bluuebunny/crossref_metadata_2025 huggingface dataset:
Subcellular localization of gonadotropins and testosterone in the developing fetal rat testis.
Authors: Childs G V , Hon C , Russell L R , Gardner P J
Published: April 2011

Gonadotropins and testosterone were immunocytochemically localized in the fetal rat testes 16-18 days of gestation with the unlabeled antibody-peroxidase anti-peroxidase complex technique. Maximum staining for gonadotropins with antiserum to the beta chain of human chorionic gonadotropin (anti-hCGbeta) occurred at 16 days gestation in the seminferous tubule and 17 days gestation in interstitial (Leydig) cells. Anti hCGbeta sites were on the plasma membranes at the luminal aspects of Sertoli cells at 16 days gestation. In addition, intracellular hCGbeta sites were evident including the nucleus, nucleolus, ribosomes, some vesicles, lysosomes and centrioles. The stain for hCGbeta disappeared rapidly and by 17 days was limited to patches in the cytoplasm and nuclei. In the fetal testes, staining for anti-testosterone binding sites was most intense at 18 days of gestation either in lipid droplets or on nuclei of Leydig and Sertoli cells. Very little testosterone stain was observed before 18 days of gestation. These findings agree with physiologic data that suggest that gonadotropins bind to receptors and stimulate testicular development and the capacity for testosterone production.
Autofluorescence of viable cultured mammalian cells.
Authors: Aubin J E
Published: April 2011

The autofluorescence other than intrinsic protein emission of viable cultured mammalian cells has been investigated. The fluorescence was found to originate in discrete cytoplasmic vesicle-like regions and to be absent from the nucleus. Excitation and emission spectra of viable cells revealed at least two distinct fluorescent species. Comparison of cell spectra with spectra of known cellular metabolites suggested that most, if not all, of the fluorescence arises from intracellular nicotinamide adenine dinucleotide (NADH) and riboflavin and flavin coenzymes. Various changes in culture conditions did not affect the observed autofluorescence intensity. A multiparameter flow system (MACCS) was used to compare the fluorescence intensities of numerous cultured mammalian cells.
Immobilization of neuraminidase and its potential application in the modification of cell surfaces.
Authors: Bazarian E R , Wingard L B
Published: April 2011

Vibrio cholerae neuraminidase was immobilized on the inside of 1.0 mm inner diameter nylon tubing with retention of enzyme activity, when assayed at 37 degrees C and pH 5.5 with mucin as substrate. The stabilities of the immobilized and soluble enzymes were similar for up to 3 hr at 37 degrees C. Preliminary data indicated that immobilized neuraminidase will release sialic acid from the surface of leukemic AKR mouse thymus and spleen lymphocytes; however, the level of immobilized enzyme activity needs to be increased for practical applications. With this improvement immobilized neuraminidase could become a novel preparation for carrying out cell surface modifications with minimal enzyme contamination of the cell.
The development of a new technology for controlled cell fixation. A methodological pilot study.
Authors: Schwetje N , Thiessen G
Published: April 2011

Fixation in a traditional sense means the immersion of biological material into a chemical fluid. For permanent preservation (1) the fixative is always supplied in excess of the cell sample, and (2) the process of fixation is influenced by chemical impurities of the fixative fluid. Both factors influence the subsequent staining of cells. In order to avoid these uncontrolled influences, a new technology for controlled cell fixation has to be developed, whereby freshly prepared formaldehyde gas in an "inert" gas-flow of helium was applied to thin membranes by use of a capillary flow-in technique. The amount of fixative gas supplied, adsorbed, absorbed, diffused, and desorbed after saturation of the membranes could be reliably measured with an on-line operating "inert" mass spectrometer of the Omegatron type.
Flow cytometric analysis of adriamycin-perturbed mouse mammary tumors.
Authors: Dethlefsen L A , Riley R M , Roti Roti J L
Published: April 2011

The effects of a single intraperitoneal injection of adriamycin (10 mg/kg) on a fast-growing C3H mouse mammary tumor (S102F) have been analyzed volumetrically, biochemically, autoradiographically and flow cytometrically. Mathematical simulation of the data was also used to aid in the interpretation of the recovery kinetics. This dose of adriamycin did not induce regression in tumor volume but did inhibit the growth rate for 4-5 days. 3H-TdR incorporation was gradually inhibited to reach a low of 20% of control at 24 and 36 hr and then recovered back to control by 96 hr after adriamycin treatment. The flow cytometric analysis also showed a marked reduction in the relative fraction of cells in the S-phase with a minimum of 23% of control at 72 hr; however, in contrast to the 3H-TdR incorporation data, the fraction of cells in the S-phase was only at 39% of control at 96 hr after the adriamycin injection. Since the 3H-TdR incorporation data disagreed with the flow cytometry data, autoradiographic analysis was also done at selected times after the adriamycin injections, and qualitatively, this analysis confirms the flow cytometry data in that the labeling index was 29% of control at 96 hr after adriamycin. The mitotic index also dropped from 8 to 1%, respectively, for controls and at 96 hr posttreatment. The degenerate index was about 1% in control tumors and no increase was observed in treated tumors. Adriamycin-induced cell-cycle delay occurs predominately in G1 and G2 but there is also an apparent minor delay in the transit across the S-phase and some apparent cytotoxicity in G2 and/or M. The long delay in volumetric growth appears to be due to the extended cell-cycle delay rather than extensive cell killing.
Multiparameter kinetic analysis of killer cell initiation by using immune RNA.
Authors: Kmieck P J , Bagwell C B , Hudson J L , Irvin G L
Published: April 2011

By treating nonsensitized C57BL/6J spleen derived lymphocytes with EL-4 tumor cell directed xenogeneic extracted RNA we were able to monitor early changes in cellular DNA content by flow cytometric (FCM) analysis and 3H-thymidine uptake. These kinetic parameters were correlated with cell mediated cytotoxicity which appeared as early as 8 hr after activation as measured by release of chromium-51 from labeled EL-4 target cells. Flow cytometric analysis and 3H-thymidine uptake data shown peak S phase activity at 72 hr. Maximum cytotoxicity was observed at 48 hr. Cell cycle kinetic parameters were correlated with the appearance of cell mediated cytotoxicity.
Automatic cell identification and enrichment in lung cancer. I. Light scatter and fluorescence parameters.
Authors: Frost J K , Tyrer H W , Pressman N J , Albright C D , Vansickel M H , Gill G W
Published: April 2011

Two physical parameters were investigated to automatically recognize cells in sputum from human squamous cell carcinoma of the lung and to separate them for preparation by the Papanicolaou methods, for human interactive identification and for automated high resolution image analysis. The two parameters, 0.5-15.0 degrees forward argon-ion laser light scatter to estimate total cell size and 546 nm Acridine orange fluorescence to approximate total cell DNA content, were measured in a flow-through fluorescence activated cell sorting system. Enrichment for neoplastic cells in three cases of squamous cell carcinoma of the lung averaged 7.8-fold over the original sputum when only green fluorescence was used and 10.5-fold using green fluorescence and forward light scatter. The average enrichment for neoplastic cells was 65.6-fold relative to polymorphonuclear deenrichment.
Automatic cell identification and enrichment in lung cancer. III. Light scatter and two fluorescence parameters.
Authors: Frost J K , Tyrer H W , Pressman N J , Adams L A , Vansickel M H , Albright C D , Gill G W , Tiffany S M
Published: April 2011

Two fluorescence parameters and size are used in a flow through system to enrich sputum specimens for cancer cells. Human cells in sputum which are stained with acridine orange show a characteristic distribution of red and green fluorescence from which cancer cells can be localized. The peak enrichment is obtained by selectively sorting cells with the largest values of red and green fluorescence. Cancer cells located in other distribution regions having smaller fluorescence intensities show progressively diminished nuclear and cytoplasmic tinctorial features by Papanicolaou stain, consistent with the decreased intensity of red and green fluorescence.
Cytochemical bioassay and radioimmunoassay of ACTH in noise stressed rats.
Authors: Anthony A , Brister N W , Colurso G J
Published: April 2011

Serum corticosterone and ACTH levels were measured in rats subjected to one and two 30 min intervals of noise (100 dB, 250--20,000 Hz). Both hormones were elevated immediately after noise exposure, dropped below pre-exposure levels after a 2 hr recovery interval and were again elevated with a second exposure. The ratio of immunoactive to bioactive ACTH was slightly depressed after one exposure and markedly depressed after two successive noise exposures (relative to controls) indicating that multiple exposure to auditory stimulation effects a proportionally greater output of bioactive ACTH.
Localization of adenylate cyclase in unfixed sections of cardiac muscle.
Authors: Revis N W
Published: April 2011

Previous investigators have shown that prefixation and lead staining completely inhibit the activity of adenylate cyclase. Lead has also been shown to stimulate the nonenzymatic hydrolysis of AMP-PMP (the substrate for adenylate cyclase) after 30 min incubation. The present studies were performed to determine if the omission of prefixation would provide a better method for localizing adenylate cyclase in cardiac muscle. These studies were also performed to determine the effect of short incubation on the lead-induced nonenzymatic hydrolysis of AMP-PNP. In prefixed sections the reaction product was diffusely localized over the section. However, in unfixed sections the reaction product appeared only on the sarcolemma and sarcotubule system. Results are presented showing that short incubation (i.e., 5 min) prevents the nonenzymatic hydrolysis of AMP-PMP by lead. In biochemical studies lead (10(-3) M) was shown to completely inhibit the activity of this enzyme. However, in the presence of 4 micrograms phosphatidylinositol, lead inhibition of this enzyme was reduced to 50% of the control value. Based on this observation, it is suggested that approximately 50% of adenylate cyclase is present in sections of cardiac muscle exposed to 2 x 10(-3) M lead, which is presumably enough activity for demonstration of adenylate cyclase activity.
Morphometry of the chinchilla organ of Corti and stria vascularis.
Authors: Santi P A , Muchow D C
Published: April 2011

This research describes a procedure for a morphometric analysis of the organ of Corti and stria vascularis in the chinchilla. In nine normal cochleae the length of the basilar membrane and the stria vascularis measured 18.47 and 25.22 mm, respectively. An average of 1910 inner and 7501 outer hair cells were present while an average of 15 inner and 90 outer hair cells were absent. In all cochleae examined there were always some missing hair cells in varying numbers even though the animals had no known ototoxic exposure. Stria area, width and thickness increased from the cochlear apex toward the base. Consistency of changes in stria dimensions among animals was enhanced by expressing position in terms of percentage stria length rather than distance as such. Total stria volume was estimated at 0.15 microliter.
Cytochemical demonstration of adenylate cyclase in cardiac muscle: effect of dimethyl sulfoxide.
Authors: Slezak J , Geller S A
Published: April 2011

Adenylate cyclase (AC) activity was evaluated after perfusion fixation of rat and dog myocardium with 4% paraformaldehyde (PFA), 2% glutaraldehyde (GA) or a combination of both, in cacodylate buffer. Dimethyl sulfoxide (DMSO) was added to the fixatives and its effect on the preservation of cell organelles and enzyme activity was determined. Adenylate cyclase activity was preserved best after fixation with 4% paraformaldehyde but this fixative did not provide for optimal maintenance of structure. Prefixation with 2% glutaraldehyde and 5% dimethyl sulfoxide provided the most effective preservation of both structural and enzymatic integrity. Precipitation of lead diphosphoimide was the morphologic indicator of sites of adenylate cyclase activity. The most intense precipitate was in the lumen of junctional sarcoplasmic reticulum in close contact with T-tubules and in subsarcolemmal cisternae. Evidence of activity was also seen on the intracellular aspect of the sarcolemmal membrane and in the nexus segment of the intercalated discs. Alloxan was effective as an inhibitor of adenylate cyclase activity only if the concentration of the activating substance sodium fluoride (NaF) was 20 mM or lower.
The immunohistolocalization of carbonic anhydrase in rodent tissues.
Authors: Spicer S S , Stoward P J , Tashian R E
Published: April 2011

Carbonic anhydrase has been localized with an immunoenzyme bridge technique in the following sites in paraffin sections of fixed rodent tissues: gastric parietal cells, the brush border of enterocytes in the small intestine, superficial nongoblet cells of the colon, selective segments of the nephron, glial cells, erythrocytes and adipose cells. Immunocytochemical localizations of carbonic anhydrase isozymes I and II in different histologic sites, by means of affinity column purified antibodies, agreed with the distribution of these enzymes in the various sites, as indicated by immunologic assays. The immunocytochemical results are compared with those reported for the cobalt-bicarbonate cytochemical method and with biochemical knowledge of the occurence of carbonic anhydrase.
Preservation of alveolar type II pneumocyte lamellar bodies for electron microscopic studies.
Authors: Collet A J
Published: April 2011

Simultaneous fixation with glutaraldehyde and osmium tetroxide, followed by an uranyl acetate (UA) treatment before dehydration and embedding (Hirsch and Fedorko 1968) ensures a very good preservation of lamellar bodies (LB's) as well as of the cellular membranes in type II pneumocyte. The uranyl acetate treatment appeared to be the most efficient step of the procedure. The morphological aspect of lamellar bodies after such a preparation was similar to that observed after freeze-etching of lipid retaining methods. Moreover, the Hirsch-Fedorko procedure is very simple and can easily be used for routine ultrastructural and radioautographic studies. On the other hand, it appeared that the uranyl acetate phospholipid "complex" is very sensitive to the pH of chemical solutions used after sectioning. The "complex" is variously dissolved by alkaline solutions, photographic developers or stains. The best preservation of ultrastructure was obtained with neutral or acidic developers and acidic stains.
Immunohistologic studies of lymphomas. New methodology yields new information and poses new problems.
Authors: Taylor C R
Published: April 2011

There occur tumors corresponding to almost all kinds of normal tissues, and clearly our classification of tumors will run parallel with our classification of normal tissues.
Use of osmium-ferrocyanide treatment for improved lysosomal acid trimetaphosphatase reaction and subcellular detail in thyroid follicular cells.
Authors: Payer A F , Battle C L , Peake R L
Published: April 2011

The histochemical reaction for acid trimetaphosphatase in addition to secondary tissue treatment with an osmium-ferrocyanide mixture was used to study lysosomes and phagolysosomes in the mouse thyroid gland. The osmium-ferrocyanide postfixation enhanced reaction product localization, reduced diffuse reaction, and improved membrane contrast. In addition, the ultrathin tissue sections did not require heavy metal staining, thus eliminating potential stain artifacts due to precipitation. In view of the improved tissue preservation and enzyme localization, it is suggested that osmium-ferrocyanide postfixation be used after the acid trimetaphosphatase method.
Embedment in glycol methacrylate at low temperature allows immunofluorescent localization of a labile tissue protein.
Authors: Carnegie J A , McCully M E , Robertson H A
Published: April 2011

The antigenic activity of a labile protein postulated to the hormone ovine chorionic somatomammotropin (OcS) is preserved in tissue fixed in either glutaraldehyde or paraformaldehyde and low temperature-embedded in the water-soluble plastic glycol methacrylate. Immunofluorescence techniques used on 1--2 micrometer thick sections show that this protein is located in the cytoplasm of certain fetal chorionic cells in association with small spherical bodies, which may be lipid. The use of borohydrate reduction or treatment with Schiff's reagent to reduce glutaraldehyde-induced background fluorescence is described.
Glutaraldehyde nonfixation of isolated viral and yeast RNAs.
Authors: Langenburg W G
Published: April 2011

The RNAs of brome mosaic (BMV), barley stripe mosaic (BSMV), and tobacco mosaic (TMV) viruses were inactivated by reaction with buffered glutaraldehyde. Glutaraldehyde did not fix 4% BMV-RNA, 20% t-RNA, 5% polyadenylic acid, or 5% adenosine monophosphate into water-insoluble precipitates, or gels, in distilled water or in low or high ionic strength buffers nor did it change their ultraviolet (UV) spectra. Two SDS- and phenol-purified commercial yeast RNA preparations from different sources gave UV spectra typical of pure RNA, but could not be freed of a contaminant that reacted with glutaraldehyde by forming a precipitate. The yeast RNAs did not become water-insoluble after glutaraldehyde reaction. BMV-RNA precipitated by Mg2+ could not be cross-linked into an insoluble form by glutaraldehyde. Nonfixation of RNA by glutaraldehyde must be considered in interpretation of attempts to localize RNA by electron microscopy.
Assay of succinate dehydrogenase activity by the tetrazolium method: evaluation of an improved technique in skeletal muscle fractions.
Authors: Green J D , Narahara H T
Published: April 2011

An improved spectrophotometric method for measuring succinate dehydrogenase (EC 1.3.99.1) activity with the use of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) is described. The procedure has been evaluated in mitochondrial fractions and homogenates of frog skeletal muscle. For mitochondrial suspensions, extraction of formazan with alcohol was found to be superior to extraction with ethyl acetate. For homogenates, complete extraction of formazan required sequential treatment with alcohol and ethyl acetate; the generally employed procedure of extracting once with ethyl acetate alone led to serious underestimation of the amount of formazan in the tissue. Observations of mitochondrial suspension incubated with various concentrations of INT led to the selection of 0.8 mM INT for optimal results. Higher concentrations, although commonly used, can exert undesirable inhibitory effects on succinate dehydrogenase activity, especially at low concentrations of mitochondria and after longer periods of incubation. The problem of instability of succinate dehydrogenase was solved by the addition of buffer at pH 7.5.
A novel technique for quantitative autoradiography of labeled histological specimens.
Authors: Partlow L M , Bush L G , Stensaas L J , Kesner R P
Published: April 2011

A novel quantitative method is described that makes it possible to routinely and accurately quantify contact autoradiographs of tissue sections in terms of actual amounts of radioactivity per square millimeter. Such determinations can either be made on the entire specimen or on any selected area of arbitrary shape that might correspond to a particular histological structure. This method utilizes the tissue specimens themselves as internal standards and requires the availability of a computerized image analyzer.
Lactoferrin: nuclear localization in the human neutrophilic granulocyte?
Authors: Briggs R C , Glass W F , Montiel M M , Hnilica L S
Published: April 2011

Conditions were established whereby nuclear or cytoplasmic immunocytochemical localization of lactoferrin was observed in the human peripheral blood granulocyte. A positive nuclear staining reaction was obtained when cells were either not fixed or treated with most fixatives. However, treatment of blood smears with formaldehydeacetone prior to the immunocytochemical localization gave a cytoplasmic staining reaction. A method was developed that allowed us to examine proteins obtained from granulocyte nuclei isolated from fixed cells. Only a trace amount of lactoferrin was detected in the electrophoretically separated nuclear proteins obtained from granulocytes treated for 1 min with formaldehyde-acetone. However, lactoferrin was major protein component in nuclei isolated from untreated cells, cells treated with other fixatives, or cells preincubated in buffered saline prior to formaldehyde-acetone fixation. formaldehyde-acetone treatment of nuclear materials containing lactoferrin did not extract lactoferrin or mask it from detection, thus indicating that lactoferrin found in the mature neutrophilic granulocyte nucleus is most likely acquired from other cellular organelles during tissue processing.
Autoradiographic demonstration of uptake and retention of 3H-estradiol after in vitro incubation.
Authors: Buell R H , Tremblay G
Published: April 2011

An in vitro incubation method is described for the demonstration of 3H-estradiol in sections of mouse uterus by thaw-mount autoradiography. The method involves the incubation of tissue sections in 5 nM 3H-estradiol with a subsequent 2 hr chase in medium containing 3.5 g% bovine serum albumin. The distribution of the silver grains observed compares favorably to that seen by others with dry-mount autoradiography after in vivo injection. The labeling is inhibited by excess estradiol or diethylstilbesterol, but not by progesterone or hydrocortisone. Its subcellular distribution appears predominantly nuclear in presumptive target cells. Some regional variability in degree of labeling is present throughout the sections, but is far less marked within a given area. Because the observed labeling has been retained during a 2 hr chase and can be inhibited, it is likely to represent physiologically significant uptake and retention of 3H-estradiol in target cells. Preliminary results with human mammary carcinoma suggest this method may be applicable to the investigation of estrogen target cells in human tissue.
Demonstration of membrane-bound proteolytic activity on the surface of mononuclear leukocytes
Authors: Zucker-Franklin D , Lavie G , Franklin EC
Published: April 2011

The existence of proteolytic enzymes bound to the surface of migrating cells has often been surmised. That such enzymes are present on mononuclear leukocytes was suggested by studies showing that serum amyloid A (SAA), the presumed precursor of amyloid protein A, is degraded in the presence of monocytes without endocytosis and with only negligible activity in the cells' supernates. Experiments using immunofluorescence were designed to support this view. It was shown that SAA binds to the cells' surface at low temperatures, whereas binding at 37 degrees C could only be demonstrated when the cells were pretreated with the serine protease inhibitor, diisopropyl-fluorophosphate (DFP) or the elastase inhibitor Ac-Ala-Ala-Pro-Val-CH2Cl. Exposure of the cells to these inhibitors before incubation with SAA at 0 degrees C permitted detection of the protein for an indefinite period of time. At 37 degrees C the DFP-treated cells polarized and eventually lost the surface-bound protein. No interiorized SAA could be demonstrated. Radioautography of cells that had been treated with 3H-DFP revealed grains on the plasma membrane and in the cytoplasm of sectioned monocytes, whereas only 8-15% of lymphocytes were labeled. In lymphocytes radioactivity was restricted to the surface membrane. Additional experiments showed that alpha-naphthyl acetate esterase activity is also located on the surface of monocytes and a subpopulation of lymphocytes. These observations have led to the conclusion that some functions of mononuclear leukocytes may be mediated by enzymes associated with the external surface of these cells.
Localization of thiamine pyrophosphatase activity in motor end plates.
Authors: László I , Bodor T
Published: April 2011

Thiamine pyrophosphatase (TPPase) activity was localized in the terminal arborization of the motor end plates both in rat and frog striated muscles. Electron microscopically the end product of the enzyme was seen in synaptic vesicles. That TPPase was axonally transported was indicated by its localization following experimental ligation of peripheral nerves. It is thus possible that TPPase may play a role in the synthesis of the synaptic mediator substance of the neuromuscular junction.
Fluorescence demonstration of cathepsin B activity in skeletal, cardiac, and vascular smooth muscle.
Authors: Stauber W T , Ong S H
Published: April 2011

Histochemical demonstration of cathepsin B activity was performed for the soleus, extensor digitorum longus, cardiac and vascular smooth muscle tissues of the rat using CBZ-Arg-Arg-4-methoxy-beta-naphthylamide or CBZ-Ala-Arg-Arg-4-methoxy-beta-naphthylamide as the substrate. The enzyme varied in its apparent activity but was localized in discrete granules in all muscle types. Cathepsin B was most active in cardiac muscle and least active in extensor digitorum longus muscles in between these extremes similar to another lysosomal protease, dipeptidyl peptidase II. However, in both types of skeletal muscle, the granules were observed more frequently at the periphery of the muscle cell just beneath the sarcolemma. Since cathepsin B is found only in lysosomes, this subsarcolemmal predominence may indicate that only one population of lysosomes in muscle contains active cathepsin B. All cathepsin B activity was abolished in the presence of the protease inhibitor, leupeptin.
Immunocytochemical localization of fibronectin in limb tissues of the adult newt, Notophthalmus viridescens.
Authors: Repesh L A , Furcht L T , Smith D
Published: April 2011

This study describes the immunocytochemical localization of fibronectin, a defined connective tissue and plasma glycoprotein, and its relationship to collagen and reticulin in adult newt limb tissues. We have also isolated the plasma form of fibronectin in a related species, the adult mudpuppy. The insoluble form of fibronectin was detected with immunoperoxidase stain in basement membranes and loose connective tissue. The endoneurium and perineurium of nerve bundles and the connective tissue elements of striated muscle stained heavily for fibronectin. The dermis and blood vessel walls also reached positively with the immunoperoxidase stain. A similar distribution was observed for reticulin with conventional histologic techniques with the exception of the dermis where only trace amounts of the protein were observed. Fibronectin and collagen were codistributed in the tissues studies. Fibronectin appeared to be intercalated among larger collagenous fibers. Collagen and fibronectin form an extracellular connective tissue scaffold that abuts against many types of adherent cells in different tissues. This supports the possible role of fibronectin in cell-matrix interactions and normal cell and tissue organization.
Interaction of DEAP fluoranthene (RMI 9563 DA) with DNA and metaphase chromosomes.
Authors: Miller F D , Lin C C , Van de Sande J H
Published: April 2011

A fluorescent compound, DEAP fluoranthene (a tilorone cogener), has been suggested to bind to DNA by intercalation. The interaction of this drug with natural and synthetic DNAs and human metaphase chromosomes has been investigated in detail. DEAP fluoranthene shows a slight A-T preference in binding to DNA and was also found to change the topological winding number of superhelical DNA. These data, and the determined unwinding angle of 27 degree for DEAP fluoranthene, strongly support an intercalation mode of binding for this drug to DNA. DEAP fluorescence is minimally quenched when intercalated between A-T base pairs and maximally quenched when interacting with G-C base pairs. DEAP fluoranthene was found to produce bright fluorescent bands on human metaphase chromosomes identical to those produced by quinacrine. The solution data on DEAP fluoranthene interaction with DNA can be used to rationalize the production of differential banding patterns on chromosomes by DEAP fluoranthene in which the drug reports underlying DNA sequence arrangement of the metaphase chromosome.
COMPARATIVE HISTOCHEMICAL AND QUANTITATIVE STUDY OF ADRENAL AND KIDNEY TISSUE BY TETRAZOLIUM TECHNIQUE
Authors: CASCARANO J. , ZWEIFACH B. W.
Published: April 2011

A study was made of the influence of experimental variables, including electrolyte balance, pH, tonicity, length of incubation and dimensions of tissue slice, on the in vitro reduction of tetrazolium.

The most discriminating approach included examination of both the histochemical and quantitative expressions of the deposition of formazan.

Some of the limitations of neotetrazolium (NT) and 2,3,5 triphenyl tetrazolium chloride (TTC) are discussed, NT being less suitable for quantitative estimates of reduction values.
Scanning microdensitometry of objects small relative to the wavelength of light.
Authors: Goldstein D J
Published: April 2011

Diffraction effects may have to be taken into account in microdensitometry when dealing with relatively dark specimens even an order of magnitude larger than the wavelength of light, and become progressively more important with smaller objects. According to geometrical optical theory, when scanning across the straight edge of a uniformly absorbing, semi-infinite object the distribution error per scan line is directly proportional to the diameter of the measuring-spot. Diffraction theory predicts similar results for measuring-spots larger than about 3 times the wavelength of light, but a significant error per scan line with very small or even infinitesimal measuring-spots. Diffraction theory further indicates that point absorbance measurements can be 95 + % accurate in the centers of 6.25, 2.5, and 2.0 microns diameter disks with absorbances respectively up to about 1.0, 0.39, and 0.25, but that this accuracy is unattainable with any object less than 1.25 microns in diameter. Scanning, integrating absorbance measurements are of somewhat lower accuracy than central point measurements with relatively large objects, e.g., they are only about 89% accurate with a 6 micron diameter object of absorbance 1.0. With very small objects, diffraction theory shows distribution error to be almost independent of the size of the scanning spot, and with an object of less than about 0.125 micron diameter the apparent integrated absorbance predicted by diffraction theory is effectively identical with that predicted by geometrical theory for an infinitesimal object scanned with a finite measuring-spot, i.e., it is the product of the object area and 0.4343 (1-It), where It is the true transmission. Scanning microdensitometry of objects of very low true absorbance is effectively free from distribution error. In practice, distribution error can be reduced by using an off-peak wavelength, by reducing the area illuminated, and by routine measurement and offsetting of apparent glare (some of which is actually due to diffraction). Little or nothing is to be gained by using a measuring-spot smaller than about one-quarter the wavelength of light.
Preparation of peroxidase: antiperoxidase (PAP) complexes for immunohistological labeling of monoclonal antibodies.
Authors: Mason D Y , Cordell J L , Abdulaziz Z , Naiem M , Bordenave G
Published: April 2011

The production of mouse peroxidase:antiperoxidase (PAP) complexes suitable for immunohistological use in conjunction with monoclonal antibodies is described. Three approaches were explored: 1) production of conventional polyclonal PAP complexes; 2) conversion of rabbit PAP to "pseudo-mouse PAP" by incubation with monoclonal mouse anti-rabbit immunoglobulin; 3) formation of PAP complexes from monoclonal mouse antiperoxidase. PAP complexes prepared by the latter technique gave the best immunohistological labeling reactions, being stable on storage and compatible with a wide range of human monoclonal antibodies. Gel filtration revealed that monoclonal PAP is of lower molecular weight than conventional PAP complexes (fulfilling theoretical predictions based on the monospecificity of monoclonal antibodies).
On the origin of alpha-glucosidase in human urine.
Authors: Nishinaka H , Minamiura N , Matoba K , Furusawa M , Yamamoto T
Published: April 2011

The recent findings that alpha-glucosidase from human kidney was identical with one component (F1) of the alpha-glucosidases found in human urine suggested the idea that this enzyme might originate in the kidneys. The present study was performed to test this idea by immunological methods. Urine alpha-glucosidase F1 was isolated in the electrophoretically homogeneous state, and the antibody prepared in rabbits was purified by affinity chromatography after the antisera were fractionally precipitated with ammonium sulfate and chromatographed on diethylamino ethyl (DEAE)-cellulose. The staining of human kidney tissue sections was performed by the indirect method, using alpha-glucosidase F1 antibody and fluorescein-conjugated anti-rabbit immunoglobulin goat sera. The proximal convoluted portion (proximal tubules) with brush border and Henle's loops (late proximal) were stained clearly. Preincubation of intact antibody with purified antigen prevented specific staining of the proximal convoluted portion and Henle's loops. In contrast, all other tissues of kidney were stained less positively or negatively. These results indicate that alpha-glucosidase F1 originates in the kidney, and that glucosidase is specifically localized in the proximal convoluted portion and Henle's loops.
Analytical electron microscopic evaluation of the effects of paraformaldehyde pretreatment on the reaction of glutaraldehyde with biogenic amines.
Authors: McClung R E , Wood J
Published: April 2011

Analytical electron microscopy was used to determine the quantitative effects of paraformaldehyde pretreatment on the formation of the biogenic amine-glutaraldehyde-chrome complex. Pretreatment with paraformaldehyde prevented the glutaraldehyde-chrome reaction with norepinephrine in the rat adrenal medulla. In contrast to the effect of paraformaldehyde on norepinephrine, pretreatment did not prevent the chrome reaction in serotonin-containing argentaffin cells of the gut. X-Ray energy spectrographic analysis revealed a significant decrease in chrome content in the paraformaldehyde treated tissue, but sufficient chrome did react to produce an electron-dense product. Thus by treating tissue with paraformaldehyde prior to the glutaraldehyde chrome procedure, serotonergic sites may be differentiated from catecholaminergic areas at the electron microscopic level.
Electron immunocytochemical localization of enkephalin-like material in catecholamine-containing cells of the carotid body, the adrenal medulla, and in pheochromocytomas of man and other mammals.
Authors: Varndell I M , Tapia F J , De Mey J , Rush R A , Bloom S R , Polak J M
Published: April 2011

Enkephalin-like immunoreactivity has been localized to electron-dense secretory granules of cat and piglet carotid bodies and adrenal medullae, horse adrenal medulla, and also to human adrenal medulla and pheochromocytomas using a gold-labeled antibody technique performed at the electron microscopic level. The same granules were also demonstrated to exhibit dopamine-beta-hydroxylase-like immunoreactivity, which suggests a granular colocalization of amines and peptides in catecholamine-storing cells.
Monocytic origin of human alveolar macrophages.
Authors: Radzun H J , Parwaresch M R , Kreipe H
Published: April 2011

The monocytic lysosomal acid esterase (AcE; EC 3.1.1.6) comprises five isoenzymes, each having specific isoelectric points (pI) as well as antigenicity. In the present study attempts were made to retrace the monocytic origin of human alveolar macrophages (AM) by comparison of their isoenzyme patterns with those of blood monocytes. Resident AM obtained from bronchial lavages lacking any neutrophils and unstimulated monocyte admixture showed in addition to the five monocytic isoenzymes nine additional isoenzyme loci. In vitro stimulation of blood monocytes (BM) using lymphokine-conditioned media led to a gradual transition of the typical monocytic isoenzyme pattern into that of AM. It is concluded that AM originates from blood monocytes by tissue-specific stimulation. This cellular transformation can be modeled in vitro as far as morphology, cytochemistry, and isoenzyme pattern are concerned.
Soluble compound electron microscope (EM) autoradiography: a resolution source to test redistribution of soluble tritiated compounds during processing.
Authors: Harris W V , Salpeter M M
Published: April 2011

The development of a resolution source that can be labeled with either a soluble or insoluble tritiated compound, and of a method for applying a dry, uniform monolayer of emulsion is reported. Influences due to redistribution of the soluble isotope during emulsion coating were measured by comparing the grain density distributions around the resolution source for soluble tritiated proline (3H-PRO) with that obtained for cross-linked tritiated bovine serum albumin (3H-BSA). The grain density distributions resulting from a standard method of emulsion application (partly gelled/loop method) are compared to that obtained from a dry stripping film. It was found that only the dry stripping film gave a grain distribution which was statistically not different for the soluble and insoluble specimens.
A flow cytometric assay of neutrophil degranulation.
Authors: Abrams W R , Diamond L W , Kane A B
Published: April 2011

A quantitative assay of neutrophil degranulation was developed using flow cytometry. Dog neutrophils were purified to greater than 95% purity and viability by isopyknic density centrifugation in an isosmotic medium. These cells concentrated the fluorochrome acridine orange (AO) in their azurophilic granules, but not in specific granules. Also contained in the azurophilic granules are elastase, myeloperoxidase, and approximately 50% of the lysozyme activity. The fluorochrome was released concomitantly with elastase activity, as shown by flow cytometry, fluorescence microscopy, and biochemical assay in response to the ionophore A23187. By flow cytometry, unstimulated cells are distributed in a single broad peak of high fluorescence intensity. With increasing concentrations of A23187 (0.48-4.80 microM), a greater proportion of the cells shifted to a single peak of low fluorescence intensity. Few cells with intermediate fluorescence were observed. These analyses revealed that the neutrophils degranulated in a quantal, all-or-none response.
Resolution and limitations of the immunoperoxidase procedure in the localization of extracellular matrix antigens.
Authors: Courtoy P J , Picton D H , Farquhar M G
Published: April 2011

A double labeling system was used to test the resolution of the indirect immunoperoxidase procedure in the localization of extracellular matrix components. A recognizable antigen, cationized ferritin, was first implanted at specific anionic sites (approximately 60 nm periodicity) in the lamina rara interna and externa of the glomerular basement membrane (GBM) and subsequently localized by immunoperoxidase. The coincidence between the location of reaction product and the ferritin clusters was assessed. When the amount of immunoadsorbed peroxidase and time of exposure to the 3,3'-diaminobenzidine (DAB)-containing medium were limited, discrete deposits of reaction product were observed around individual ferritin clusters. When immunolabeling was increased, the whole GBM was stained, and DAB staining was also found along the endothelial plasmalemma and the epithelial plasmalemma at the base of the foot processes at some distance (greater than 100 nm) from the ferritin clusters in the laminae rarae. These findings indicate that oxidized DAB reaction product can diffuse over long distances and be reabsorbed onto cell membranes. Even under limited incubation conditions some diffusion of DAB reaction product was encountered. The value and limitations of the DAB-peroxidase procedures are discussed.
Immunocytochemical localization of rat intestinal vitamin D-dependent calcium-binding protein.
Authors: Taylor A N , Gleason W A , Lankford G L
Published: April 2011

Vitamin D-dependent calcium-binding protein (CaBP) was localized in intestinal tissue sections obtained from rats raised under three different nutritional conditions: a normal vitamin D-replete diet, a vitamin D-free diet followed by supplementation with vitamin D3, or a vitamin D-free diet without additional supplementation. An indirect immunoperoxidase technique, with immunocontrols, was used to visualize the specific sites of CaBP. CaBP was visualized only in the cytoplasm of absorptive cells. In the duodenum of animals raised on a normal diet, CaBP was present in absorptive cells from the upper crypt region to the villus tips. In the jejunum, many fewer absorptive cells contained CaBP, while in the ileum only random absorptive cells near the villus tips contained CaBP. In rats raised on a vitamin D-deficient diet then supplemented with vitamin D3, CaBP was present in cells at the full depth of the crypts and in absorptive cells along the total villus length in the duodenum. Rats raised on the same deficient diet but without supplementation with additional vitamin D exhibited no CaBP in crypt cells nor in absorptive cells more than half way up the villi. Absorptive cells higher on the villi contained immunoreactive CaBP but the intensity of immunostaining and number of CaBP-containing cells was markedly reduced compared to the vitamin D-supplemented group.
Immunohistochemical and biochemical demonstration of the change in glycolipid composition of the intestinal epithelial cell surface in mice in relation to epithelial cell differentiation and bacterial association.
Authors: Umesaki Y
Published: April 2011

We have previously demonstrated the appearance of fucosyl asialo-GM1 (FGA1) in the small-intestinal epithelial cells of germ-free mice via the induction of GDP-fucose: asialo-GM1 (GA1) alpha(1 leads to 2) fucosyltransferase (FT) after the conventionalization of these animals (Umesaki Y, Sakata T, Yajima T: Biochem Biophys Res Commun 105:439, 1982). The present study, based on this earlier work, demonstrates the changes in the glycolipid antigens of the small-intestinal epithelial-cell membrane as shown immunohistochemically with specific antibodies raised against asialo GM1 (GA1) and FGA1. In germ-free mice, GA1 was localized both in the villus cells and in the crypt cells. In the process of conventionalization, FGA1 appeared in the villus cells while the GA1 content of these cells was decreased. Four to 5 days after the conventionalization procedure, the fluorescence produced by anti-FGA1 was strongest in the villus cells, while that produced by anti-GA1 was detected only in the crypt cells. At this same time the FT activity of the small-intestinal mucosa was highest, with most of the GA1 apparently being converted into FGA1, as shown in the paper cited above. Thereafter, the GA1 content of both the villus and crypt cells again increased greatly. On the other hand, the fluorescence produced with anti-FGA1 decreased, and could no longer be detected 14 days after conventionalization. The activity of FT, measured biochemically in epithelial cells differentially isolated from the villus tip to the crypt, was greater in the villus than in the crypt region. This confirmed the intense staining with anti-FGA1 that was seen in villus cells. The fluorescence produced by the two anti-glycolipid antibodies used in the study distributed not only in the microvillus membrane but also to some extent in the basolateral membrane. The localization of the respective glycolipids contrasted with that of the glycoprotein sucrase--isomaltase enzyme complex, the fluorescence of which was exclusively confined to the microvillus-membrane side of the villus cells.
Adenosine analogue induced ultrastructural changes in the nucleolus that correlate with inhibition of ribosomal RNA processing.
Authors: Tunkel A R , Studzinski G P
Published: April 2011

The aminonucleotide of puromycin (AMS) is known to have a differential effect on ribosomal RNA (rRNA) synthesis and the cell-cycle traverse in normal as compared to neoplastic or virally transformed cells. In this study, AMS and its demethylated derivative, 3'-amino-3'-deoxyadenosine (3'-AmA) have been used to compare their effects on normal (IMR 90) versus transformed (AG 2804) human fibroblasts with regard to preribosomal RNA (pre-rRNA) transcription, the processing of this RNA, and structural changes in the nucleolus. The processing of pre-rRNA in normal and transformed fibroblasts treated with 3'-AmA for 4 hr was markedly depressed. However, this process did not appear to be affected by the AMS treatment of normal cells, while in transformed cells it was maximally inhibited within 4 hr of exposure to this drug. Ultrastructural changes were observed in the nucleoli of normal and transformed cells treated with 3'-AmA, whereas treatment of these cells with AMS produced alterations of nucleolar structure only in the transformed cells. These changes correlated with the onset of inhibition of pre-rRNA processing. Our findings suggest that the impairment of pre-rRNA processing produced by AMS and 3'-AmA in transformed cells and by 3'-AmA in normal cells may be due to structural disorganization of the nucleolus produced by these antimetabolites.
Comparison of muscle fiber typing by quantitative enzyme assays and by myosin ATPase staining.
Authors: Hintz C S , Coyle E F , Kaiser K K , Chi M M , Lowry O H
Published: April 2011

Fibers in cross sections of human and rat muscle were typed by using histochemical ATPase stains, and the results were compared with those of quantitative enzyme assays of fragments of the same fibers dissected from serial freeze-dried sections. Two enzymes previously used to assess the metabolic type were measured in each case: lactate dehydrogenase and either adenylokinase (human fibers) or malate dehydrogenase (rat fibers). With human fibers there was essentially complete agreement between ATPase staining and the metabolic enzyme assays in distinguishing types I and II fibers. The agreement was less consistent with regard to type IIA and IIB fibers. A number of ATPase type IIC fibers were identified in one human muscle, and were found to fall between ATPase types I and IIA on the basis of metabolic enzyme assay results. Rat-fiber ATPase types I, IIA, and IIB from the plantaris muscle were rather well segregated on a two-dimensional lactate dehydrogenase-malate dehydrogenase grid. In the rat soleus muscle, ATPase types I and IIA fibers were shifted to lower lactate dehydrogenase levels, with IIC fibers interposed between them.