
THE ROLE OF HISTOCHEMICAL AND BIOCHEMICAL PREPARATION METHODS FOR THE DETECTION OF GLYCOGEN
Authors: LESKE R. , MAYERSBACH H. v.
Published: April 2011

Block fixation for the histochemical demonstration does not safely preserve glycogen. Even when using "glycogen fixatives" and low temperatures the staining measured intensity does not correspond to the amount of glycogen measured chemically. Adequate preservation is obtained only with quenching.

Lyo- and desmoglycogen fractions in biochemical assays are obtained when coarse tissue homogenates are extracted with TCA, while from fine homogenates the same glycogen amount can be extracted with TCA as with KOH digestion of the tissues.

The histochemical demonstrability of glycogen in fixed tissue blocks does not depend on the presence of lyoglycogen.
THE REDUCING ACTION OF ELASTIC TISSUE ON FERRIC FERRICYANIDE A HISTOCHEMICAL ANALYSIS
Authors: COOPER JOHN H.
Published: April 2011

Exposure of tissue sections to the original Unna ferric ferricyanide reagent for 10 min produces strong blue staining of elastica, demonstrating a reducing activity of this substance. This activity was analyzed histochemically in sections of human skin, kidney and spleen by observing its resistance to various blocking techniques, correlated with the results of corresponding end group methods. For comparative purposes the properties of four other reducing components (keratin, melanin, fibrin and proximal tubule cytoplasm) were studied. The results indicate that the reducing activity of the elastica is largely a function of phenolic groups, presumably in the tyrosine residues of elastin. In view of the inordinate resistance of this activity to preliminary treatment with aqueous periodic acid, the presence of additional minor reducing components, such as lipochrome or polytyrosines, cannot be excluded. This distinctive histochemical property should prove useful in studies of pathologically altered elastica and other "elastotic" material.
LIGHT MICROSCOPIC STUDY OF THE PEROXIDATIC ACTIVITY OF CATALASE IN FORMALDEHYDE-FIXED RAT LIVER
Authors: HIRAI KEI-ICHI
Published: April 2011

The peroxidatic activity of rat liver catalase was demonstrated by histochemical staining with 3,3'-diaminobenzidine as hydrogen donor. The activity was so weak that its location was hard to identify in formaldehyde-fixed cells, although high catalatic activity was present, as evidenced by the production of bubbles upon the addition of hydrogen peroxide to the incubation medium. Pretreatment of fixed sections for 60 min at 37°C with formamide, urea or trypsin enhanced the peroxidatic activity significantly. The reaction granules scattered throughout the cytoplasm of the parenchymal cells probably correspond to the peroxisomes.
A NEW ELECTRON-DENSE STAIN FOR ELASTIC TISSUE
Authors: ALBERT ERNEST N. , FLEISCHER EVERLY
Published: April 2011

Tetraphenylporphine sulfonate has been shown to have a specific affinity for elastic tissues of the body in fluorescence microscopy. However, tetraphenylporphine sulfonate does not impart electron density to this tissue and thus is not suitable for electron microscopic studies. Therefore, tetraphenylporphine sulfonate was complexed with various heavy metals in order to use it as a specific stain for elastic tissues in electron microscopy. The silver and gold metallic complexes gave the most consistent and specific staining reactions. These compounds were prepared in this laboratory. The synthesis and staining procedures are described in detail.