bluuebunny/crossref_metadata_2025 huggingface dataset:
THE BIOCHEMICAL GENETICS OF RABBIT ERYTHROCYTE ESTERASES HISTOCHEMICAL CLASSIFICATION
Authors: SCHIFF ROBERT
Published: April 2011

Esterases found in rabbit erythrocytes and platelets were separated by zone electrophoresis in starch gels. Activity tests in various substrates were used to classify the esterase bands into four major categories. The bands of each category were further subdivided into groups by the effects of various inhibiting and activating agents. These experiments showed that the genetic loci which control the enzymes of systems 1, 2 and 3 control isozymes that respond identically with the test reagents in their respective systems. Diisopropylfluorophosphate and eserine sulfate inhibition indicated that the active sites of systems 1 and 2 enzymes are different. This supported the conclusion that the esterases of systems 1 and 2 are the products of closely linked loci and not multiple alleles at a single locus. Carbonic anhydrases which migrated in the cathodal portion of the gel were identified by their sensitivity to acetazolamide and more intense staining with β- rather than α-naphthyl acetate as substrate.
FINE STRUCTURAL LOCALIZATION OF CHOLINESTERASE ACTIVITY IN THE RAT SUBMANDIBULAR GLAND
Authors: BOGART BRUCE I.
Published: April 2011

The innervation of the rat submandibular gland was investigated by means of electron microscopy and cytochemical techniques. Axonal swellings partially depleted of their Schwann cell investment were observed in relationship to myoepithelial cells, acinar cells, ducts, capillaries and smooth muscle cells of arterioles. No direct contacts were found to exist between these elements and the nerve fibers. Axonal swellings containing mitochondria and vesicles fell into two distinct types: those characterized by small agranular vesicles and larger vesicles with pale cores and those characterized by small agranular vesicles and small vesicles with dense cores. Reaction product due to acetylcholinesterase activity was observed to be associated with the axolemma and the Schwann cell membrane at the axon-Schwann cell interface of many unmyelinated nerves found in relationship to both the vascular and parenchymal elements of the gland. Activity was found in association with the surface pits and vesicles of myoepithelial cells and arteriolar smooth muscle cells and with the rough endoplasmic reticulum and the nuclear envelope of the myoepithelial cells. Axonal swellings devoid of activity were observed particularly in relationship to arterioles. Reaction product was found only at the axon-Schwann cell interface when sections were incubated in medium for the demonstration of butyrylcholinesterase activity, while no reaction product was found upon addition of eserine sulfate to any of the media. The possibility that the reaction product due to acetylcholinesterase activity observed in association with the axolemma and the surface vesicles and pits of the myoepithelial cells and the arteriolar smooth muscle cells may define the autonomic cholinergic junction in the rat submandibular gland was explored.
FLUORESCENT-PROTEIN STAINING OF NUCLEOLI IN MONOLAYER CELL CULTURES: DETERMINANTS AND SIGNIFICANCE
Authors: MAISEL JOHN C.
Published: April 2011

In order to distinguish whether fluorescent-protein staining of nucleoli in acetone-fixed cell culture monolayers is a useless artifact or whether nucleolus-globulin affinity reflects a physiologic interaction between cells and serum, the phenomenon was investigated by further characterizing the interacting components and the conditions required. Staining proved to be a time-dependent and temperature-dependent, two-step procedure: initial modification of nucleoli by smaller serum γ-globulins is required for later ionic binding to nucleoli of fluorescein-labeled, nonimmune α-, β- or larger α-globulins. The initial modification is essential only in cells grown in minimal serum and harvested toward the end of the logarithmic growth phase of the culture. The nucleolar component binding the conjugate is both heat- and pH-stable, is maintained in situ apparently by ionic bonds and most likely includes a saline-insoluble, nonhistone basic protein. Previous serum modification of nucleoli in living primarily explanted monkey kidney cells, detected with this technique, could not be demonstrated with established H-Ep 2 or HeLa cell lines of human tumor origin. These data suggest that serum-dependent nucleolar fluorescent protein staining might be a useful artifact, signifying something about nucleolar organization or content of unexplained relation to cell growth.
THE CYTOCHEMICAL LOCALIZATION OF GLYCOLYTIC AND OXIDATIVE ENZYMES WITHIN MITOCHONDRIA OF SPERMATOZOA OF SOME PULMONATE GASTROPODS
Authors: ANDERSON WINSTON A. , PERSONNE PAUL
Published: April 2011

In this electron microscopic cytochemical study, the periodic acid-thiosemicarbazide-silver proteinate procedure was used to demonstrate glycogen stores within the mitochondrial derivative of sperm of pulmonate gastropods. In the presence of phenazine methosulfate and tetrazolium salt, enzymatic activity for glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase is shown in the matrix and in the compartment containing glycogen, but in the absence of phenazine methosulfate, tetrazolium reductase activity in the matrix is emphasized. Activity for NADH2-tetrazolium reductase and succinate dehydrogenase is also demonstrated in the matrix. Using 3,3'-diaminobenzidine tetra-HCl, cytochrome c oxidase activity is shown in the paracrystalline mitochondrial structure. The interrelation between glycolytic and oxidative pathways in this highly compartmentalized mitochondrion is considered.
HISTOCHEMICAL EVIDENCE AND CONSEQUENCES OF THE OCCURRENCE OF ISOENZYMES OF ACETYLCHOLINESTERASE
Authors: KOELLE WINIFRED A. , HOSSAINI KHADIJEH SHARIFI , AKBARZADEH PARI , KOELLE GEORGE B.
Published: April 2011

Frozen sections of unfixed cat stellate (StG) and ciliary (CG) ganglia and intercostal muscle (ICM) and mouse ICM were exposed to saline (0.9% NaCl) solution and to 0.3% Triton X-100 in saline solution, and then stained for acetylcholinesterase (AChE) by a modification of the standard copper-thiocholine method. In the StG and CG, treatment with saline solution resulted in marked diffusion of AChE, and Triton X-100 caused its marked loss from the CG and nearly complete loss from the StG. In ICM, in contrast, both treatments caused increased intensity of staining of AChE at the motor end plates (MEP's). All of the foregoing effects were reversed progressively, with concomitant reduction in AChE activity, by prior fixation in cold formaldehyde (4%)-sucrose (7.5%)-maleate buffer (pH 7.3) solution for 2-24 hr. Results are interpreted on the basis that the AChE of the ganglia and MEP's exists predominantly in different isoenzymatic forms, the possible natures of which are discussed.
HISTOCHEMICAL INDICATIONS FOR LYSOSOMAL LOCALIZATION OF HEAVY METALS IN NORMAL RAT BRAIN AND LIVER
Authors: BRUN ARNE , BRUNK ULF
Published: April 2011

With a modified sulfide-silver method for the demonstration of heavy metals, hepatic parenchymal cells, Kupffer cells, neurons and glial cells were found normally to contain stainable cytoplasmic granules with a shape, size and distribution identical with that of lysosomes in the various cells studied. Previous studies have shown the lysosomes of mast cells and eosinophilic leukocytes normally to contain zinc. Iron has also been demonstrated in residual bodies in different tissues. Under pathologic conditions, such as Wilson's disease and experimentally induced intoxications, lysosomes have been shown to take up copper, mercury and lead. Our results suggest that heavy metals are normal constituents of lysosomes in a more general way than hitherto assumed.
ACID PHOSPHATASE ACTIVITY IN INDIVIDUAL NEURONS DURING CHROMATOLYSIS: A QUANTITATIVE HISTOCHEMICAL STUDY
Authors: HIRSCH HILDE E. , OBENCHAIN THEODORE
Published: April 2011

Two fluorogenic substrates, α-naphthyl phosphate and 4-methylumbelliferyl phosphate, were used to measure acid phosphatase activities in individual anterior horn neurons of the monkey spinal cord after section of the sciatic nerve. Although many studies utilizing staining methods have reported a striking increase in acid phosphatase activity during chromatolysis, no significant differences were observed here between the neurons of the operated and unoperated side, despite widespread chromatolysis. β-galactosidase activity was also unchanged, but the marked elevation of glucose 6-phosphate dehydrogenase during the reparative phase was confirmed.
ADRENERGIC FIBERS IN BROWN FAT OF COLD-ACCLIMATED RATS
Authors: COTTLE MERVA K. W. , COTTLE W. H.
Published: April 2011

The adrenergic nerve fibers of brown adipose tissue of warm- and cold-acclimated rats were visualized using the fluorescence histochemical technique. Small arteries and arterioles evidenced by their rich adrenergic innervation appeared more abundant in tissue from cold-acclimated animals. Lengths of fine fibers between the fat cells were demonstrable in the brown fat tissue from cold-acclimated rats because of their abundant, strongly fluorescent varicosities whereas such portions of fine fibers were only occasionally seen in the tissue of warm-acclimated rats.
A METHOD FOR IN SITU CYTOPHOTOMETRIC ESTIMATION OF ABSOLUTE AMOUNT OF RIBONUCLEIC ACID USING AZURE B
Authors: SHEA J. R.
Published: April 2011

A colorimetric method is proposed for in situ cytophotometric estimation of ribonucleic acid (RNA) in biologic material, using a gelatin-RNA model system as a reference. Both specificity and stoichiometry of azure B dye binding to RNA in this model system were demonstrated. As the amount of dye binding is stoichiometric with the concentration of RNA, the regression equation generated from cytophotometric data of relative concentration ( E/d) of azure B-RNA complex as a function of concentration of RNA (picograms per cubic micron) can be used to calculate the absolute concentration (picograms per cubic micron) of RNA in biologic material. Comparison of such an estimate for nucleolar RNA in Purkinje cell nuclei of the mouse with an estimate derived from available experimental data in the literature is made to assess the validity of the reference. These estimates are in agreement, supporting the applicability of the proposed method.
HISTOCHEMICAL SEPARATION OF CONJUGATED AND UNCONJUGATED BILIRUBIN AND ITS ASSESSMENT BY THIN LAYER CHROMATOGRAPHY
Authors: RAIA SILVANO
Published: April 2011

Conjugated and unconjugated bilirubin can be stained and separated histochemically by a method using diazotized 2,4-dichloroaniline. In cholestatic human liver both pigments are demonstrable. Icteric human kidney in which bilirubin is predominantly conjugated and Gunn rat papillae containing only unconjugated pigment were used to confirm the validity of the histochemical method. Furthermore, homogenates of cholestatic liver were treated as in the histochemical method; the azo pigments formed were identified by thin layer chromatography and correspond to azo pigments formed from known conjugated and unconjugated bilirubin.
HISTOCHEMICAL AZO COUPLING REACTIONS OF THE PIGMENTS OF OBSTRUCTIVE ICTERUS AND OF HEMATOIDIN
Authors: LILLIE R. D. , PIZZOLATO P.
Published: April 2011

Azo bilirubins and azohematoidins resist alcohol dehydration and mounting in resinous media after adequate coupling reactions. The azo colors are resistant to hydrochloric acid extraction (0.1 N 30 min routinely) but are bleached at varying rates according to the specific dye by sodium dithionite solution. Fresh dithionite is required. The azo coupling reaction of bile casts is prevented by ethanol acetic anhydride acetylation, restored by alcoholic KOH saponification and prevented completely by oxidation with 0.2% CrO3 (4 hr) or by a diammine silver, iodine, thiosulfate sequence. These oxidations do not alter the alkaline azo coupling reactions of tissue protein. Azo coupling of bile casts is unaltered by a 24-hr 10% iodine-methanol, 2-min thiosulfate sequence or by a 24-hr 0.1 M bisulfite reduction. The reactive bilirubin is resistant to hot methanol chloroform primary fixation as well as to exhaustive extraction by this solvent after formol fixation, although both of these procedures extract readily visible amounts of yellow material into the solvent. It appears that the azo-reactive material must be protein bound. Alkaline azo coupling with diazosafranin is weakened by lesser amounts and completely prevented by addition of equimolar or greater amounts of uric acid to the diazo.
OBSERVATIONS ON HEPATIC AND RENAL PEROXISOMES (MICROBODIES) IN THE DEVELOPING CHICK
Authors: ESSNER EDWARD
Published: April 2011

Peroxisomes (microbodies) in developing liver and metanephric kidney of the chick were studied with a staining method that visualizes these organelles by light and electron microscopy. In both tissues nucleoid and anucleoid peroxisomes were evident at all stages studied (15th day of incubation to the adult). Hepatic peroxisomes showed no special topologic relationship to glycogen areas and were mainly confined to other areas of the cytoplasm. Nucleoid type peroxisomes were more apparent before and after hatching and rarely observed in the adult. Peroxisomes were present in kidney proximal tubule cells, but absent from cells of distal convoluted tubules. They appeared to increase in number during development and were most conspicuous in the adult. The structure of the nucleoid in renal peroxisomes resembled that of the hepatocyte but contained a more distinct series of parallel dense laminae. Diaminobenzidine (DAB) oxidative activity, although relatively weak, visualized nucleoid and anucleoid peroxisomes in liver and kidney at all stages of development. Anucleoid peroxisomes could be distinguished from various dense bodies in hepatocytes and from droplets in proximal convoluted tubule cells, both of which lacked DAB oxidative activity. The DAB reaction in peroxisomes was not suppressed in the absence of hydrogen peroxide or in the presence of the catalase inhibitor, 3-amino-1,2,4 triazole. DAB reaction, sensitive to potassium cyanide, was localized to cristae, but not to the outer membrane, of hepatic and renal mitochondria. Mitochondria of distal tubule cells showed a stronger reaction. The possible significance of nucleoid-bearing peroxisomes in the developing chick is discussed in terms of nitrogen metabolism, uric acid excretion and the presence of the uricolytic enzymes. The nature of the DAB reaction in peroxisomes and in mitochondria is considered.
LOCALIZATION OF TISSUE ANTIGENS ON THE ULTRATHIN SECTIONS WITH PEROXIDASE-LABELED ANTIBODY METHOD
Authors: KAWARAI YASUYUKI , NAKANE PAUL K.
Published: April 2011

Peroxidase-labeled antibody method was used to localize tissue antigens directly on ultrathin sections. For the demonstration of the method, luteinizing hormone, growth hormone and prolactin were localized on ultrathin sections of methacrylate-embedded anterior pituitary gland of the rat. The hormones were localized in secretion granules and in endoplasmic reticulum, but not in nucleus or mitochondria. This approach totally eliminates the problem of penetration of antisera through tissues and permits better immunologically controlled experiments by using serially sectioned materials.
A NEW FLUORESCENT STAIN WITH o-PHTHALALDEHYDE FOR A CELLS OF THE PANCREATIC ISLETS
Authors: TAKAYA KENICHI
Published: April 2011

Pancreatic A cells of several vertebrate species (rat, mouse, pig, hamster, rabbit, dog, guinea pig, hen, pigeon, newt and crucian carp) selectively fluoresced after staining the fresh frozen cryostat sections of the pancreas with 1% o-phthalaldehyde (Carbiochem) in physiologic saline (dissolved at 58°C). The pertinent cells could be identified as A cells in the same sections observed under dark field illumination or phosphotungstic acid-hematoxylin staining. The histamine assay and some experiments using reserpine, compound 48/80 and cobaltous chloride indicated that the fluorogenic material is neither histamine, catecholamine nor tryptamine. This o-phthalaldehyde stain is a simple, rapid and specific method to disclose A cells of the pancreatic islet, although the nature of responsible substance is yet unknown.
THE LOCALIZATION OF CYTOCHROME c OXIDASE ACTIVITY DURING MITOCHONDRIAL SPECIALIZATION IN SPERMIOGENESIS OF PROSOBRANCH SNAILS
Authors: ANDERSON WINSTON A.
Published: April 2011

This cytochemical study of differentiating prosobranch spermatids demonstrates changes in the localization of intramitochondrial cytochrome c oxidase activity during the course of mitochondrial fusion and internal reorganization. Using the osmiophilic compound 3,3'-diaminobenzidine, activity for cytochrome c oxidase is localized in or on the internal mitochondrial membranes and within the intracristal compartment of metamorphosing mitochondria. However, some enzyme activity consistently appears in the matrix. Enzyme reaction product in the matrix disappears as the internal membranes become organized, and in mature spermatozoa activity for cytochrome c oxidase appears only on cristae and in the intracristal space. It is hypothesized that positive matrix material could either represent enzyme molecules not yet incorporated into membranes or disaggregated products of membrane breakdown associated with remodeling of the cristae.
THE DISTRIBUTION OF CHOLINESTERASE IN THE RABBIT EAR ARTERY
Authors: WATERSON J. G. , HUME W. R. , DE LA LANDE I. S.
Published: April 2011

Acetylcholinesterase and butyrylcholinesterase activities were demonstrated in transverse sections of the rabbit ear artery using a "direct coloring" thiocholine technique. BW 284C51, iso-OMPA and DFP were used as inhibitors, and acetyl and butyrylthiocholine iodides as substrates. Both enzymes caused stain deposits at the medial-adventitial border of the artery, in a region where sympathetic nerves are present. Sections treated to demonstrate acetylcholinesterase showed staining which was lighter than that for butyrylcholinesterase. Degeneration of the sympathetic nerves following superior cervical ganglionectomy virtually abolished stain at the medial-adventitial border. These results provide some support for the evidence of other workers that a cholinergic mechanism may be linked to sympathetic nerve transmission in the rabbit ear artery.
MODULATION IN NUCLEOSIDE DIPHOSPHATASE ACTIVITY OF MAMMOTROPHIC CELLS OF THE RAT ADENOHYPOPHYSIS DURING SECRETION
Authors: SMITH ROBERT E. , FARQUHAR MARILYN G.
Published: April 2011

The modulations in nucleoside diphosphatase (NDPase) activity which occur during protein secretion have been investigated in mammotrophic hormone-producing cells (MT) of the female rat adenohypophysis. Pituitaries were fixed by perfusion or immersion in glutaraldehyde, and nonfrozen sections were incubated for NDPase. Tissue was examined from lactating and estrogen-treated animals in which mammotrophic hormone secretion (MTH) is high and from postlactating rats in which secretion of MTH is suppressed. In all experimental groups, NDPase reaction product was found: ( a) in Golgi cisternae, ( b) around forming and mature secretion granules, ( c) in pericapillary and intercellular spaces and ( d) along the outer surface of endothelial and MT cell membranes. The staining intensity of both the intracellular and extracellular sites paralleled the level of MTH secretion; it was greatest after estrogen treatment, moderate during lactation and minimal in the postlactating animal. In animals given estrogen, the Golgi apparatus was increased in size and up to six successive cisternae along the concave Golgi face were filled with reaction product in virtually every cell. In postlactating animals, the Golgi apparatus was small, and there was only patchy staining of a few cisternae in some cells. From its location and fluctuations with secretion, it seems likely that NDPase activity may be associated with the condensation and/or discharge of secretory granules.
PRESERVATION OF Na-K-ACTIVATED AND Mg-ACTIVATED ADENOSINE TRIPHOSPHATASE ACTIVITIES OF AVIAN SALT GLAND AND TELEOST GILL WITH FORMALDEHYDE AS FIXATIVE
Authors: ERNST STEPHEN A. , PHILPOTT CHARLES W.
Published: April 2011

The effect of glutaraldehyde and formaldehyde fixation on the level of biochemically demonstrable Na-K-adenosine triphosphatase (Na-K-ATPase) and Mg-ATPase of avian salt glands and teleost gill filaments was studied. Sections, 100-200 µ, prepared with the Smith-Farquhar tissue chopper, were fixed for varying periods, homogenized and assayed for ATPase activity. Fixation of salt gland tissue with 0.5% glutaraldehyde for 40-60 min completely inhibited the Na-K-ATPase activity and reduced the level of Mg-ATPase by 85%. In contrast, fixation with 2 or 3% formaldehyde, prepared from paraformaldehyde, for 60-90 min resulted in a loss of only 30% of the Na-K-ATPase activity and 65% of the Mg-ATPase activity. Similar results were obtained with gill filaments. In addition, Na-K-ATPase of formaldehyde-fixed tissue retained an obligatory requirement for Na+ and K+ and was fully sensitive to ouabain. Electron microscopic examination of formaldehyde-fixed tissue, sectioned with either the tissue chopper or in the cryostat and incubated in the Wachstein-Meisel medium, showed excellent morphologic preservation. Reaction product deposition (presumably due to Mg-ATPase) was associated with the extracellular side of the plasma membrane in the secretory cells of the salt gland and over the mitochondrial matrix of chloride cells present in the gill epithelium.
A QUANTITATIVE HISTOCHEMICAL STUDY OF INTESTINAL MUCOSA AFTER X-IRRADIATION
Authors: GALJAARD H. , BUYS J. , VAN DUUREN M. , GIESEN J.
Published: April 2011

The effect of low doses of x-irradiation (50-400 R) on the activity of various enzymes in rat intestinal epithelium has been investigated by histochemical staining methods and quantitative microchemical analyses of crypts and villi dissected from frozen-dried sections. Irradiation had no effect on the activities of enzymes which in nonirradiated animals are present exclusively or mainly in the villus epithelium (aminopeptidase, various phosphatases) or are evenly distributed along the epithelium of crypts and villi (various dehydrogenases). However, nonspecific esterase activity decreased markedly both in crypt epithelium and villus epithelium. This occurred 36 hr after irradiation, independent of the radiation dose. The number of crypt cells with reduced esterase activity and the duration of the effect increased with higher radiation doses. These results were confirmed by quantitative analyses which also showed that esterase activity is 5 times higher in the villus than in the crypt. The remarkable correspondance between the period of reduced esterase activity in the crypt and the period of increased proliferative activity after various radiation doses suggest a relationship between changes in crypt cell population dynamics and esterase activity; the functional consequences for the villus epithelium of changes in the crypt cells after irradiation are discussed.
THE UNLABELED ANTIBODY ENZYME METHOD OF IMMUNOHISTOCHEMISTRY PREPARATION AND PROPERTIES OF SOLUBLE ANTIGEN-ANTIBODY COMPLEX (HORSERADISH PEROXIDASE-ANTIHORSERADISH PEROXIDASE) AND ITS USE IN IDENTIFICATION OF SPIROCHETES
Authors: STERNBERGER LUDWIG A. , HARDY PAUL H. , CUCULIS JOHN J. , MEYER HOWARD G.
Published: April 2011

Antigen was identified histochemically without the use of labeled antibodies by the sequential application of (a) specific rabbit antiserum, (b) sheep antiserum to rabbit immunoglobulin G, (c) specifically purified, soluble horseradish peroxidase-anti-horseradish peroxidase complex (PAP), (d) 3,3'-diaminobenzidine and hydrogen peroxide and (e) osmium tetroxide. A simple method for preparation of high yields of PAP consisted of precipitation of antibody from specific rabbit antiserum with horseradish peroxidase (PO) at equivalence, solubilization of the washed precipitate with excess PO at pH 2.3, 1°C, followed by immediate neutralization and separation of PAP from PO by half-saturation with ammonium sulfate. The ratio of PO to anti-PO in PAP was 3:2 irrespective of the source of antiserum. PAP was heterogeneous on electrophoresis, homogeneous on sedimentation, diffusion and electron microscopy and consisted of pentagons with diameters of 205 Å. s20, w, 11.98 x 10–13; d20, w, 2.48 x 10–7; molecular weight by sedimentation velocity, 429,000, and equilibrium, 413,000. Sensitivity and specificity of immunohistochemical staining of spirochetes was about 100- to 1000-fold that of immunofluorescence. The unexpected ratio of PO to anti-PO is presumed to be due to stabilization by the pentagonal shape in which three corners are suspected to be PO and two antibody fragment Fc.
A HISTOCHEMICAL STUDY OF ADENOSINE TRIPHOSPHATASE IN THE TOAD (BUFO SPINULOSUS) GASTRIC MUCOSA
Authors: S. CECILIA KOENIG , C. JUAN D. VIAL
Published: April 2011

Adenosine triphosphatase (ATPase) activity was studied by histochemical methods in the gastric mucosa of Bufo spinulosus. Two types of activity were established. One is activated by Mg++, and is localized mainly at the intercellular boundaries and the basal zone of the oxyntic-peptic cells; the reaction products are found only on the outer side of the cells. The other is activated by HCO3– and is mainly localized at the microvilli at the apical zone of the oxyntic-peptic cells. The intensity and/or distribution of the reactions are influenced by histamine stimulation. Control experiments demonstrated that: the microsomal fraction of gastric mucosa contained a Mg++-requiring ATPase activity which was enhanced by addition of HCO3–; 25% of the ATPase activity of fresh tissue was maintained after fixation and incubation in presence of lead ion; the medium employed in the histochemical studies did not enhance the lead-catalyzed, nonenzymatic hydrolysis of ATP by more than 20% when compared with spontaneous ATP hydrolysis; and incubation in media with different ATP-Pb ratios did not significantly alter the location of the staining.
INCORPORATION OF 3H-THYMIDINE INTO MITOCHONDRIA LOCALIZATION OF AUTORADIOGRAPHIC LABEL TO INDIVIDUAL MITOCHONDRIA
Authors: GREEN M. R. , BAHR G. F.
Published: April 2011

A simple method is described and tested which lends itself to the quantitative evaluation of radioactive labeling in small biologic objects requiring the use of electron microscopy. The practicality of the method is demonstrated with isolated liver mitochondria and tritiated thymidine.
STAINING OF TISSUE POLYCATIONS BY ANIONIC DYES IN SALT SOLUTIONS WITH SPECIAL REFERENCE TO GINGIVA
Authors: STAPLE P. H.
Published: April 2011

Over the range pH 5.6-9.4, the presence of KCl or other common metallic chlorides in excess of 0.1 M in 0.05% solutions of Biebrich scarlet or acid fuchsin produced a colored precipitate within 24 hr. Within the same period, over the range pH 2-7, no precipitate resulted when up to 5 M choline chloride (highest concentration tested) was present in 0.01% solutions of these and other anionic dyes used for staining tissue sections. In paraffin sections of frozen-dried tissues, previously exposed to modified Newcomer's fluid or cyanuric chloride in anhydrous reaction medium, equilibrium staining in the presence of increasing concentrations of choline chloride or potassium chloride was investigated at pH 2.28-2.70, 5.6 and 6.18-6.70 using 0.01% or 0.001% solutions of acid fuchsin, Biebrich scarlet or its isomer crocein scarlet, fast green FCF, naphthol yellow S. The tissues studied were: prevertebral structures included in a horizontal section through the cervical region of a mouse, rat abdominal skin and gingiva, human gingiva. In general, increasing salt concentration progressively inhibited staining, but over a limited range could increase staining, e. g., in cartilage, by unmasking additional dye-binding groups through the dissociation of macromolecular polyionic complexes. Sequential staining with the same or two different dyes in presence of different salt concentrations showed that inhibition of straining by salt was not due to elution of reactive material from sections but to competition between salt and dye anions for cationic sites in tissues. The biologic polyanions heparinate and hyaluronate were found to compete with Biebrich scarlet for tissue cationic sites. The concept of critical electrolyte concentration (CEC) proposed for staining with cationic dyes in salt solutions was found to be applicable to staining with anionic dyes and thus to be generally valid for staining that involves polar interactions. Rat gingival keratin, previously shown to have only a low CEC for the cationic dye Alcian Blue, displayed a high CEC for all of the anionic dyes investigated. Some sites in mouse arterial wall and laryngeal cartilage known to have a high CEC for Alcian Blue showed high CEC for Biebrich scarlet. It is suggested that the high CEC of human gingival parakeratin for anionic dyes depends on the presence of arginine, histidine and lysine protein residues and those few α-amino groups capable of protonation.
A COMPARISON OF ALCIAN BLUE-ALDEHYDE FUCHSIN AND PEROXIDASE-LABELED ANTIBODY STAINING TECHNIQUES IN THE RAT ADENOHYPOPHYSIS
Authors: SWOPE ALICE E. , KAHN RAYMOND H. , CONKLIN JAMES L.
Published: April 2011

The peroxidase-labeled antibody (P-Ab) technique was compared on adjacent sections with a permanganate-Alcian Blue (AB)-aldehyde fuchsin (AF) procedure on the anterior pituitary gland of young adult, female rats. The cells that stained with both AB and AF (AB, AF+) were large and polygonal and frequently possessed long processes; these cells correspond to those which reacted with the TSH antibody. The cells that stained only with AF reacted with the FSH antibody in the P-Ab technique and the cells which reacted with the LH antibody were not stained with either AB or AF.AB,AF+ cells ("TSH" cells) were distributed throughout the anterior lobe except along the lateral and dorsal peripheries of the gland and adjacent to the intermediate lobe, while both the AF+ ("FSH" cells) and the "LH" cells were distributed throughout the anterior lobe.
SELECTIVE REMOVAL OF RIBONUCLEIC ACID RESPONSIBLE FOR HYPERBASOPHILIA IN RAT LIVER PARENCHYMA DURING AZO DYE CARCINOGENESIS
Authors: BRIÈRE NORMAND
Published: April 2011

Certain areas in preneoplastic livers of rats fed the azo dye 4-dimethylaminoazobenzene are characterized by intense ribonucleic acid (RNA) staining and possibly represent the sites of neoplastic transformation. Cytoplasmic RNA responsible for increased basophilia in the hyperbasophilic foci and hepatomas was selectively extracted by incubating liver sections for short periods in solutions containing low concentrations of ribonuclease, without apparently altering basophilia in surrounding tissue. Moreover, experiments on protein staining confirmed that the RNA responsible for hyperbasophilia is the one that binds to and masks the cytoplasmic proteins in these sites. Since hyperbasophilia in sites of neoplastic transformation and liver tumors results from the presence of some RNA which is more sensitive to mild ribonuclease treatment than normally occurring RNA, it is suggested that it might be either a normal type or an altered form of RNA present in excess.
STUDY ON THE SITE OF BIOSYNTHESIS OF β-GLUCURONIDASE AND ITS APPEARANCE IN LYSOSOME IN NORMAL AND HYPOXIC RATS
Authors: VAN LANCKER JULIEN L. , LENTZ PATRICK L.
Published: April 2011

For the purpose of investigating the site of synthesis of β-glucuronidase, the enzyme was purified, after injection of labeled amino acids, from various cell fractions of regenerating rat liver. Enzyme preparations purified from microsomal, lysosomal, mitochondrial, nuclear and supernatant fractions had identical catalytic and electrophoretic properties. In nonhypoxic rats, incorporation was detectable only in the microsomal enzymes and maximum labeling occurred 30 min after the injection of the labeled amino acid. No label was detectable in the enzyme purified from the lysosomal fraction of nonhypoxic animals. Labeling of enzyme purified from lysosomal fraction was, however, significant after 2 hr of hypoxia.
MEMBRANEOUS ULTRASTRUCTURAL DEMONSTRATION OF AMINOPEPTIDASE AND γ-GLUTAMYL TRANSPEPTIDASE ACTIVITIES WITH A NEW DIAZONIUM SALT THAT YIELDS A LIPOPHOBIC, OSMIOPHILIC AZO DYE
Authors: SELIGMAN ARNOLD M. , WASSERKRUG HANNAH L. , PLAPINGER ROBERT E. , SEITO TAKASHI , HANKER JACOB S.
Published: April 2011

When 4-aminophthalhydrazide is freshly diazotized and used as the coupling agent in demonstrating aminopeptidase activity or γ-glutamyl transpeptidase activity, with substrates that yield 4-methoxy-2-naphthylamine or even 2-naphthylamine, azo dyes are produced which are readily osmiophilic. Although the distribution of the reddish azo dyes is similar to that noted with earlier methods in light microscopy, the distribution of the deposits in electron microscopic preparations is distinctly membraneous in location in contrast to the droplet distribution of previous methods. We attribute membraneous localization of enzymatic activity with the new methods presented here to the lipophobic properties of the azo dyes provided by their 4-aminophthalhydrazide (4-APH) moiety. The new agent 4-aminophthalhydrazide was designed with this in mind.
LOSS OF PROTEINS AND OTHER MACROMOLECULES DURING PREPARATION OF CELL CULTURES FOR HIGH RESOLUTION AUTORADIOGRAPHY QUANTITATION BY A MICROMETHOD
Authors: VANHA-PERTTULA TAPANI , GRIMLEY PHILIP M.
Published: April 2011

A reproducible procedure was devised in order to measure the extraction of 3H-labeled cellular products during aldehyde fixation and subsequent processing for electron microscopic autoradiography. Human carcinoma cell monolayers were cultivated in combustible plastic wells, so that all label could be counted as 3H2O. Radioisotope extraction during individual steps of processing could be analyzed and separate groups of experiments were directly comparable. Initial aldehyde fixation and subsequent buffer washes caused the major loss of radiolabeled amino acids, but this never exceeded 15% under conventional conditions. Radioisotope losses were influenced by the relative duration of fixation and buffer washes, fixation temperature and fixative concentration. Formaldehyde and glutaraldehyde both produced a nonspecific, time-related binding effect when 3H-labeled amino acids were introduced along with the fixative. Less significant nonspecific binding was observed when 3H-mannose, 3H-uridine or 3H-thymidine was added. Extraction of radioisotopes during formaldehyde fixation of cell cultures labeled with protein precursors was consistently greater than during glutaraldehyde fixation. Differences were less marked with the other precursors. Evaluation of the total protein extraction is complex, since the net losses observed were apparently the sum of precursor extraction, nonspecific amino acid binding and real molecular extraction. The implications for quantitative interpretative interpretation of high resolution autoradiography must be considered.
A STUDY OF MASS LOSS AND PRODUCT FORMATION AFTER IRRADIATION OF SOME DRY AMINO ACIDS, PEPTIDES, POLYPEPTIDES AND PROTEINS WITH AN ELECTRON BEAM OF LOW CURRENT DENSITY
Authors: STENN K. S. , BAHR G. F.
Published: April 2011

Amino acids, peptides, polypeptides and proteins were irradiated with electrons at 70-kv accelerating potential in an electron microscope mockup. Loss of mass and chemical changes occur almost instantaneously. For a given dose rate an organic object is converted to a steady state product. This is characterized by a poorly structured infrared pattern, unchanging elemental composition. The higher the polymerization of the target the smaller was the effect of the irradiation. Such studies conducted outside of the electron microscope reveal only qualitatively what might occur to the electron microscopic specimen during observation.
AN AUTOMATIC METHOD FOR VIABILITY ASSAY OF CULTURED CELLS
Authors: O'BRIEN REGINA , GOTTLIEB-ROSENKRANTZ PHYLLIS
Published: April 2011

An automatic method for viability assay of a population of cells is described. Cell populations are stained with trypan blue (for dead cells) and fluorescein diacetate (for live cells). A rapid cell spectrophotometer measures changes in light intensity which occur when individual cells pass through the photometric field. Tallies of the number of decreases (absorption and scatter by dead cells) and the number of increases (fluorescence by live cells) in light intensity are recorded and viability percentages are calculated. These automatically determined viability percentages are compared with viability percentages determined by visual counting and classifying of cells from the same populations.
CYTOCHEMICAL LOCALIZATION OF NUCLEIC ACIDS BY AN ACRIFLAVINE-PHOSPHOTUNGSTATE COMPLEX FOR FLUORESCENCE MICROSCOPY AND ELECTRON MICROSCOPY
Authors: CHAN-CURTIS VICTORIA , BELT W. DUANE , LADOULIS CHARLES T.
Published: April 2011

An acriflavine-phosphotungstate complex (A-PTA) was synthesized from a reaction between acriflavine and phosphotungstic acid, with the former in excess. The purified product is fluorescent and electron-dense, and was studied for its use as a cytochemical reagent for fluorescence microscopy and electron microscopy. A selectivity for nucleic acids and sulfated cerebroside esters that excludes sulfated glycoaminoglycans is proposed. The reaction for fluorescence microscopy is rapid, sensitive and reproducible. The electron-dense A-PTA complex localized nucleic acids for electron microscopy. Known sites of DNA and RNA in cells of pancreatic, cerebellar and testicular tissue have been demonstrated with the reaction. The reaction is administered as a single step, interposed in the electron microscopic preparative procedure, and can be used in either an alcoholic or an aqueous system. The alcoholic A-PTA reaction produced adequate contrast on short duration (2 hr); the aqueous block reaction was found most satisfactory when prolonged to 48 hr. Uranyl acetate, phosphomolybdic acid and phosphotungstic acid can be used as supplementary stains in addition to the A-PTA reaction. Under these conditions, DNP and RNP sites were demonstrated.

The A-PTA complex is a prototype of the coupled dye-metal histochemical reagents, and its selectivity for nucleic acids represents a new approach in cytochemistry. Its reactions can be investigated by fluorescence microscopy and electron microscopy, and in solution. Other dye-metal reagents can probably be formulated on a similar design as detection systems for lipids, polysaccharide or specific groups in proteins.
CHEMICAL STUDIES ON AN ACRIFLAVINE-PHOSPHOTUNGSTATE COMPLEX; STAINING OF PHAGE DEOXYRIBONUCLEIC ACID MOLECULES FOR THE ELECTRON MICROSCOPE
Authors: CHAN-CURTIS VICTORIA , BEER MICHAEL , KOLLER THEODOR
Published: April 2011

Electrophoretic studies showed that the acriflavine-phosphotungstate (A-PTA) complex, which was formed by the reaction of acriflavine with phosphotungstic acid (PTA), contained a lower net positive charge than neutral acriflavine alone. Spectral studies from the titrations of specified aliquots of acriflavine with increasing volumes of PTA showed that the formation of A-PTA was characterized by a shift of the absorption peak of acriflavine from 452 mµ to a lower wavelength, at 446 mµ. The saturation of PTA binding sites on the acriflavine occurred when 3 molecules of dye reacted with 1 molecule of PTA. Binding curves of both neutral acriflavine and of A-PTA with "native" and with heat-denatured DNA showed shifts of λmax to longer wavelengths. A-PTA bound less native and less denatured DNA than the dye alone. The coupling of acriflavine with PTA caused a decrease in the net ability of the A-PTA complex to bind DNA. Double stranded DNA molecules from T3 bacteriophage were stained with the A-PTA complex for the electron microscope.
AN ANALYSIS OF THE HISTOCHEMICAL PROCEDURE FOR SODIUM ION DETECTION
Authors: SHIINA SHIN-ICHI , MIZUHIRA VINCI , AMAKAWA TAKANORI , FUTAESAKU YUTAKA
Published: April 2011

Some aspects of the histochemical technique for the detection of sodium ions with potassium antimonate have been analyzed. When potassium antimonate is used for the detection of sodium ions the pH should not decrease below 7.2 during fixation. Potassium antimonate was also precipitated by ethanol used for dehydration, resulting in dilution of the water used as solvent. This should not be confused with the histochemical product. Precipitate may be formed with calcium ions and magnesium ions. Potassium phosphate buffer used to control the pH seems to inhibit the precipitation reaction of potassium antimonate with sodium ions; the nonbuffered fixative produces a good result at the electron microscopic level.
STUDIES OF ACID PHOSPHATASE AND NONSPECIFIC ESTERASE ACTIVITIES IN RAT ADRENAL GLANDS FOLLOWING OPERATIVE STRESS
Authors: CHOUDHURY S. R. , LUNDY A. M.
Published: April 2011

Acid phosphatase and esterase activities were studied in adrenal glands obtained from rats killed at regular intervals following surgical stress (cauda equina transection). Zymograms of acid phosphatase produced by starch gel electrophoresis revealed increased reactivity in the operated samples. With esterases, a slightly different pattern was observed in the operated group, which exhibited a few additional bands particularly in the cathode region. This was confirmed by densitometric analysis of the gel strips. Two of these additional bands were organophosphate-sensitive and the remaining few were activated by p-chloromercuribenzoate. These latter bands appeared to arise from splitting of the preexisting organophosphate-resistant bands present in control zymograms. Biochemical assay of the two hydrolytic enzymes demonstrated a remarkable similarity in their responses to operative stress—probably implying a general lysosomal activation. Both enzymes exhibited a peak activity 8 hr after operation, followed by a gradual decline. Both organophosphate-sensitive and organophosphate-resistant esterases contributed toward the rise in total esterase activity. Histochemical studies on tissue sections revealed a more reactive adrenal cortex in the operated group, but were of little help in localizing the additional esterase activity observed in gel strips.
INTRALAMINAR DISTRIBUTION OF ACID RIBONUCLEASE IN RABBIT CEREBRAL CORTEX
Authors: TAKAHASHI Y. , SUZUKI Y.
Published: April 2011

Acid ribonuclease activity of the cortical layer and the subcortical white matter of area parietalis of rabbit brain was measured microchemically and the distribution of enzyme activity was related to the histologic composition. The cytoarchitectonic distribution pattern of acid ribonuclease showed it to be higher in layers III, IV, V and VI than in layers I and II, and especially high in layer V. The activity in white matter was the lowest among all examined layers.
DETERMINATION OF TRITIUM IN MICROGRAM TISSUE SAMPLES: DISTRIBUTION OF EXOGENOUS 3H-CHOLESTEROL IN THE RAT ADRENAL CORTEX
Authors: PARVINEN MARTTI , NIEMI MIKKO
Published: April 2011

An internal sample metal cathode proportional counter for measurements of low tritium activity in biologic samples of microgram size has been designed. Using a guard detector operating in anticoincidence together with a thick steel castle, a background of 0.5 cpm and an efficiency of 54.6% for tritium were realized. Radioactivities down to 2 pC (4.4 dpm) can be measured with an accuracy of ±16%. Tritium activity was measured in single adrenal cortical sections of 7 µm in thickness and 0.4 mm in diameter having a mean dry weight of 1.2 µg. The cortical layers were identified histologically from alternate sections. Two days after the injection of tritiated cholesterol the highest radioactivity was encountered in the outer fascicular zone (39 dpm/µg). The distribution of the radioactivity within the cortex was equal in all animals, although the mean level varied individually. The glomerulosa and reticularis contained usually about one-third of the activity in the outer fasciculata. Ether anesthesia and unilateral adrenalectomy caused significant decrease in the incorporation of the cholesterol, which was not further reduced by an injection of adrenocorticotropin. Ninety-five per cent of the radioactivity in the adrenals was bound to cholesterol or its esters 2 days after the injection.
MONOAMINES IN THE CAROTID BODY CELLS OF THE CAT
Authors: CHIOCCHIO S. R. , KING M. P. , CARBALLO L. , ANGELAKOS E. T.
Published: April 2011

The presence of monoamines in the carotid body cells of the cat was investigated using histochemical methods, microspectrofluorometry and chemical analyses. Large amounts of norepinephrine (31 to 51 ng/carotid body), dopamine (122 to 131 ng/carotid body) and DOPA (86 to 150 ng/carotid body) were found in pooled tissues analyzed with specific chemical techniques. The majority of the glomus cells exhibited an intense fluorescence after formaldehyde treatment, having a spectrum characteristic of catecholamines. Some, but not all, of these cells gave a fluorescence reaction with the trihydroxyindole histochemical method. It is concluded that most of the glomus cells contain norepinephrine or DOPA or a combination of these two compounds while some cells contain only dopamine. A few yellow fluorescent cells were also found after formaldehyde treatment which probably contain 5-hydroxytryptamine.
ON PHOSPHOTUNGSTIC ACID STAINING. I
Authors: QUINTARELLI G. , ZITO R. , CIFONELLI J. A.
Published: April 2011

In this study histochemical experiments have been carried out in order to understand the "staining mechanism" of phosphotungstic acid (PTA). One of the main objectives of this project was to investigate the mode of interaction of the heavy metal and to define the type of functional groups in the substrates responsible for PTA binding. Therefore, tissues containing known macromolecules were selected and utilized as model systems. Epiphyseal cartilage, rat sublingual glands, human bone and purified collagen were used to study the interaction of the polyacid with chondroitin sulfate (cartilage), sialic acid (sublingual gland) and collagen (purified collagen and bone). The results obtained suggested that PTA does not interact with chondroitin sulfate, with sialic acid or with the hydroxyl groups of the sugar moieties of these macromolecules. Rather, the binding appeared to be selective for positively charged groups. Since PTA interaction to organic cations was pH-dependent, it is suggested that the heavy metal binds by means of coulombic forces and that no hydrogen bonds are involved.
ULTRASTRUCTURAL LOCALIZATION OF A SOLUBLE COLLAGEN ANTIGEN
Authors: LUSTIG LIVIA
Published: April 2011

Soluble collagen antigen was localized at the ultrastructural level using an enzyme-labeled antibody technique. Specific antiserum against neutral salt-soluble collagen was conjugated with horseradish peroxidase. Tissue culture of fibroblasts, sections of chick embryo connective tissue and skin and tendon of newborn and adult chickens were incubated with the conjugated antiserum and processed for light and electron microscopy. Light microscopic observations showed a localization of the collagen antigen in the cytoplasm of fibroblasts and in the extracellular collagen fibers. Ultrastructurally, the antigen was found on the ribosomes, on the membranes of the rough endoplasmic reticulum, in the intracisternal material of fibroblasts and extracellularly in the collagen fibrils. The collagen fibrils of chick embryo cartilage were intensely stained while the matrix itself remained unstained. Controls performed with absorbed antiserum, conjugated normal rabbit serum or different antigens failed to react with the tissues.