bluuebunny/crossref_metadata_2025 huggingface dataset:
AMYLOID. VI. A COMPARISON OF TWO MORPHOLOGIC COMPONENTS OF HUMAN AMYLOID DEPOSITS
Authors: GLENNER G. G. , KEISER H. R. , BLADEN H. A. , CUATRECASAS P. , EANES E. D. , RAM J. S. , KANFER J. N. , DELELLIS R. A.
Published: April 2011

Two morphologic components of human amyloid deposits, the periodic rod and the fibril, have been prepared free of cross-contamination. On tryptic digestion of guanidine-denatured material, peptide maps indicate these two components to have characteristic patterns with no detectable peptide fragments in common. Additional studies on ultrastructural morphology, physical characteristics and chemical composition reveal that the two components are clearly distinct entities with no evidence that one is a derivative of the other. The periodic rod, composed of stacked pentagonal unit structures or "doughnuts," is an antigenic globular glycoprotein deficient in tryptophan and methionine. The fibril, composed of laterally aggregated unit structures or filaments, is a poorly or nonantigenic fibrous glycoprotein of "pleated sheet" type containing both tryptophan and methionine. Congo red staining and birefringence, a dual characteristic of the fibrils and filaments, is dependent upon the physical integrity of these structures.
METACHROMATIC PROPERTIES OF AMYLOID IN SOLUTION
Authors: PRAS MORDECHAI , SCHUBERT MAXWELL
Published: April 2011

The chromotropic properties of water-soluble amyloid are quantitatively studied in solution with the metachromatic dyes, crystal violet and toluidine blue and compared with the properties of chondroitin sulfate of bovine nasal cartilage. The stabilities of the metachromatic compounds are measured in terms of the ethanol concentration required to cause the return of the depressed α band to half its peak value in water alone. The metachromatic properties of amyloid stand in sharp contrast to those of chondroitin sulfate: amyloid compounds of toluidine blue and crystal violet are about equally stable, but the chondroitin sulfate compound with toluidine blue is far more stable than either, and the chondroitin sulfate compound with crystal violet is far less stable than either. An attempt to measure the equivalent weight of amyloid by studying the depression of the α band of each of these dyes, as well as of acridine orange, as a function of amyloid concentration gave values of 7000, 6000 and 3000 for three different preparations.
Displaced scaphoid waist fractures: the use of a week 4 CT scan to predict the likelihood of union with nonoperative treatment
Authors: Amirfeyz R. , Bebbington A. , Downing N. D. , Oni J. A. , Davis T. R. C.
Published: April 2011

This study assessed whether nonunion of displaced scaphoid waist fractures with nonoperative treatment could be predicted from 4 week CT scans. Thirty-one patients with unilateral displaced scaphoid waist fractures and adequate follow-up were included. CT scans in the longitudinal axis of the scaphoid with sagittal and coronal slices were done 4 weeks after the index injury. The effects of fracture gap, sclerosis and bone resorption on union were assessed. Fracture union was observed in all 13 displaced fractures with a <2 mm gap, four of the seven with a gap of 2–3 mm and only four of the 11 with a gap >3 mm ( p = 0.01). Bone resorption involving more than 50% of the fracture cross-section was also associated with nonunion, but sclerosis was not.
ISOLATED HISTONE FRACTIONS AND THE ALKALINE FAST GREEN REACTION
Authors: BERLOWITZ LAURENCE , PALLOTTA DOMINICK , PAWLOWSKI PHILIP
Published: April 2011

When the alkaline fast green reaction is applied to calf thymus histone fractions f1, f2a, f2b and f3 on Millipore filter paper it is found to stain equal amounts of these fractions equally well. The dye reaction, therefore, seems to depend on the over-all charge and not exclusively on the per cent arginine plus lysine in the histone fractions. This staining reaction has been applied to histone fractions separated by acrylamide gel disc electrophoresis. Deamination of the histone fractions on both paper and acrylamide gel resulted in a reduction in the intensity of the stain. The stain that remained followed the relative amount of arginine in each fraction or band. In the gels, the f1 band was removed completely. This confirms previous interpretations of the cytochemical deamination reaction. The stain reactions have been utilized as a means of comparing electrophoretically resolved histone bands of similar mobility from crickets and mealy bugs.
DISTRIBUTION OF LEUCYL NAPHTHYLAMIDASE AND ALKALINE PHOSPHATASE ON THE VILLI OF THE CHICK DUODENUM
Authors: GREY ROBERT D. , LECOUNT THOMAS S.
Published: April 2011

Serial samples from successive levels of the villi and crypts were collected by sectioning a flattened piece of frozen duodenum parallel to the gut wall. Hatched chicks of 1 and 4 weeks of age were studied. Groups of sections were pooled for assay of enzyme activity and protein content. In both age groups, leucyl naphthylamidase was present in the crypts and highest at the bases of the villi while alkaline phosphatase (EC 3.1.3.1) was low or absent in the crypts and highest at the villi tips. Alkaline phosphatase at all levels of the villi hydrolyzed phenyl phosphate faster than β-glycerophosphate; the ratio of activities on these substrates was uniform along the length of the villi, but differed between age groups.
ULTRASTRUCTURAL LOCALIZATION OF OXIDASE ACTIVITIES IN CORN ROOT TIP CELLS WITH TWO NEW OSMIOPHILIC REAGENTS COMPARED TO DIAMINOBENZIDINE
Authors: NIR IZHAK , SELIGMAN ARNOLD M.
Published: April 2011

The ultracytochemical potential of N,N'-bis(4-aminophenyl)-1,3-xylylene-diamine (BAXD) and N,N'-bis(4-aminophenyl)-N,N'-dimethylethylenediamine (BED) as electron donors for plant oxidases was tested and compared to 3,3'-diaminobenzidine (DAB). Incubations were 50-60 min, and osmication was for 60 min. Root meristematic cells which are rich in a variety of organelles and enzyme activities were used. Upon oxidation, BAXD produced intense staining of mitochondrial, amyloplast, Golgi and vacuolar membranes. Very intense homogenous staining of vesicles which varied in size and shape was also noted. BED in general was less readily enzymatically oxidized. This was seen as less intense or less frequent staining, particularly on mitochondrial cristae, when compared to BAXD. However, endoplasmic reticulum, plasmalemma and nuclear membrane were better stained with BED. Counterstaining was not used. In control experiments, inactivation by heat and by 2.5 hr of glutaraldehyde fixation were strong indications for the enzymatic nature of the oxidation. Experiments with catalase and peroxidase inhibitors provided evidence against the participation of these enzymes in the staining reactions. On the other hand, the oxidations were sensitive to cyanide (10–3 M). This concentration prevented almost completely the oxidation of BAXD, BED and DAB. Azide (10–2 M) was also an effective inhibitor, although less than 10–3 M cyanide. Although the precise nature of BED oxidase is not yet known, it may be of interest to compare these results in plants with those already published for rat liver and rat heart (Seligman AM, Wasserkrug HL, Plapinger RE: Histochemie 23:63, 1970).
STANDARDS FOR ELECTRON PROBE MICROANALYSIS OF BIOLOGIC SPECIMENS
Authors: LEHRER G. M. , BERKLEY C.
Published: April 2011

A procedure is described for the preparation and application of gelatin standards in the quantitative elemental analysis of microscopic portions of tissue sections in the electron microprobe x-ray spectrometer. Ten to fifteen per cent gelatin solutions containing varying concentrations of the elements to be measured are quick-frozen in capsules, sectioned in a cryostat at the same thickness as the tissue and freeze-dried. Standard sections are mounted with each tissue section, carbon-coated and analyzed simultaneously. Concentrations of the elements in portions of the tissue as small as 10–12 liters are then determined directly from a regression curve or equation derived from the standards.
STAINING OF BLOOD CELL SURFACES WITH A LECTIN-HORSERADISH PEROXIDASE METHOD
Authors: PARMLEY RICHARD T. , MARTIN B. J. , SPICER S. S.
Published: April 2011

Human and rabbit blood cell surfaces were stained by a lectin-horseradish peroxidase (HRP) technique using either concanavalin A (Con A) or Lens culinaris hemagglutinins (LcH). Intensity of staining of the human cells with the Con A-HRP method decreased in the order: platelets, mononuclear cells, granulocytes, erythrocytes. Cell surface staining of mature cells was similar to the bone marrow precursor cells as well as to leukemic blast cells. Surface staining of erythrocytes correlated with agglutinability of these cells with Con A or LcH. Differing ability of Con A and LcH to allow penetration of diaminobenzidine into myeloid cells indicated stearic differences in their binding to the cell surface. The Con A-HRP staining of cell surfaces was prevented on all cells by prior periodate oxidation and on cells other than erythrocytes by prior acetylation but was not altered by methylation or sialidase digestion.
HISTOCHEMICAL CHARACTERIZATION OF ADENOSINE TRIPHOSPHATASES IN SKELETAL MUSCLE FIBERS BY SELECTIVE EXTRACTION PROCEDURES
Authors: SCHIAFFINO STEFANO , BORMIOLI SANDRA PIEROBON
Published: April 2011

Selective extraction procedures have been applied to frozen sections of rat skeletal muscles to determine the relative contribution of myosin adenosine triphosphatase (ATPase) and of membrane-bound (mitochondrial and sarcotubular) ATPases to the histochemical ATPase reactions demonstrated in muscle fibers by the calcium precipitatation mehod (Padykula-Herman procedure) and by the lead precipitation medium (Wachstein-Meisel procedure). A detergent treatment was used to solubilize membrane-bound enzymes and hypertonic KCl solution to extract myosin. Pretreatment with the non-ionic detergent, Triton X-100, severely depressed the ATPase activity demonstrated by the lead method, as well as several membrane-bound dehydrogenase activities, but did not affect the intensity of the ATPase reaction demonstrated in the muscle fibers by the calcium method. After detergent treatment, however, the end product of the latter reaction showed a network distribution which contrasted with the characteristic myofibrillar localization observed in untreated sections. Hypertonic KCl pretreatment, when applied to undried sections, abolished the ATPase reaction demonstrated in the muscle fibers by the calcium method; the ATPase demonstrated by the lead method was slightly depressed and the dehydrogenase activities unmodified by the same treatment. The results indicate that the ATPase activity demonstrated histochemically in skeletal muscle fibers by the calcium method is due to myosin ATPase, and suggest that the intermyofibrillar localization of the end product which occurs under certain conditions is artifactual.
A DECISIONAL MODEL OF RECOGNITION APPLIED TO THE CHROMOSOME BOUNDARIES
Authors: GALLUS GIUSEPPE , REGOLIOSI GIUSEPPE
Published: April 2011

A new approach is presented for the analysis of the chromosome boundary, based on the following philosophy: starting from the most relevant information of the boundary, the program follows the most reliable path of recognition (out of a large number) shifting continuously between local and contextual criteria. Feedback procedures allow the modification of decisions already taken, on the bases of results of computations carried out at a higher level. The presented results concern the analysis of 40 unbanded cells and 20 banded ones. The error rate, less than 5%, represents a sharp improvement in comparison with our previous methods.
HISTOCHEMISTRY OF MUSCLE SPINDLES IN SNAKES OF NATRIX SPECIES
Authors: PALLOT D. J. , TABERNER JANIS
Published: April 2011

The muscle spindles of snakes consist of a single intrafusal fiber; in addition to this, two types of spindles are found. We have studied the histochemistry of the snake intrafusal fibers. One type of spindle, the long capsule spindle intrafusal fiber, is characterized by high levels of the enzymes myosin adenosine triphosphatase, succinic dehydrogenase and phosphorylase; the other type, the short capsule spindle intrafusal fiber, is characterized by low levels of myosin adenosine triphosphatase and phosphorylase and an intermediate level of succinic dehydrogenase. The short capsule spindle intrafusal fiber is thus histochemically similar to the tonic extrafusal fibers, whereas the long capsule spindle intrafusal fiber is similar to the twitch extrafusal muscle fibers. The long capsule spindle is concerned mainly with monitoring static length, the short capsule spindle with monitoring changes in length. It is interesting that the histochemical profiles of long and short capsule spindle intrafusal fibers are similar to the mammalian bag and chain fibers, respectively.
The cytochemical localization of succinic dehydrogenase in the sperm mitochondria of the gastropod Biomphalaria glabrata. A comparison of two methods.
Authors: Bogitsh B J
Published: April 2011

The mitochondrial derivative of the sperm of the gastropod pulmonate Biomphalaria glabrata was studies to ascertain succinic dehydrogenase localization cytochemically. Two techniques were compared. One technique depends on a tetrazolium salt that yields an osmiophilic formazan upon reduction. The other technique is dependent on the reduction of copper ferricyanide. The effects of several electron transport inhibitors were studied. The reaction product observed in the matrix of the mitochondrial derivative using the former technique is sensitive to rotenone and is believed to be nicotinamide adenine dinucleotide-dependent. The reaction product observed in the intracristal spaces using the copper ferricyanide method is insensitive to rotenone and is believed to cytochemically demonstrate succinic dehydrogenase in this material.
7-Amino-actinomycin D as a cytochemical probe. I. Spectral properties.
Authors: Gill J E , Jotz M M , Young S G , Modest E J , Sengupta S K
Published: April 2011

The optical absorption and fluorescence characteristics of 7-animo-actinomycin D were determined to evaluate its potential as a fluorescent cytochemical probe. At pH 7.0, the absorption maximum and fluorescence excitation maximum are both at 503 nm; the fluorescence emission is at 675 nm. When this compound forms complexes with DNA in solution, the absorption and fluorescence excitation maxima shift to 543 nm and the fluorescence emission shifts to 655 nm. The fluorescence quantum yield is 0.016 for 7-amino-actinomycin D free in solution and 0.01-0.02 for complexes with native DNA. The 7-amino-actinomycin D also exhibits fluorescence shifts characteristic of binding when put into solution with poly(dG-dC) poly(dG-dC), but not with poly(dI-dC) poly(dI-dC). The spectral characteristics are the same at pH 7.0 whether the solvent is 0.01 M PO4 with 0.0001 M EDTA or Earle's salts with 0.025 M N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid.
Localization of a thrombin-sensitive calcium pool in platelets.
Authors: Sato T , Herman L , Chandler J A , Stracher A , Detwiler T C
Published: April 2011

Electron microscopic x-ray microprobe analysis of pyroantimonate precipitates in platelets fixed in osmium tetroxide-pyroantimonate revealed calcium localization in the nucleoids of alpha-granules. This pool of calcium had largely disappeared within 10 sec after stimulation of platelets by thrombin. Such a rapid change suggests that this calcium pool may have a regulatory role in stimulus-response coupling.
Dipotassium tetramethyl osmate: a stain for electron microscopy.
Authors: Hinckley C C , Murphy J A
Published: April 2011

Methanol solutions of dipotassium tetramethyl osmate (DTMO) have been found to be useful as general stains in electron microscopic studies of plant and fungal ultrastructure. The stain solutions are easy to prepare, stable when anhydrous and convenient to use. Although generally similar in staining to lead citrate stains, some elements of cell ultrastructure appear different with dipotassium tetramethyl osmate staining, particularly the outer cell walls of fungi. Indications of specific precipitate-producing reactions in cell storage areas are observed.
Agarose beads as matrices for proteins in cytophotometric investigations of immunohistoperoxidase procedures;.
Authors: Streefkerk J G , van der Ploeg M , van Duijn P
Published: April 2011

Quantitative aspects of direct immunohistoperoxidase procedures were studied in a model system consisting of agarose beads to which antigens or antibodies had been coupled. It could be proven that the final amount of reaction product resulting from the histoperoxidase reaction with 3,3-diaminobenzidine-tetra HCl in a bead was linearly related to the volume of the beads and to the staining time. This implies that protein-coupled agarose beads are a suitable model for the study of stoichiometric aspects of immunologic reactions in immunohistochemistry as well as in general immunologic methods when peroxidase is used as the protein marker.
Selective subcellular localization of cations with variants of the potassium (pyro)antimonate technique.
Authors: Simson J A , Spicer S S
Published: April 2011

Fixation of rat parotid with an unbuffered osmium tetroxide solution containing nearly saturated potassium (pyro)antimonate resulted in abundant deposition of cation-antimonate precipatates in acinar cells. Altering the antimonate concentration, including buffers or chelators in the solution or changing the primary fixative resulted in an altered intensity and distribution of the precipitates formed in the tissue, apparently reflecting a degree of selectivity in ion localization. Decreasing the concentration of pyroantimonate to about half-saturation preserved predominantly the less soluble antimonate salts (e.g., Na+, Ca++) and resulted in preferential retention of deposits along the plasmalemma and in mitochondrial "dense bodies," with loss of most cytoplasmic and nuclear precipitates. A similar pattern was seen if fixation with the high concentration antimonate-osmium procedure was followed by a prolonged rinse. Adding phosphate or collidine buffers markedly decreased precipitates in the nuclei and on granular reticulum as well. Phosphate buffer or ehtyleneglycoltetraacetate inhibited in vitro precipitation of calcium and sodium and decreased or abolished plasmalemmal deposits. Glutaraldehyde fixation, either in the presence of antimonate or prior to antimonate-containing osmium tetroxide, abolished heterochromatin deposits. Mitochondrial dense bodies were of two types, one containing precipitate and the other inherently osmiophilic. The latter were also observed in pyrophosphate-osmium controls. Results from in vitro titrations of cations with the various antimonate methods and from neutron activation analyses of fixed tissues supported conclusions drawn from fine structural distribution patterns and were interpreted as follows. In rat parotid acinar cells, deposits in heterochromatin and on granular reticulum probably arose from precipitation in sites of high K+ and H+ as well as--NH3+-rich histones. Plasmalemmal antimonate deposits demonstrated sites of sodium and/or calcium accumulation. Some mitochondrial dense bodies contained Ca++ whereas others were inherently osmiophilic. Large, extracellular deposits were probably predominantly sodium precipitates.
Cytochemical localization of peroxisomes in Tetrahymena pyriformis.
Authors: Fok A K , Allen R D
Published: April 2011

Pronounced staining of Tetrahymena pyriformis peroxisomes was demonstrated when glutaraldehyde-fixed cells were incubated in an alkaline medium containing 3,3'-diaminobenzidine and hydrogen peroxide. A variable amount of electron-opague deposit was observed when cells were incubated in diaminobenzidine and H2O2 for 1 hr while an intense deposit followed incubation for 4 hr in the same medium. The staining was abolished completely when 3-amino-1,2,4-triazole, KCN or NaN3 was added to the incubation medium. Based on these cytochemical observations and the morphologic identification by size, shape and other ultrastructural details, it is suggested that this study presents evidence for a conclusive morphologic identification of Tetrahymena peroxisomes.
Separation and partial characterization of surface-active fractions from mouse and rat lung homogenates. Identification of a possible marker system for pulmonary surfactant.
Authors: Hitchcock-O'Hare K , Meymaris E , Bonaccorso J , Vanburen S B
Published: April 2011

The purpose of this study was to identify a "marker system" for pulmonary surfactant. A density gradient centrifugation procedure was developed to concentrate the surface-active material in mouse and rat lung homogenates. These fractions contained morphologic variants of surfactant such as lamellar bodies and tubular myelin. Lipid and protein analyses were consistent with other techniques indicating that these preparations were surface active. A comparison of the nonspecific esterases in these and other surfact-active preparations (i.e., pulmonary lavage fluid and isolated type II cells) allowed the identification of certain surfactant-associated esterases which are distinguishable electrophoretically and cytochemically from the other pulmonary esterases and which, therefore, may be used as "markers" for surfactant. Possible uses for these enzyme markers in studies of (a) the sites of synthesis and routes of clearance of surfactant and (b) the assessment of variations in surface activity due to either diseases such as the respiratory distress syndrome or to experimental manipulation are discussed.
Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.
Authors: Shepard N , Mitchell N
Published: April 2011

The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.
A phenomenon of photoenhancement of acridine orange-induced fluorescence.
Authors: De D N , Bhanja P
Published: April 2011

It is a common experience that, with exposure to exciting radiation of the fluorescence microscope, the acridine orange-induced red fluorescence of the nucleus, produced by Feulgen hydrolysis, fades with a concomitant shift to green. The present investigation reports a phenomenon of photoenhancement observed in the hydrolyzed cytoplasm where pale green fluorescence increases in intensity with exposure to exciting radiation. The phenomenon has been noticed in Rhizobium, Oscillatoria, tomato root tip cells and human buccal epithelial cells. It is tentatively concluded that the gain in fluorescence yield is due to certain conformational changes of the acridine orange-protein complex induced by ultraviolet light flux.
Organization of the hypothalamic-pituitary system: current concepts from immunohistochemical studies.
Authors: Zimmerman E A , Antunes J L
Published: April 2011

Immunoperoxidase technique and light microscopy have been used to localize neurosecretory systems for vasopressin, oxytocin and related neurophysins (neurohypophysial peptides)) and gonadotropin-releasing hormone (Gn-RH) in the rhesus monkey brain. All the neurohypophysial peptides were found in the magnocellular nuclei (suproptic and paraventricular) of the hypothalamus and in their projections to the posterior pituitary gland, the zona externa of the median eminence and the organum vasculosum of the lamina terminalis (OVLT). Gn-RH was found in smaller cell bodies which were widely scattered in the hypothalamus. Some of these were found in the media-basal hypothalamus in the infundibular nucleus and lateral and dorsal to it, while others were found in dorsal hypothalamus. Numerous cells were also located in the preoptic area close to the OLVT. Gn-RH-containing fibers projected to the OVLT and the zona externa of the median eminence. The two neurosecretory systems studied have two common features: magnocellular perikarya containing the neurohypophysial peptides and smaller elements containing Gn-RH are found near and appear to terminate around the fine vessels of the OVLT. In addition, cells of both systems send fibers to the hypophysial portal capillary system in the zona externa of the median eminence. Many more vasopressin-than oxytocin-containing fibers end in the entire expanse of the zona externa, where they are mainly concentrated in the anterior and middle parts, while Gn-RH fibers project to all portions.
A cytochemical study of the bag cell organs of Aplysia californica.
Authors: Smock T , Arch S
Published: April 2011

Egg-laying hormone in Aplysia californica is synthesized and secreted by cells that seem to be homogeneous ultrastructurally and electrophysiologically. Several conventional methods have been used to demonstrate histochemical homogeneity and special staining techniques based on the known properties of the hormone show the neuroendocrine organ to be uniform in appearance. Furthermore, since stain specificity for egg-laying hormone is demonstrable using release and biochemical studies, the authors concluded that the organ consists of a population of biochemically homogeneous neurons.
Alkaline phosphatase activity in human bladder tumor cell lines.
Authors: Benham F , Cottell D C , Franks L M , Wilson P D
Published: April 2011

The cellular localization and isoenzyme pattern of alkaline phosphatase in five cell lines derived from human bladder carcinomas (T24, RT4, RT112, J82, EJ) shown not to be HeLa cells has been established. RT112 cells had a high level of alkaline phosphatase. RT4 had a moderate amount of alkaline phosphatase but in the other three lines, levels were extremely low. Prednisolone caused a small (2 to 3-fold) increase in total alkaline phosphatase in T24 and RT112 lines only. Electrophoretic separation of isoenzymes showed that RT112 and RT4 cells (derived from more highly differentiated tumor types) had three heat stable bands equivalent to placental alkaline phosphatase and three slower bands of a modified placental type. Prednisolone increased only the former. In T24 cells the enzyme resembled the liver-type alkaline phosphatase in electrophoretic mobility and sensitivity to heat denaturation. Cytochemical studies confirmed the presence of cell surface-associated extramembraneous placental type enzyme in RT112 cells. All five cell lines had small deposits of intramembraneous alkaline phosphatase in the plasma membrane and deposits associated tith the mitochondrial membranes and the endoplasmic reticulum that were not completely inhibited by phenylalanine or Levamisole.
Automated classification of cells in electron microscopic images of lymphoreticular tissue.
Authors: Pfoch M , Kade W
Published: April 2011

In classifying cells in tissue sections, one must consider the fact that only random projections of cells and of subcellular structures are available in the two-dimensional image. Therefore, measurement values that solely reflect the size of such projections cannot be taken on their own as a basis for cell classification. More complex morphologic features such as shape, texture and distribution pattern of cells and their components should be analyzed. Using cell nuclei as an example, the relationship between such features and geometric measurement values is evaluated. It can be shown that a well balanced combination of geometric parameters provides a suitable basis for reproducing the visual preclassification of lymphocytes in tissue sections. Moreover, using a cluster algorithm, which allows different levels of similarity to be defined, a hierarchical sequence of subclusters turns out, indicating the heterogeneity of the visually determined cell classes. Whether or not these subclusters can be correlated to functionally defined subpopulations of lymphocytes remains a matter for further investigation.
Imaging systems for correlation of false alarms in flow.
Authors: Wheeless L L , Kay D B , Cambier M A , Cambier J L , Patten S F
Published: April 2011

False alarms, arising from a variety of sources, are the greatest remaining obstacle to development of an automated prescreening system for gynecologic cytology. This paper describes two correlation systems under development at the University of Rochester and discusses their utilization in the study of false alarms in slit-scan cytofluorometry. Both systems permit imaging of objects in flow and correlation between images and corresponding slit-scan contours. Correlation systems will permit a detailed study of false alarm causes and aid in the search for new features to assist in their recognition.
The application of cytochemistry to automation.
Authors: Kaplow L S
Published: April 2011

The application of cytochemical techniques for use with automated instruments introduces a variety of problems not encountered with traditional cytochemical stains prepared for human visualization. This is particularly true for flow-cytophotometers, which require that cells be stained in suspension, making it difficult to remove reagents once they are added to a staining mixture. It is much less a problem for pattern recognition instruments. The major difficulties relate to cell preparation, fixation, staining, controls and interpretation of instrumental data. The new cytochemical techniques or modifications of existing methods that are required for use with such instruments present a fresh challenge to cytochemists. The need for such methods is great and the rewards may be equivalent.
Cytochemical demonstration of sodium, potassium-adenosine triphosphatase by a hemepeptide derivative of ouabain.
Authors: Mazurkiewicz J E , Hossler F E , Barrnett R J
Published: April 2011

A cytochemical probe for the ultrastructural localization of the NaKATPase was devised, which utilizes the biological affinity of the noncompetitive inhibitor, ouabain (ouab) to which was coupled a hemepeptide (H11P) which possesses peroxidatic activity. The conjugate, ouab-H11P, had an apparent Ki of approximately 8 x 10(-7) M. When reacted with fixed tissue from the salt gland of osmotically stressed ducklings, the NaKATPase was localized to the basal and lateral infoldings of the plasma membranes of secretory epithelial cells. Reaction product consisted of fine textured deposits distributed in focal patches on the outer aspects of the membrane. Apical membranes were negative, as were intracellular membrane components. Preincubation of tissue with unlabeled ouabain or binding of ouab-H11P in the presence of 10 mM K+, no ATP and no Mg++, resulted in the absence or diminution of reaction product.
Use of hydrogen peroxide-egg albumin to eliminate nonspecific staining in immunoperoxidase techniques.
Authors: Zehr D R
Published: April 2011

The application of hydrogen peroxide and egg albumin to paraffin sections before immunostaining prevents nonspecific staining by immunoperoxidase techniques. This method is more effective than pretreating secions with normal sera, or using either egg albumin or hydrogen peroxide separately, or using diluted antisera with prolonged incuabations in the staining procedure.
DNA cytophotometry of human chromosomes.
Authors: Groen F C , Van der Ploeg M
Published: April 2011

The applicability of Feulgen-based parameters to detect variant metaphase chromosomes involved in deletions or translocations, was investigated and algorithms developed to compute such parameters. This report is focused primarily on the magnitude of the errors involved during the prerequisite procedures of photography, measurement and computation. Measurements were performed by stage-scanning of photographic negatives of Feulgen-stained metaphases. In the scanned images the initial chromosome boundaries were obtained by thresholding, while definite chromosomal areas and local background values were obtained by expansion of the initial boundaries. The integrated density profiles and the relative DNA content were computed for the individual chromosomes (straight as well as bent). Total DNA content, DNA arm ratio, as well as length and centromere index can be obtained from the profile. It was shown that under such conditions the experimental errors associated with the measurements are small compared to biologic variations (e.g., differences between homologues) and that the procedures applied allow to detect polymorphisms. In addition to this, mean and standard deviations of both DNA and length parameters are given for metaphases of five subjects. Comparison of the applicability of DNA and length parameters is realized by a classification experiment.
Cell-cycle phase-dependence of drug-induced cycle progression delay.
Authors: Göhde W , Meistrich M , Meyn R , Schumann J , Johnston D , Barlogie B
Published: April 2011

The effect of adriamycin on cell cycle phase progression of CHO cells synchronized into the various phases of the cell cycle by elutriation was investigated by high resolution pulse cytophotometry. Cells treated in all phases of the cell cycle showed delay in their subsequent progression. In addition to the wellknown block of cells in the G2-phase, a delay in passage of cells from G1 to S and a decreased rate of transit through the S-phase were observed. A broadening of the DNA distributions of the treated cells was observed after cell division indicating induction of chromosomal abnormalities.
Fluorescence background discrimination by prebleaching.
Authors: Hirschfeld T
Published: April 2011

A number of electrooptical techniques are described that discriminate against background fluorescence in biologic staining, whether from sample background or unbound excess stain. These techniques are based on the fluorescent decay lifetime difference between bound stain and the sample background or between the bound stain its free form. The fluorescence decay lifetimes may be measured either directly or in a combination gated photometry scheme to substantially enhance the sample background contrast. An alternative procedure uses the photochemical bleaching of fluorescent dyes under intense exposure to time discriminate with higher selectivity, sensitivity and in a more convenient fashion between diverse fluorescent molecules.
Reproductive tract peroxidases as endproducts of estrogen-specific gene expression.
Authors: Anderson W A , Burnett C
Published: April 2011

Estrogen is an essential requirement for the postpubertal trophic development and maintenance of the differentiated state of the oviduct, uterus, cervix, vagina and mammary glands of mammals. Estrogen, apparently functioning through its specific cytoplasmic receptor protein via a multistep interaction pathway induces gene expression of specific biochemical events leading to growth and differentiation of target tissues (Jensen et al., Proc Natl Acad Sci, 59:632, 1968; Gorski et al., Recent Prog Horm Res 24:45, 1968). One biochemical expression of the estrogen gene is the synthesis of specific mRNA transcripts for certain specific marker proteins, including ovalbumin, lysozyme and ovomucoid in the chick oviduct (O'Malley and McGuire, Proc Natl Acad Sci 60:1527, 1968; Palmiter and Schimke, J Biol Chem 248:1502, 1973), tubulin in the mammalian oviduct (Brenner and Anderson, Handbook of Physiology 7(2):123, 1973; Brenner et al., Endocrinology 95:1094, 1974) and peroxidase (EC 1,11.1.7) in the rodent uterus (Brockelmann and Fawcett, Biol Reprod 1:59, 1969; Churg and Anderson, J Cell Biol 62:449, 1974; Anderson et al., J Cell Biol 64:668, 1975).
Comparison between peroxidase-conjugated antigen or antibody and peroxidase-anti-peroxidase complex in a postembedding procedure.
Authors: Tougard C , Tixier-Vidal A , Avrameas S
Published: April 2011

The staining efficiency of peroxidase labeled immunoglobulin conjugate, used either as antigen or as antibody, has been compared with that of peroxidase-anti-peroxidase complex (PAP) on ultrathin sections of araldite embedded material. The conjugate gave positive results in a two layer method as well as in a three layer method when used as antibody. No staining was observed when it was used as antigen. The conjugation seemed to impair the antigenic reactivity of immunoglobulin. The conjugate when used as antibody in the three layer method gave approximately the same staining efficiency as PAP.
Horseradish peroxidase labeled antisera--a diagnostic method for evaluating the percentages of B lymphocytes in peripheral blood.
Authors: Middleditch P R , Minard B J , Cawley L P , Bastian F O
Published: April 2011

Horseradish peroxidase labeled antisera supplied commercially was used to evaluate B lymphocytes in peripheral blood. This technique not only gave comparable results to the conventional fluorescein method, but also proved to be more advantageous.
Immunohistochemical localization of exocrine enzymes in normal rat pancreas.
Authors: Bendayan M , Ito S
Published: April 2011

On the basis of morphological and biochemical differences, the exocrine pancreatic tissue has been divided in peri- and teleinsular regions. In the present study the enzymatic profile of these regions has been investigated by the immunofluorescent technique using antibodies against nine pancreatic enzymes (alpha-amylase, lipase, chymotrypsinogen A, trypsinogen, elastase, carboxypeptidase A and B, DNase and RNase A). These antibodies were specific to their antigens without cross reaction. By immunofluorescence, most acinar cells of the normal rat pancreas were positive to the nine enzymes tested. However, an inhomogeneity in the staining pattern was found; specifically, the cells located in the periinsular region of many islets showed a brighter fluorescence than acinar cells in the teleinsular tissue. These data add a new parameter to describe the inhomogeneity of the exocrine pancreas.
Immunohistochemical localization of procollagens. II. Electron microscopic distribution of procollagen I antigenicity in the odontoblasts and predentin of rat incisor teeth by a direct method using peroxidase linked antibodies.
Authors: Karim A , Cournil I , Leblond C P
Published: April 2011

In an attempt to locate procollagen I in rats odontoblasts, antibodies raised in rabbits were purified by affinity methods and linked to peroxidase. They were then incubated with chopped slices from the growing end of rat incisor teeth. The antibodies binding to the antigens in the slices were visualized by reacting the peroxidase moiety with diaminobenzidine in the presence of hydrogen peroxide. The slices were then embedded in Epon and sectioned for ultrastructural study. Within odontoblasts, the immunostaining indicative of procollagen I antigenicity is moderate in rough endoplasmic reticulum cisternae, strong in spherical and cylindrical Golgi distensions, intense in secretory granules, and variable in lysosomal structures. In predentin, immunostaining is intense close to the odontoblast layer, but decreases gradually in a distal direction. Hence, procollagen I (and/or substances endowed with similar antigenicity such as pro alpha (I) chains and procollagen fragments) is present: 1) along the intracellular pathway of collagen precursors where its concentration gradually increases to reach a maximum in secretory granules; 2) in predentin, into which it is released from the granules for transformation into nonimmunoreactive collagen I; and 3) in lysosomal structures where some of it is hydrolyzed.
Alteration of immunoglobulin-bearing lymphoma cells by fixation.
Authors: Warnke R
Published: April 2011

Four large cell lymphomas known to be monoclonal B-cell proliferations were studied with immunofluorescent and immunohistochemical methods for the detection of kappa- and lambda-light chains. Frozen sections of lymphoma tissues as well as formalin and B-5-fixed tissues embedded in paraffin were studied. Both immunofluorescent and immunohistochemical methods gave similar results on frozen sections; however, a number of discrepancies were noted between the results obtained on fixed tissues and those obtained on frozen tissues. In an effort to identify a fixative which did not alter immunoglobulin (Ig), mouse lymph nodes were fixed in different fixatives before Ig detection; but all of the fixatives tested destroyed the Ig present on normal cortical B lymphocytes. Immunoglobulin-bearing normal and neoplastic lymphocytes are better detected on frozen sections than on paraffin sections after routine fixation.
Heterogeneous enzyme immunoassay.
Authors: O'Beirne A J , Cooper H R
Published: April 2011

During the past 10 to 15 years immunoassays have gained acceptance as the methods of choice in the diagnosis of a number of disease states. At present the immunodiagnostic techniques employed range from radioimmunoassay for haptens through immunofluorescence for autoimmune diseases to complement fixation for viral infections. All of these assays have their own individual limitations such as: safety, short shelf life and sensitivity. The development of enzyme immunoassays, in particular enzyme linked immunosorbent assay (ELISA), has led to a substantial literature which offers the view that enzyme immunoassays provide a safe, sensitive and specific alternative to standard methods for the detection of antibodies or antigens. The application of heterogeneous enzyme linked immunosorbent assays for the quantitation of haptens, macromolecular antigens and antibodies is reviewed.
Selective localization of a Golgi apparatus acid phosphatase isoenzyme in bone using pyridoxal-5'-phosphate.
Authors: Coleman R A , Schofield B H , McDonald D F
Published: April 2011

Substrates commonly used for localizing bone Golgi apparatus (GA) acid phosphatase (AcPase), e.g., beta-glycerophosphate, p-nitrophenylphosphate, cytidine-5'-monophosphate, and di(dicyclohexylammonium)-2-napthylthiolphosphate, give strong staining not only of GA but also of lysosomes. Thiamine pyrophosphate and inosine-5'-monophosphate--substrates that give good GA staining in some soft tissues--give only lysosomal staining in bone. No previously used substrate or substrate-effector combination has selectively localized the GA acid phosphatase in bone. This article describes results using a new AcPase medium having pyridoxal-5'-phosphate (PLP) as substrate. In bone this medium produced strong staining of the osteoblast GA, but relatively little staining of lysosomes, including lysosomes in osteoclasts. The weak lysosomal staining was almost totally eliminated, without affecting the GA reaction, by pretreating the tissue in 0.3% NH3 solution. Conversely, elevated ionic strength of the substrate medium eliminated the GA reaction, while having little effect on lysosomal staining. The GA enzyme was very sensitive to 1 mM tartrate whereas the lysosomal enzyme was not. These differences suggest the presence of distinct isoenzymes in the two locations. The distribution of osteoblasts with stained GA coincided with the distribution of strongest alkaline phosphatase activity and rapid bone mineralization, supporting previous suggestions that osteoblast GA AcPase is involved in the processing of one or more newly synthesized bone matrix components.
A comparative study of new substrates for the histochemical demonstration of acid phosphomonoesterase activity in tissues which secrete acid phosphatase.
Authors: McDonald D F , Schofield B H , Geffert M A , Coleman R A
Published: April 2011

The histochemical demonstration of acid phosphatase activities against phosphoethanolamine (PEA), phosphorylcholine (PC), and D-ephedrine phosphate (DEP) are reported for a variety of rat tissues and are compared to acid beta-glycerophosphatase (beta GPase) activity. Intense acid beta GPase activity was demonstrated in all tissues examined. However, liver, kidney, intestine, spleen and bone marrow cells failed to exhibit any enzyme activity against PEA, PC, or DEP. In addition, significant differences in the hydrolysis of these substrates were noted among the tissues that did demonstrate activity (bone, tooth, oral mucosa, sebaceous gland, and prostate gland). These observations suggest that PEA, PC, and DEP are more specific substrates for acid phosphatase than beta GP and permit the differential localization of several distinct acid phosphatase isoenzymes.
The ultracytochemical localization of cytidine-5'-monophosphatase in normal human thyroid follicle cells.
Authors: Shannon W A , Roth S I
Published: April 2011

The properties and distribution of an enzyme specifically hydrolyzing cytidine-5'-monophosphate and the possible relationship of the enzyme to the synthesis and secretion of thyroid hormone in man were investigated using cytochemical methods. Activity due to cytidine-5'-monophosphatase was separated from that due to acid or alkaline phosphatase, both of which are also capable of hydrolyzing cytidine-5'-monophosphate. This distinction was established on the basis of manganese ion stimulation and differences in localization, levels of activity, and pH optimums. The localization of the enzyme along the face of Golgi apparatus involved in the formation of thyroglobulin suggests an association of the enzyme with the glycosylation of thyroglobulin.
Immunocytochemical localization of amylase in the parotid gland of developing and adult rats.
Authors: Tanaka T , Gresik E W , Barka T
Published: April 2011

An antiserum against purified rat parotid amylase was used to localize the protein in parotid glands of developing and adult rats. The unlabeled antibody peroxidase-antiperoxidase method and the protein A-gold colloid technique were used at the light and electron microscope levels, respectively. Immunoreactive amylase was detected in a few scattered cells in the glands of 2-day-old rats. During the following days the number of cells stained immunocytochemically for amylase increased rapidly; at 15 days of age all acinar cells revealed amylase, but the intensity of immunostaining varied from cell to cell. Electron microscopically, amylase was localized in the secretory granules, and by using a more concentrated antiserum, in the rough endoplasmic reticulum and Golgi complex. At early stages of development the acinar cells contained fewer and smaller secretory granules than in adult animals; the gold particles indicative of amylase were randomly distributed over the secretory granules. In the glands of adult rats, amylase was distributed inhomogeneously within the secretory granules. In the majority of secretory granules gold colloid particles were located over the electron-dense portions of the granules. However, secretory granules in which an amylase-rich shell surrounded an amylase-poor or amylase-negative "core" were not infrequent.