bluuebunny/crossref_metadata_2025 huggingface dataset:
QUANTITATIVE HISTOCHEMISTRY OF URIDINE DIPHOSPHOGLUCOSE-PYROPHOSPHATASE AND URIDINE DIPHOSPHOGLUCOSE-PYROPHOSPHORYLASE IN DEVELOPING RAT KIDNEY
Authors: CORDER CLINTON N. , BERGER MARK L. , LOWRY OLIVER H.
Published: April 2011

Quantitative histochemical measurements of two enzymes of uridine diphosphoglucose (UDPG) metabolism have been made in the developing rat kidney nephron. Kidneys were examined from -4 days to 44 days of age. In the adult kidney, UDPG-pyrophosphatase was concentrated in proximal convoluted and straight tubules. During maturation, activity decreased in glomeruli, increased in the proximal tubule and changed little elsewhere in the nephron. UDPG-pyrophosphorylase revealed a different pattern. Activity was more nearly uniformly distributed throughout the nephron but was highest in the proximal straight tubule and ascending limb of Henle. During development, activity was unchanged or increased in glomeruli and small arteries and increased elsewhere, particularly in the proximal straight tubule and ascending limb of Henle.
ELECTRON MICROSCOPIC AND KINETIC STUDIES DEALING WITH AN ARTIFICIAL ENZYME MEMBRANE APPLICATION TO A CYTOCHEMICAL MODEL WITH THE HORSERADISH PEROXIDASE-3,3'-DIAMINOBENZIDINE SYSTEM
Authors: BARBOTIN J. N. , THOMAS D.
Published: April 2011

A new colorimetric method for measuring horseradish peroxidase (HRP) activity, in solution, with 3,3'-diaminobenzidine (DAB) as the hydrogen donor is presented. The same method was also used to test HRP activity when immobilized in an artificial proteic membrane. The reaction was shown to be of first order in both cases. Direct spectrophotometric measurements dealing with HRP membrane show that the total amount of insoluble polymer produced does not increase linearly with the quantity of enzyme introduced in the membrane. Moreover, a study of the DAB permeability through membranes with or without peroxidase activity was performed to allow the study of diffusion limitations. Membranes have also been studied by electron microscopy. We have especially visualized the existence of product concentration profiles inside the membrane. The results show that in the membrane there is no geometrical similarity between the distribution profiles of the enzyme and of the insoluble product.
PERIODATE-LYSINE-PARAFORMALDEHYDE FIXATIVE A NEW FIXATIVE FOR IMMUNOELECTRON MICROSCOPY
Authors: MCLEAN IAN W. , NAKANE PAUL K.
Published: April 2011

A new fixative which primarily stabilizes carbohydrate moieties was developed for immunoelectron microscopy. It contains periodate, lysine and paraformaldehyde. Theoretically, the carbohydrates are oxidized by periodate and cross-linked by lysine. The fixative can preserve antigenicity as well as paraformaldehyde and ultrastructure as well as glutaraldehyde. Using this fixative and the peroxidase-labeled antibody technique, basement membrane antigen was localized within the cisternae of endoplasmic reticulum of parietal yolk sac cells and in extracellular basement membranes with adequate tissue preservation, a task which has not been successfully accomplished by conventional fixatives.
ULTRASTRUCTURAL LOCALIZATION OF A CHARACTERISTIC ACID PHOSPHATASE IN GRANULES OF RABBIT BASOPHILS
Authors: KOMIYAMA ATSUSHI , SPICER SAMUEL S.
Published: April 2011

Bone marrow basophils incubated in Gomori medium at pH 6.0-6.8 exhibited strong acid phosphatase activity suggestive of a potential role in endocytosis in one-third of the cytoplasmic granules and also in Golgi elements. Buffy coat basophils contained about one-third as many reactive granules. Reaction product was confined to the threadlike component of the larger granules predominant in early basophils and was absent from the denser-type granules predominant in late basophils. In centrioles of basophils acid phosphatase appeared localized between triplet fibers. Reactivity with the Gomori medium was diminished at pH 5.0, absent at pH 8.0 and only slightly decreased with p-nitrophenyl phosphate as substrate. Basophils incubated in Barka-Anderson medium at pH 5.0-6.8 revealed light acid phosphatase activity in the Golgi lamellae but essentially none in cytoplasmic granules. Tris-maleate buffer of the Barka-Anderson medium replacing the sodium acetate of the Gomori medium inhibited the reactivity in the granules. Incubation in media containing NaF, or lacking substrate, eliminated the heavy precipitates in granules and Golgi elements but yielded light, nonenzymatic lead staining in Golgi and tubulovesicular structures and atypical granules present only in buffy coat basophils.
ALKALINE PHOSPHATASE ACTIVITY ASSOCIATED WITH THE NUCLEAR PORE IN NORMAL AND NEOPLASTIC SALIVARY GLAND TISSUE
Authors: CUTLER LESLIE S. , CHAUDHRY ANAND P. , MONTES MARIO
Published: April 2011

During the cytochemical investigation of human parotid mixed tumors, focal alkaline phosphatase activity was observed along the nuclear envelope of many neoplastic epithelial cells. Further investigation localized the enzyme reaction product within the nuclear pore and in relation to the pore-associated material. Commonly, the reaction product assumed a geometric pattern and measurements of the reaction product deposits (761 ± 8 Å) conformed to the size of the nuclear pore. The possible significance of the presence of the enzyme in the nuclear pore is discussed.
A MICROSPECTROPHOTOMETRIC STUDY OF METACHROMASIA
Authors: TROYER HENRY
Published: April 2011

From hyaluronidase digestion studies of costal cartilage it was determined that stoichiometric relationships do not exist between metachromatic dyes and anionic groups on chondroitin sulfate of low concentrations or low molecular weight. Therefore, it may not always be possible to obtain a true estimate of chondroitin sulfate in tissue with metachromatic stains.
DEMONSTRATION OF HISTONASE WITH FLAVIANIC ACID
Authors: KASS LAWRENCE
Published: April 2011

A method was developed to demonstrate histonase activity in the cytoplasm of polymorphonuclear leukocytes, eosinophils and monocytes. The technique was based on the ability of flavianic acid (2,4-dinitro-l-naphthol-7-sulfonic acid) to form highly insoluble salts with arginine. Presumably, the source of the free arginine was from histone proteolysis as a result of the action of histonase.
DEOXYRIBONUCLEIC ACID CYTOPHOTOMETRY OF STAINED HUMAN LEUKOCYTES III. THERMAL DENATURATION OF CHROMATIN
Authors: DRESKIN STEPHEN C. , MAYALL BRIAN H.
Published: April 2011

Thermal denaturation of chromatin has been monitored in situ in single cells using absorption cytophotometry on gallocyanin-chrome alum (GCA)-stained preparations. Spreads of human lymphocytes were fixed by ethanol freeze-substitution, digested with ribonuclease and treated at different temperatures prior to staining. Treatment in 0.15 M NaCl-0.0l5 M Na citrate (pH 7.0) for 10 min was followed by 0.15 M NaCl-0.0l5 M Na citrate (pH 7.0) with 10% formalin at the same temperature for 20 min. The GCA staining was localized to nuclei and was measured cell by cell with an integrating scanning cytophotometer. Cellular GCA stain content increased progressively with treatment temperatures above 60°C until 90°C, when it was about 1.5 times that of cells treated at 22°C. Formation of single strand deoxyribonucleic acid at different temperatures was monitored by digesting slides with an endonuclease that is specific for single strand nucleic acids and then staining with GCA. Digestion had little effect on slides that had been treated at less than 70°C, but above this temperature it caused a progressive decrease in stain content up to 90°C, when the stain content was reduced to about one-half that of undigested cells. Both with and without enzymatic digestion, these new approaches to monitoring chromatin denaturation yield preparations that are stable and can be measured on any visible light absorption cytophotometer; in addition, the enzymatic approach exemplifies a class of potential methods in which biochemical procedures are adapted to cytophotometry.
CYTOPEMPSIS OF HORSERADISH PEROXIDASE IN THE HEPATOCYTE
Authors: MA M. H. , LAIRD W. A. , SCOTT H.
Published: April 2011

An excretory role of pericanalicular vesicles was studied by following the uptake and transport of horseradish peroxidase in rabbit hepatocytes. The observations are interpreted to indicate uptake of horseradish peroxidase by pinocytotic vesicles which then move toward the biliary pole, thus becoming pericanalicular vesicles which discharge their contents directly into the bile canaliculi. With small loads, lysosomes other than multivesicular bodies are not involved in this route of transport. Although multivesicular bodies have also been shown to be active in the uptake of horseradish peroxidase from the sinusoidal surface, their subsequent fate is not known.
THE ULTRASTRUCTURAL LOCALIZATION OF MONOAMINE OXIDASE (MAO) WITH TRYPTAMINE AND A NEW TETRAZOLIUM SALT, 2-(2'-BENZOTHIAZOLYL)-5-STYRYL-3-(4'-PHTHALHYDRAZIDYL) TETRAZOLIUM CHLORIDE (BSPT)
Authors: SHANNON W. ALLEN , WASSERKRUG HANNAH L. , SELIGMAN ARNOLD M.
Published: April 2011

The ultrastructural localization of monoamine oxidase (MAO) is demonstrated with tryptamine and a new nonosmiophilic tetrazolium salt, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT), which is reduced to an osmiophilic formazan. Under the cytochemical conditions for conducting osmication, the styryl group is not osmicated. MAO is localized in guinea pig myocardial, renal and hepatic tissues and rat myocardium. Enzymatic activity is demonstrated in endoplasmic reticulum, nuclear envelope and mitochondrial outer compartment, specifically in the outer membrane. Sensitivity to formaldehyde fixation and specific MAO inhibitors indicates that the enzymatic activity is due to MAO and possibly other amine oxidases.
QUANTITATIVE HISTOCHEMICAL STUDIES OF THE HYPOTHALAMUS DEHYDROGENASE ENZYMES
Authors: PACKMAN P. M. , BOSHANS R. L. , ROBINS E.
Published: April 2011

Levels of glucose 6-phosphate dehydrogenase, glutamic dehydrogenase, lactic dehydrogenase and malic dehydrogenase in the adult male rat hypothalamus were measured. There was no one consistent pattern for all enzymes; each had its own distinct distribution. There were many significant differences between hypothalamic nuclei and these differences would be masked when one makes comparisons of the major anatomical portions of the hypothalamus (anterior, middle and posterior) which are composed of these specific nuclei. Reports showing no differences in the metabolism of various portions of the hypothalamus might be accounted for on this basis. No apparent correlation between blood supply and oxidative or glycolytic pathways is noted.
REFINEMENT OF THE BIS-(THIOACETOXY) AURATE (I) METHOD FOR THE ELECTRON MICROSCOPIC LOCALIZATION OF ACETYLCHOLINESTERASE AND NONSPECIFIC CHOLINESTERASE
Authors: KOELLE GEORGE B. , DAVIS RICHARD , SMYRL ELOISE GABEL , FINE ASHLEY V.
Published: April 2011

The bis-(thioacetoxy) aurate (I) histochemical method has been refined to permit reliable electron microscopic localization of acetylcholinesterase and nonspecific cholinesterase in autonomic ganglia and other mammalian and submammalian tissues. The detailed procedure is presented, along with illustrations of its specificity by light microscopy and high degree of resolution by electron microscopy.
TISSUE-SPECIFIC GLUCOSE 6-PHOSPHATE DEHYDROGENASES IN ANOPHELINE MOSQUITOES
Authors: BIANCHI UMBERTO , RINALDI ANTONIETTINA
Published: April 2011

Glucose 6-phosphate dehydrogenase (G6PD) electrophoretic variants have been detected in single adult homogenates by screening laboratory strains of Anopheles atroparvus, Anopheles labranchiae and Anopheles stephensi. Pair mating crosses of A. atroparvus individuals set up to study the inheritance mechanism of this apparent polymorphism failed to show Mendelian segregation. Furthermore, monomorphic and tissue-specific G6PD bands were obtained from single adult "midgut" and single adult "skin" homogenates and the apparent polymorphism disappeared. However, the electrophoretic heterogeneity reappeared when 10 µl of the gut homogenate were added to an equal volume of the skin homogenate and permitted to interact in vitro at room temperature (20-25°C) for 4-5 min. Bovine trypsin greatly modified the anodical mobility of the skin isoenzyme. Single whole homogenates, prepared in buffers containing soybean (trypsin inhibitor), partially retained the electrophoretic heterogeneity. On this experimental background it is possible to draw the following conclusions: (a) at least two monomorphic and tissue-specific (gut and skin) G6PD isoenzymes are present in the anopheline species studied by us; (b) a factor (or factors) possessing a trypsin-like action seems to be present in the whole body homogenate, this factor seems to be particularly active in interacting with the skin enzyme; and (c) the occurrence of a similar interaction could facilitate the formation of G6PD catalytically active molecular artifacts. These data and analogous results obtained by other authors permitted us to conclude that if genetic analysis has not been performed it is very hazardous to interpret zymograms simply by assuming that any electrophoretic heterogeneity necessarily represents a genetic polymorphism.
THE EFFECT OF DIGESTION WITH CHONDROITINASES UPON CERTAIN HISTOCHEMICAL REACTIONS OF MUCOSACCHARIDE-CONTAINING TISSUES
Authors: YAMADA KAZUYORI
Published: April 2011

The effect of digestion with chondroitinases ABC and AC upon certain histochemical reactions of mucosaccharide-containing vertebrate tissues was studied in various species including man. Differing degrees of diminution or abolition in five histochemical staining reactions used for acid mucosaccharides were observed in all of the tissues following digestion with either of the two mucosaccharidases. The histochemical reactions for vicinal hydroxyl groups of mucosaccharides in these tissues were either unaffected or increased following the same digestion procedures. The results of this study indicate the possible usefulness of these two chondroitinases in providing a means of histochemically demonstrating isomeric chondroitin sulfates and related acid mucosaccharides.
COLLOIDAL IRON USED AT pH'S LOWER THAN 1 AS ELECTRON STAIN FOR SURFACE PROTEINS
Authors: BLANQUET PIERRE R. , LOIEZ ANNIE
Published: April 2011

The effects of hydrochloric acid on stability, charge and selective affinity of the colloidal ferric hydroxide were studied. The charge was tested electrophoretically. The stability was controlled by measuring the turbidity. The affinity was determined by applying colloid to gelled agarose sections containing hyaluronic acid, polyvinyl sulfate or polylysine. Affinity was also determined by applying the colloid to free tumor cells previously submitted to various types of chemical and enzymatic treatments (esterification, acetylation, periodic acid-hydroxylamine method; neuraminidase, phospholipase C, hyaluronidase) and to isolated rat liver surface membranes pretreated by lipid extraction or incubated with phospholipase C. It was found that the chloride ions at pH's lower than 1 bring about the recharging of the conventional positively charged colloid to a negative form. This negative colloid can be used as a new cytochemical method at the electron microscopic level, to visualize with relative specificity positively ionized groups such as the basic amino groups of protein side chains in the outer and inner hydrophilic leaflets of the cell surface membrane.
QUANTITATIVE HISTOLOGIC DISTRIBUTION OF ADENOSINE-3',5'-MONOPHOSPHATE IN THE RAT STOMACH IMPROVED PROCEDURE FOR THE LUMINESCENCE ASSAY
Authors: GLICK DAVID , KATSUMATA YOSHINAO , VON REDLICH DOROTHY
Published: April 2011

Procedural improvements of the luminescence method for determination of adenosine-3',5'-monophosphate (cAMP) in submilligram samples, e.g., fresh-frozen microtome sections of tissue or l0-µl volumes of homogenate, were developed for technical simplification, time-saving and increased analytical precision and then applied to determination of the quantitative histologic distribution of cAMP in the body of the glandular stomach of the fed and fasted rat. In fasted rats, a sharp peak in cAMP concentration (0.12 pmole/µg protein-nitrogen, approximately ≈ 3.3 pmoles/mg wet weight) was observed in the parietal mucous neck cell zone and the values fell off rapidly to about one-half peak value in the chief cell region and remained in this range into the muscle layer. When fed, additional second and third peaks appeared in the chief cell and muscle zones, each about l0% less than the first peak. The histologic position and the height of the first peak did not change significantly from that in the fasted state. For comparison, homogenates of glandular stomach of fed rats were found to have cAMP values in the range, 1.9-2.4 pmoles/mg wet weight. The findings are discussed in relation to quantitative distribution and function of histamine and histidine decarboxylase and to acid secretion.
LOCALIZATION OF α-FETOPROTEIN IN HUMAN AND RAT FETAL LIVERS AN IMMUNOHISTOCHEMICAL METHOD USING HORSERADISH PEROXIDASE
Authors: NAYAK N. C. , DAS P. K. , BHUYAN U. N. , MITTAL ASHA
Published: April 2011

α-Fetoprotein (AFP) was successfully demonstrated in paraffin-embedded sections of human and rat fetal livers, by a multilayering immunohistochemical technique using the peroxidase-antiperoxidase system. Serial sections simultaneously subjected to immunofluorescence showed identical sites of localization in hepatocytic cytoplasm and sometimes in perivenous connective tissue. AFP-containing hepatocytes were located around efferent veins as well as randomly in the lobule. The immunoperoxidase technique has certain advantages over the immunofluorescence method which would justify the former's application in studies on the dynamics of AFP synthesis.
DIFFUSION OF H36Cl IN DECALCIFIED IVORY DENTINE
Authors: BIRKEDAL-HANSEN HENNING
Published: April 2011

The diffusion coefficient for diffusion of HCl in the organic matrix of elephant's ivory dentine was determined for various concentrations by measuring the fractional uptake of H36Cl in decalcified, cylindrical specimens with concealed end surfaces using a Geiger-Müller counter. Values for the effective diffusion coefficient for sorption of HCl were greatly dependent on concentration (6.1 x l0–6 cm2/sec for 1.0 N HCl to 0.l8-10–6 cm2/sec for 0.01 N). On the basis of an approximate kinetic model the diffusion coefficient for simple diffusion was estimated (9-12 x 10–6 cm2/sec). The effect of stirring on the rate of sorption was negligible.
KINETICS OF ACID DEMINERALIZATION IN HISTOLOGIC TECHNIQUE
Authors: BIRKEDAL-HANSEN HENNING
Published: April 2011

The rate of progression of the demineralization front during acid demineralization is investigated for standardized elephant's ivory dentine specimens. A theoretical model for the penetration based upon a combined diffusion with reaction process is set up. It is demonstrated that the results are in accordance with the proposed model. For specimens of certain geometrical shapes (semi-infinite or plane sheet) the distance penetrated by the front is directly proportional to the square root of the time.
SPECTRAL MATCHING OF CLASSICAL CYTOCHEMISTRY TO AUTOMATED CYTOLOGY
Authors: ORNSTEIN LEONARD , ANSLEY HUDSON R.
Published: April 2011

Aspects of the history of automated cytology and a brief comparison of present and anticipated future levels of performance of flow-through and pattern recognition technologies for white blood cell recognition and counting are briefly presented. The relationship of the spectral properties of cytochemical preparations to machine performance are discussed. Some advantages of matching the spectral properties to hardware and software capabilities are illustrated. Some kinds of difficulties in achieving such a match are reviewed and examples of present levels of achievement are illustrated.
DEOXYRIBONUCLEIC ACID CYTOCHEMISTRY FOR AUTOMATED CYTOLOGY
Authors: GILL J. E. , JOTZ M. M.
Published: April 2011

Fluorescent staining for intracellular deoxyribonucleic acid content is being investigated as a means of making automated measurements quantitative and reliable. Ambiguities regarding the role of sulfur dioxide in Feulgen staining chemistry have been resolved showing that: (a) SO2 does not covalently bind to the anilinium moieties of pararosaniline; (b) SO2 does promote the binding of pararosaniline within the nuclei of hydrolyzed cells; (c) SO2 also does not bind to dyes such as acriflavine used in fluorescent-"Feulgen" staining and (d) SO2 is not required for strong, nucleus-specific acriflavine staining of nuclei of hydrolyzed cells. These findings permit simplifications of the usual fluorescent-"Feulgen" procedures. Attempts to use fluorescent intercalating dyes as vital stains for deoxyribonucleic acid content have been unsuccessful.
MICROFLUOROMETRIC ANALYSIS OF DEOXYRIBONUCLEIC ACID REPLICATION KINETICS AND SISTER CHROMATID EXCHANGES IN HUMAN CHROMOSOMES
Authors: LATT SAMUEL A.
Published: April 2011

Fluorescence of the dye 33258 Hoechst, when bound to chromosomes, is partially quenched by the incorporation of 5-bromodeoxyuridine into chromosomal deoxyribonucleic acid (DNA). This effect allows microfluorometric analysis of DNA synthesis. Metaphase chromosomes from cultured human leukocytes which have incorporated 5-bromodeoxyuridine for a portion of the DNA synthesis period exhibit reduced 33258 Hoechst fluorescence in 5-bromodeoxyuridine-containing regions. Regions synthesizing DNA during a particular interval can thus be highlighted by the appropriate protocol of 5-bromodeoxyuridine administration. Chromosomes from cells which have replicated twice in medium containing 5-bromodeoxyuridine exhibit one brightly and one dully fluorescing chromatid, reflecting incorporation of 5-bromodeoxyuridine into one or two chains of chromatid DNA, respectively. Sister chromatid exchanges, evident as sharply demarcated reciprocal alterations in fluorescence along chromosomes, can be located relative to quinacrine banding patterns. This fluorometric approach should be useful in many instances as a convenient, high resolution alternative to autoradiography.
COMPUTER-ORIENTED ANALYSIS OF HUMAN CHROMOSOMES IV. DEOXYRIBONUCLEIC ACID-BASED CENTROMERIC INDEX
Authors: MENDELSOHN MORTIMER L. , BENNETT DEBORAH E. , BOGART ELLIOT , MAYALL BRIAN H.
Published: April 2011

A density-oriented semiautomatic method to estimate centromeric index is described and tested on 4611 human chromosomal images. Chromosomes are stained for deoxyribonucleic acid with gallocyanin-chrome alum and their optical density is recorded by a digital scanning microscope. Shape information in the chromosome boundary leads to a provisional centromeric position. The chromosome is divided into strips 0.25-0.35 µm wide paralleling the centromere. Optical density is integrated within each strip, strips are assembled into a density profile and the definitive centromere is located at the major local minimum in the profile. Centromeric index (large arm:total) is based on optical density or area. A test set of 4611 chromosomal images is based on over 2500 chromosomes (56 metaphase cells from six individuals) and excludes overlapped and excessively bent chromosomes. The program fails in 1.2% of the test images. Standard deviation within chromosome groups of centromeric index based on density is 0.024 and replication error is 0.016. Systematic differences between area-based and density-based measurements are observed and interpreted.
TECHNIQUES APPLICABLE TO COMPUTER-AIDED CYTOPHOTOMETRY IN VIROLOGY
Authors: TER MEULEN VOLKER , KOSCHEL KLAUS , AUS HANS M. , KAEKELL MATHILDA , SCHOLZ WOLFGANG
Published: April 2011

Computer-aided cytophotometry is a new tool in the analysis of virus-infected cells. Structural changes of the cell morphology can easily be recognized and interpreted by the analytic systems available. More specific biochemical events that occur during virus replication can in certain instances be recorded and recognized if well defined trace markers are applied. This is demonstrated on poliomyelitis virus-infected HeLa cells.
TEXTURAL PARAMETERS RELATED TO NUCLEAR MATURATION IN THE GRANULOCYTIC LEUKOCYTIC SERIES
Authors: LIPKIN BERNICE SACKS , LIPKIN LEWIS E.
Published: April 2011

Texture parameters derived from on-line digitized images of 100 Giemsa-stained early and mature human marrow cells were evaluated as possible nuclear descriptors for automatic analysis. Two likely candidates are discussed. One correlates well with the progressive submembranous chromatin condensation observed with maturation. The other, from preliminary analysis, shows promise of paralleling the generally observed "fineness" or "coarseness" of nuclear appearance.
THE USE OF COHERENT OPTICAL PROCESSING TECHNIQUES FOR THE AUTOMATIC SCREENING OF CERVICAL CYTOLOGIC SAMPLES
Authors: KOPP R. E. , LISA J. , MENDELSOHN J. , PERNICK B. , STONE H. , WOHLERS R.
Published: April 2011

Coherent optical signal processing techniques applied to the screening of exfoliated cytologic cervical samples were evaluated. The two-dimensional Fourier spectrum of some 80 isolated cells was obtained from high resolution cell photographs and recorded. Quantitative spectrum data were collected on a training set consisting of 15 categorized malignant cells taken from slides diagnosed as malignant and 10 categorized normal cells taken from slides diagnosed as normal. A variety of transform parameters were measured and certain combinations of them were found which separated completely the normal from malignant cells. Certain parameters were shown to be functionally related to the cell diameter, nuclear diameter and nuclear density. Other parameters were found which appear to be related to other cell features such as clumping of nuclear deoxyribonucleic acid. When they were used in combination with the previous parameters, they greatly enhanced the discriminatory capabilities. Computer model studies formed the basis for our experimental design and parameter selection and have validated much of our experimental results. Although the sample size to date is admittedly small, our results have been very encouraging.
STATUS QUO IN FLOW-THROUGH CYTOMETRY
Authors: FULWYLER MACK J.
Published: April 2011

Flow-through systems of single cell analysis and sorting have been under development for several years. Fluorescence and volume sensors are well developed but light scatter sensors can be expected to improve. Cell sorters are in use in several laboratories and commercial versions are under development. The most serious problems in flow-through cytometry are in cell preparation and the greatest potential for advancement lies in development of new, specific stains and markers.
CAN LIGHT-SCATTERING TECHNIQUES BE APPLIED TO FLOW-THROUGH CELL ANALYSIS?
Authors: BRUNSTING ALBERT
Published: April 2011

Much of the information deduced from the scattering of light from cells is briefly reviewed with a minimum of optical theory. It is shown that many of the existing methods can possibly be applied to cells flowing one at a time through a detecting volume in automated analysis. Outlined are some potentially promising optical systems which may provide light-scattering applications for flow-through cell analysis.
FLOW MICROFLUOROMETRIC ANALYSIS OF CELL KILLING WITH CYTOTOXIC DRUGS
Authors: YATAGANAS XENOPHON , CLARKSON BAYARD D.
Published: April 2011

The relative deoxyribonucleic acid (DNA) content of cells in culture was determined in a flow microfluorometer after staining with a fluorescent DNA-specific technique. The distribution of the DNA content was used to study the effect of arabinosylcytosine (Ara-C), hydroxyurea, bleomycin and actinomycin D on the progression of the cells through the cell cycle. It was found that short exposures to 10–5 M Ara-C reversibly blocks the cells predominantly in early S phase. The cells blocked in S died after prolonged treatment at low concentrations. A small population was blocked in G1. Similar effects were observed at 10–4 M Ara-C which were irreversible even after short exposures. A block of the majority of the cells in early S and a few cells in G1 was also found with 10–3 M hydroxyurea. Bleomycin resulted in a block in G1 and G2 phases. Actinomycin D treatment at 10–8 M for 24 hr inhibited the initiation of DNA synthesis, since almost all cells were found in G1. The method is very suitable for rapid studies of drug effects on cell cycle progression.
PERFORMANCE TESTING FOR AUTOMATED PRESCREENING DEVICES IN CERVICAL CYTOLOGY
Authors: BARTELS PETER H. , WIED GEORGE L.
Published: April 2011

Performance specifications for automated prescreening devices for cervical cancer will concern rates for missed positive samples and rates for false alarms. Testing whether a given device adheres to such specifications within given confidence limits should be feasible for the missed positive rates. The test for adherence to the specified false alarm rate presents a serious sampling problem.
THE QUANTIMET CYTOSCREEN AND THE INTERACTIVE APPROACH TO CANCER SCREENING
Authors: HUSAIN O. A. N. , ALLEN R. W. B. , HAWKINS E. JUNE , TAYLOR JUDY E.
Published: April 2011

Results are presented supporting the philosophy that malignancy can be detected by studying the size/density matrix of nuclei in cell populations to form the basis of an automated screening system. Some methods of cell dispersal and staining are briefly described. A preliminary assessment of the performance of the Cytoscreen is discussed.
AN AUTOMATIC MICROSCOPE SYSTEM FOR DIFFERENTIAL LEUKOCYTE COUNTING
Authors: DEW BROCK , KING THOMAS , MIGHDOLL DAVID
Published: April 2011

Described are the operation and special purpose hardware of a practical system for an automatic differential leukocyte count. The system is based on an extended memory configuration minicomputer. Major features of the system are: a high speed, three-axis, servo-controlled microscope stage; a cell location sensor; a special slowed scan, two spectral band television camera for direct computer input; and a focus signal generated from the camera, with the loop closed through the computer. The camera and monochrome monitor scan a 256 x 256 element frame 15 times/sec. The original or modified images may be displayed from the computer memory. The performance goal is the processing of one cell/sec, with the identification of 17 or more cell types.
A REAL TIME PICTURE PROCESSOR FOR USE IN BIOLOGIC CELL IDENTIFICATION I. SYSTEM DESIGN
Authors: LEMKIN PETER , LIPKIN LEWIS , SCHULTZ MORTON , CARMAN GEORGE , SHAPIRO BRUCE , KAISER PETER
Published: April 2011

We show that the class of special purpose picture processors has common characteristics which set it apart from conventional general purpose computers. We argue for the implementation of a fast serial processor of gray scale images having easy access to a tessellated gray scale neighborhood, where the tessellation of a neighborhood is the movement of that neighborhood through the image array in some deterministic way. Such a machine would be very useful in an interactive system used for problems such as the design of effective procedures for automated cell classification.
A REAL TIME PICTURE PROCESSOR FOR USE IN BIOLOGIC CELL IDENTIFICATION II. HARDWARE IMPLEMENTATION
Authors: CARMAN GEORGE , LEMKIN PETER , LIPKIN LEWIS , SCHULTZ MORTON , SHAPIRO BRUCE , KAISER PETER
Published: April 2011

Hardware design specifications for a real time picture processor are presented. Attention is concentrated on those unique aspects of the hardware that make possible high speed storage and processing of images. The latter are the major contributors to rapidity by which various image transforms may be accomplished—speeds which are great enough to warrant the term real time.
SIMULTANEOUS DIFFERENTIAL STAINING BY A CATIONIC CARBOCYANINE DYE OF NUCLEIC ACIDS, PROTEINS AND CONJUGATED PROTEINS I. PHOSPHOPROTEINS
Authors: GREEN MARIE R. , PASTEWKA JULLIA V.
Published: April 2011

The cationic carbocyanine dye l-ethyl-2-[3-(l-ethylnaphtho[l,2d]thiazolin-2-ylidene)-2-methylpropenyl]-naphtho[1,2d]thiazolium bromide interacts in different ways with several classes of polymeric anions in solution. This results in changes in absorption maxima which differ from the absorption maximum of the dye solution. The multiple interactions with macromolecules were demonstrated in histologic sections. Under appropriate electrolyte, dye and hydrogen ion concentrations, nucleic acids, proteins and conjugated proteins were distinguished from each other by color. The phosphoproteins stained blue, proteins red, nucleic acids purple and mucoproteins and mucopolysaccharides various colors. Further discrimination was obtained by chemical procedures and enzymatic digestions. Secretions in alveolar and ductal lumina in the mammary glands of pregnant mice were shown to stain differentially from the other macromolecules. It is inferred on the basis of enzymatic and chemical procedures that the secretions stained blue because of their phosphoprotein content. Conventional staining procedures did not allow this distinction to be made.
5'-NUCLEOTIDASE AND AN ACID PHOSPHATASE OF SPINAL CORD COMPARATIVE HISTOCHEMISTRY AND SPECIFICITY OF THE ENZYMES IN MOUSE AND CAT SPINAL CORDS. CYTOLOGIC LOCALIZATION IN MOUSE SUBSTANTIA GELATINOSA
Authors: SURAN ANITA A.
Published: April 2011

Histochemical procedures show a concentrated zone of phosphomonoesterase activity in the substantia gelatinosa of mouse spinal cord which represents at least two enzymic activities. At pH 7,5'-nucleotidase activity is observed in both somata and neuropil, while at pH 5 the reaction is evident only in somata. An acid phosphatase of unknown specificity is seen in somata only and exhibits optimal activity near pH 5. No comparable enzymic reactions are observed in cat spinal cord sections. Electron microscopic studies demonstrate separate cytologic distributions of the two enzymic activities in substantia gelatinosa. With nucleotides, the reaction is confined to boundaries of processes within the neuropil and to nuclei at pH 7. At pH 5 using an acid phosphatase substrate, β-glycerol phosphate, slight reactions were noted within nerve fibers. Use of β-glycerol phosphate near pH 7 demonstrates both types of activity simultaneously, the boundary reactions typical of the nucleotidase and the reactions within axons and dendrites characteristic of the phosphatase.
FORMALDEHYDE CONDENSATION AT REDUCED TEMPERATURE INCREASED SENSITIVITY AND SPECIFICITY OF THE FLUORESCENCE MICROSCOPIC METHOD FOR DEMONSTRATING PRIMARY CATECHOLAMINES
Authors: HÅKANSON ROLF , SUNDLER FRANK
Published: April 2011

The present modification of the formaldehyde condensation method for demonstrating catecholamines and indolamines involves generation of the formaldehyde gas at + 80°C followed by reaction at +25 or 0°C. This procedure results in improved sensitivity for demonstrating noradrenaline and dopamine. Because adrenaline and peptides with NH2-terminal tryptophan give no or very weak fluorescence under these reaction conditions the modification also offers possibilities for improved discrimination between primary catecholamines on one hand and adrenaline and tryptophyl peptides on the other. Under the conditions used, the fluorescence yield of 5-hydroxytryptamine did not seem to be improved by variations in the reaction temperature.
The histochemical demonstration of hydrolase activities with films of homopolyribonucleotides.
Authors: Daoust R
Published: April 2011

Films of polycytidylic acid, polyuridylic acid and polyguanylic acid were exposed to tissue sections and the results were compared with those obtained in previous studies on polyadenylic acid and ribonucleic acid. Important variations were observed in the distribution of the hydrolases acting on the different polyribonucleotides, suggesting that a variety of nucleases with marked proclivity for particular nucleotide residues can be demonstrated by the use of films of homopolymers.
Peptides with NH2-terminal tryptophan in adrenocorticotrophic hormone and melanocyte-stimulating hormone granules of adenohypophysis.
Authors: Håkanson R , Sundler F , Larsson L I , Ekman R , Sjöberg N O
Published: April 2011

Fluorescence microscopy has demonstrated formaldehyde-ozone-induced fluorescence in the pars intermedia cells (melanocyte-stimulating hormone cells) and in certain cells of the pars distalis of the mammalian pituitary. From histochemical and chemical evidence the fluorescence is believed to reflect the presence of peptides with NH2-terminal tryptophan. In the pars distalis of hamster, cat and pig pituitary, the cells that exhibit formaldehyde-ozone-induced fluorescence have now been identified as adrenocorticotrophic hormone (ACTH) cells by immunohistochemistry. Granules from pig pituitaries were purified by passage through a succession of Millipore filters followed by centrifugation on a continuous sucrose gradient. Two granular fractions were identified by electron microscopy and found to contain high concentrations of peptides with NH2-terminal tryptophan as well as high ACTH bioactivity. These fractions, when pelleted and analyzed histochemically, displayed formaldehyde-ozone-induced fluorescence and ACTH-like immunoreactivity.
Comparison of Sirius red and Congo red as stains for amyloid in animal tissues.
Authors: Brigger D , Muckle R J
Published: April 2011

Sirius red and Congo red were compared for specificity and sensitivity of amyloid staining in animal and human material. Previously described advantages of Sirius red as an amyloid dye were confirmed, as well as its disadvantage of lack of ultraviolet fluorescence. Two further disadvantages of Sirius red were discovered, both relating to animal material: (a) its unexpectedly weak staining of early experimentally induced amyloid deposits and (b) frequent uncontrollable nonspecific staining of fibrous tissues. It is therefore concluded that, overall, Congo red used by the improved alkaline technique of Puchtler, Sweat and Levine (1962) remains the best single method for demonstration of amyloid in both human and animal tissues.
Enzyme cytochemistry of Candida albicans.
Authors: De Nollin S , Thoné F , Borgers M
Published: April 2011

The application of a new preparation method for demonstrating the activities of hydrolytic and oxidative enzymes in Candida albicans is reported. The problem of inadequate penetration of fixatives into yeast cells has been solved by sectioning solidified pellets of the cells in the presence of glutaraldehyde, a procedure that yields a fairly well preserved ultrastructure and sufficient enzyme activities. The subcellular distribution of most specific and nonspecific phosphatases and of peroxidases is at variance with that found in mammalian cells. The activities toward beta-glycerophosphate, p-nitrophenylphosphate, adenosine triphosphate, adenosine monophosphate, thiamine pyrophosphate and glucose 6-phosphate are almost exclusively confined to the central vacuolar apparatus. Oxidative and peroxidative activities are demonstrated only in mitochondria. Specific marker enzymes for endoplasmic reticulum, plasmalemma, Golgi apparatus and peroxisomes in C. albicans are not found. The possible function of the various subcellular organelles in relation to their enzymatic content is discussed.
Histochemical localization of glutathione in tissues.
Authors: Asghar K , Reddy B G , Krishna G
Published: April 2011

A histochemical method has been developed for the localization of glutathione (GSH) in frozen sections from various tissues including liver, lung, kidney, testis and eye. The reliability and specificity of the method has been investigated by comparing the rates of reaction in tissue and gelatin sections and after depletion of GSH in liver by diethyl maleate. In principle, the method is based on the formation of an irreversible complex of mercury orange with the --SH group of GSH. A 5-min staining period was found to be optimal for staining the --SH group of GSH. In brief, frozen sections 8 mu thick are stained with a 50 muM solution of mercury orange dissolved in toluene, counterstained in 0.05 per cent methylene blue and mounted in Histoclad. Pretreatment of the sections with fixatives or drying them in air completely prevented the staining. In hepatic lobules the brick red granules of the GSH mercury orange complex were distributed uniformly, whereas in other tissues they were not uniform. The GSH staining was localized in the proximal convoluted tubules in the cortex of the kidney, the interalveolar epithelial cells of lungs, the epididymis and the capsule of testis, epithelial cells of vas deferens and the periphery of the lens.
The ultracytochemical demonstration of a formaldehyde-resistant dehydrogenase of leuco nitroxyl analogues.
Authors: Shannon W A , Wasserkrug H L , Plapinger R E , Lynm D , Seligman A M
Published: April 2011

A dehydrogenase which is relatively stable in formaldehyde fixative is demonstrated ultracytochemically by the reduction of various leuco nitroxyl analogues in rat hepatic, renal, myocardial, skeletal muscle and prostatic tubuloalveolar glandular tissues. The nonosmiophilic tetrazolium salt, t-(2'-benzothiazolyl)-5-styryl-3-(4'-phtalhydrazidyl) tetrazolium chloride, is subsequently reduced to an insoluble osmiophilic formazan by the hydrogen ions resulting from the dehydrogenase activity. Exposure of the formazan to osmium tetroxide results in electron density enabling visualization of the reaction product in the electron microscope. Known inhibitors of various dehydrogenases were utilized in an attempt to determine the existence and/or extent of any specific characteristics of the dehydrogenase(s) involved.
Quantitative light microscopic autoradiography. Emulsion sensitivity and latent image fading.
Authors: Boren H G , Wright E C , Harris C C
Published: April 2011

Emulsion sensitivity, latent image fading, and the effects of temperature, humidity, radiation dose and chemography on them were measured for NTB2 autoradiographic emulsion using quantitative methods. Sensitivity of NTB2 emulsion increased as the temperature during exposure increased, with the greatest increase per degree occurring between -20 degrees C. At 4 degrees C, emulsion sensitivity remained constant with time and radiation dose. Direct measurement of latent image fading showed no latent image fading for 60 weeks on slides exposed at 4 degrees C with Drierite. Slides exposed at 27 degrees C showed significant latent image fading and great variation between samples. High humidity decreased emulsion sensitivity and increased latent image fading. No evidence of either positive or negative chemography was found. The practical use of autoradiography requires an internal standard on each slide to correct for fluctuations in temperature and humidity during exposure time.
The distribution of collagenase in normal rat tissues.
Authors: Montfort I , Pérez-Tamayo R
Published: April 2011

A rabbit monospecific anti-rat uterus collagenase antibody has been used to study the distribution of collagenase in normal rat tissues by immunohistochemical methods. Indirect staining was performed with fluorescein-conjugated goat anti-rabbit immunoglobulin G antibody. The organs studied were brain, lung, myocardium, liver, spleen, kidney, adrenal, testes, uterus, xiphoid cartilage, tail tendon, skeletal (triceps) muscle and skin. Collagenase is widely present throughout the connective tissue structures in all organs examined. The enzyme is apparently bound to collagen fibers, reticulum fibers and basement membranes. The results suggest that control of collagenase activity depends on factors other than the presence of the enzyme in tissues.
Peroxisome development in the metanephric kidney of mouse.
Authors: Goeckermann J A , Vigil E L
Published: April 2011

The relationship of enzymatic activity to organelle development and organelle number during differentiation of the metanephric kidney in the mouse was approached from several experimental directions. Biochemical analyses of marker enzymes for peroxisomes (catalase and D-amino acid oxidase), mitochondria (cytochrome oxidase) and lysosomes (acid phosphatase) were performed on kidneys at ages from 17 days prenatal to adult. These data were correlated with a morphometric analysis of populations of peroxisomes and mitochondria in differentiating cells of the proximal tubule. Postnatal development of the metanephric kidney was found to be accompanied by a rapid increase in both the specific activity of catalase and the number of peroxisomes per 100 mu2 in the proximal tubule during the first 4 weeks of postnatal growth. Elaboration of the endoplasmic reticulum (ER) was seen to parallel the increase in number of peroxisomes to which segments of ER were often in close apposition. Extensive interactions between segments of ER and peroxisomes were readily visible in 0.5-mu sections viewed in the high voltage electron microscope. In contrast to peroxisomes, neither mitochondria nor lysosomes followed a similar pattern of net organelle increase, suggesting that a defined population density of mitochondria and lysosomes may exist in the proximal tubule at birth, prior to complete development of the kidney.
Atomic absorption spectrophotometry applied to photographic densitometry.
Authors: Troyer H , Rosenquist T H
Published: April 2011

For this study of photographic densitometry, sections of cartilage stained with Alcian Blue, safranin O and high iron diamine were photographed at x40 with Nikon photomicrography equipment on Kodak Panatomic X film with appropriate filters to enhance contrast. Portions of the developed negative films were selected from intercellular matrix regions, and circles of film equivalent in diameter to a 30-mu circle of tissue were obtained with a hand-held paper punch. Silver was eluted from the circles of film with 35% nitric acid, and the quantity of silver deposited on the film was determined by atomic absorption spectrophotometry as a measure of stain intensity. The intensity determined by this analytic procedure compared favorably with results obtained previously from the same tissue with microspectrophotometry. This method of silver analysis has advantages over earlier studies which used silver elution to determine photographic densitometry in its technical ease, accuracy and sensitivity. Furthermore, this method compares well with microspectrophotometry in its results and has the advantages of relative inexpensiveness and availability of equipment.
A cytofluorometric and radiochemical analysis of the uptake and turnover of 5-hydroxytryptamine in mast cells.
Authors: Enerbäck L , Jarlstedt J
Published: April 2011

Uptake and turnover of 5-hydroxytryptamine (5-HT) after the administration of 5-hydroxytryptophan (5-HTP) has been studied in rat peritoneal mast cells using radiochemical and quantitative cytofluorometric methods. There was a close agreement between the results obtained with the two methods. The extreme sensitivity of the cytofluorometric method was indicated by the fact that 0.2 pg 5-HT contained in mast cells from control rats could be readily quantitated. The cytofluorometric analysis demonstrated a large variation in 5-HT content of individual mast cells within the mast cell populations. The storage capacity for 5-HT greatly exceeded the amount found in normal mast cells. Intraperitoncally injected 5-HTP was rapidly taken up and eliminated from the mast cells within 12 hr, while 5-HT, probably derived from intracellular decarboxylation of 5-HTP, was retained in the cells and slowly eliminated. The elimination of 5-HT followed an exponential course and the half-life was found to be about 10 days. The results indicate that the turnover of 5-HT is much slower in mast cells than in other cells which normally store 5-HT.