bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of gap junction proteins, connexins 32 and 26, in rat and guinea pig liver as revealed by quick-freeze, deep-etch immunoelectron microscopy.
Authors: Kuraoka A , Iida H , Hatae T , Shibata Y , Itoh M , Kurita T
Published: April 2011

By use of site-specific antibodies against synthetic oligopeptides, we examined the localizations of the gap junction proteins connexin 32 (Cx32) and connexin 26 (Cx26) in rat and guinea pig liver. Double-labeling immunofluorescence microscopy revealed that in guinea pig liver both proteins were spread throughout the liver lobules and seemed to localize together within the same gap junction plaque. In rat liver, co-localization of both Cx32 and Cx26 in the same plaques was also suggested in periportal zones. Quick-freeze, deep-etch immunoelectron microscopy showed that immunolabeling of isolated guinea pig liver gap junction plaques with either Cx32 or Cx26 antiserum yielded complete and dense antibody decoration of the cytoplasmic surface of the plaques. In isolated rat liver plaques, the cytoplasmic surfaces were densely decorated with Cx32 antiserum, whereas Cx26 labeling yielded diffuse decoration with variable intensity of the plaques. In both species we did not observe any focal or patchy clusters of the labeling in any plaques examined. Double-labeling immunoelectron microscopy confirmed that both Cx32 and Cx26 are co-localized in the same gap junction plaques. These results suggest that in hepatocytes expressing both Cx32 and Cx26, both types of gap junction proteins are not segregated but intermingle randomly within the same plaques.
Label-fracture of plasma membranes isolated from measles virus-infected cells.
Authors: Rutter G , Hohenberg H
Published: April 2011

We present a method that permits correlation of the intramembrane architecture of plasma membrane fracture faces with the distribution of specific molecules at the corresponding cytoplasmic or exoplasmic membrane surfaces. HeLa cells infected with measles virus were used as a model system. Large fragments of the dorsal membrane were isolated after the virus glycoproteins were tagged at the outer cell surface with immune serum and protein A-gold markers. In a second step, different virus polypeptides at the inner cell surface were also identified by a smaller gold label. Thereafter, the isolated plasma membranes were frozen and freeze-fractured. The complementary fracture faces were shadowed with heavy metals and carbon and examined in the transmission electron microscope without cleaning of remaining biological material. Thus, the micromorphology of the replicated fracture faces and the topochemistry of virus components localized at the corresponding leaflets of the plasmalemma could be seen on the same image at high resolution. Of note is that the freeze-fracture morphology of the protoplasmic face is related to the molecular composition of the cytoplasmic surface, as revealed by antibody tagging.
Localization of S1 and elongation factor-1 alpha mRNA in rat brain and liver by non-radioactive in situ hybridization.
Authors: Lee S , Stollar E , Wang E
Published: April 2011

Elongation factor-1 alpha (EF-1 alpha) is a ubiquitous, highly conserved protein that functions in peptide elongation during mRNA translation. We recently reported that, as do lower species, mammals also contain a second EF-1 alpha-like gene (S1). Unlike EF-1 alpha, which is present in all tissues, S1 mRNA is detected only in brain, heart, and muscle by Northern analysis and RNAse protection assays. In this report we present the identification of S1 and EF-1 alpha messages by non-radioactive in situ hybridization in brain and liver. We show that with this technique we can detected S1 mRNA only in certain cells in brain, mostly neurons; on the other hand, EF-1 alpha is present in all cell types that we have studied so far. We demonstrate that although EF-1 alpha mRNA can be detected in S1-negative cells it is also present in high abundance in S1-positive cells. The results presented here correlate with our previous finding that mammalian species contain a tissue-specific EF-1 alpha-like gene, S1. The presence of a second EF-1 alpha-like transcript within fully differentiated cells suggests a novel cell type-specific gene expression whose function may be related to the permanent growth-arrested state of cells in brain, heart, and muscle.
Comparison of the sensitivities of ribonucleic acid and oligonucleotide probes for in situ detection of Theiler's virus mRNA.
Authors: Aubert C , Ozden S
Published: April 2011

To evaluate the sensitivity of in situ detection of the Theiler's virus genome, we hybridized BHK-21-infected cells with antisense ribo- and oligonucleotide 35S-labeled probes. The sensitivity achieved with the anti-sense 280-nucleotide riboprobe was similar to that obtained with a 93-mer oligonucleotide probe. However, more reproducible and accurate results were obtained with the riboprobe. With long exposure times, the background was higher with the oligonucleotide probe than with the RNA probe. The background was improved by using freshly labeled oligonucleotide probe.
Effects of digitonin on the intracellular content of rat hepatocytes: implications for its use in the study of intralobular heterogeneity.
Authors: Racine L , Scoazec J Y , Moreau A , Bernuau D , Feldmann G
Published: April 2011

Anterograde or retrograde perfusion of rat liver with digitonin selectively permeabilizes the periportal or the perivenous zone of the hepatic lobule. Digitonin perfusion is used to analyze the effluents released by permeabilized hepatocytes or, combined with collagenase perfusion, to obtain cell suspensions enriched in either periportal or perivenous hepatocytes. Despite the wide use of digitonin to study lobular heterogeneity, its affects on rat hepatocytes are not well documented. We therefore analyzed the effects of digitonin perfusion on the intracellular content of rat hepatocytes by combining electron microscopy, histoenzymology, immunohistochemistry, and in situ hybridization. At the concentration currently used for the study of lobular heterogeneity, digitonin perfusion induced a marked cytosolic clarification of permeabilized hepatocytes, while most organelles except mitochondria were well preserved. In the digitonin-altered zones, there was no histochemical detection of non-membrane-bound enzymes (lactate dehydrogenase, glutamate dehydrogenase), whereas membrane-bound enzymes (succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, NADPH dehydrogenase, glucose-6-phosphatase) were still detected. Immunohistochemistry and in situ hybridization revealed significant amounts of several plasma proteins (albumin, alpha 2-macroglobulin, alpha 1-inhibitor 3, alpha 1-acid glycoprotein) and their respective mRNAs in digitonin-permeabilized hepatocytes. The demonstration that digitonin-permeabilized hepatocytes retain many intracellular constituents shows that biochemical analysis of cellular effluents released from digitonin-permeabilized hepatocytes must be interpreted with caution and that the apparent characteristics of cell suspensions obtained by the digitonin-collagenase technique might be significantly altered by contamination with permeabilized hepatocytes from the opposite zone.
Expression of tenascin in developing and adult mouse lymphoid organs.
Authors: Ocklind G , Talts J , Fässler R , Mattsson A , Ekblom P
Published: April 2011

The extracellular matrix (ECM) is essential in regulating many cell functions in non-lymphoid cells, and the ECM may also play a role in the function of the immune system. Tenascin is a hexameric glycoprotein of the ECM. In mouse, two major polypeptides of MW 210 KD and 260 KD are formed by differential splicing. Northern blot screening of various mouse tissues showed that the short 6 KB tenascin message was strongly expressed in the adult thymus, whereas very little or no tenascin mRNA could be detected in spleen. In addition, immunoblotting and histological analysis with monoclonal anti-tenascin antibodies revealed the presence of tenascin in lymph nodes and spleen. In thymus, only a short-splice variant of tenascin was detected by immunoblotting, which supported the Northern blot results. Immunohistology showed that the epithelial reticular stroma in both embryonic and adult mouse thymus expressed tenascin, as did the postnatal mesenchymal reticular stroma in lymph nodes and spleen. The distribution of tenascin in the thymus was more restricted than that of fibronectin and laminin.
Analysis of ODC and c-myc gene expression in hepatocellular carcinoma by in situ hybridization and immunohistochemistry.
Authors: Gan F Y , Gesell M S , Alousi M , Luk G D
Published: April 2011

We quantitated mRNA and protein for ornithine decarboxylase (ODC) and c-myc in formalin-fixed liver sections from 25 specimens of hepatocellular carcinoma (HCC) and seven normal livers by a non-radiolabeled in situ hybridization technique and immunohistochemistry. This non-radioactive in situ hybridization technique was highly specific, with virtually no background, and permitted quantitative analysis based on optical density. Reaction products were quantitated with computer-assisted microdensitometry. Samples were classified as normal, adjacent uninvolved, cirrhosis, well-differentiated HCC, and poorly-differentiated HCC. There was a progressive increase in all four parameters measured, ODC mRNA and protein, and c-myc mRNA and protein, from normal, to adjacent uninvolved liver, to cirrhosis, to well-differentiated HCC, to poorly-differentiated HCC. The sole exception was that ODC mRNA was lowest in cirrhosis. The patterns of ODC and c-myc gene expression are similar in HCC. The quantitative detection of ODC mRNA, c-myc mRNA, and their protein products in hepatocellular carcinoma and cirrhosis by in situ hybridization and immunohistochemical techniques may have a potential role in the study of hepatocarcinogenesis and in the diagnosis of hepatocellular carcinoma.
Avidin-EITC: an alternative to avidin-FITC in confocal scanning laser microscopy.
Authors: Hulspas R , Krijtenburg P J , Keij J F , Bauman J G
Published: April 2011

Detection of fluorescein-5-isothiocyanate (FITC)-labeled conjugates is suboptimal in two-color confocal scanning laser microscopy (CLSM). This limits the detection of small, dimly fluorescent targets. We explored the possible advantages of applying eosin-5-isothiocyanate (EITC) conjugated to avidin (Av-EITC) as an alternative for Av-FITC in CSLM. Despite the lower quantum efficiency of EITC, we found that the measured Av-EITC and Av-FITC emission intensities were similar as a result of the standard filter combinations used for simultaneous two-color detection in the Bio-Rad MRC 600 CSLM. The advantage of Av-EITC was that its fading characteristics compared very favorably to those of Av-FITC. An excitation intensity-dependent increase in Av-EITC fluorescence was observed, followed by an exponential decrease. This increase in fluorescence allows longer observation times, averaging of several scans without loss of brightness, and thus detection of dimly fluorescent targets by CSLM.
An indirect immunofluorescence procedure for staining the same cryosection with two mouse monoclonal primary antibodies.
Authors: Lewis Carl S A , Gillete-Ferguson I , Ferguson D G
Published: April 2011

Indirect immunofluorescence procedures reported thus far are not effective at localizing two antigens in the same preparation when both primary antibodies are raised in the same species. In this case, the secondary antibodies can crossreact with both primary antibodies. We report here a protocol in which mouse monoclonal antibodies (MAb) specific for actin and myosin were used sequentially to stain the same frozen section of guinea pig skeletal muscle. The myosin-specific mAb was applied first and was localized with a rabbit anti-mouse IgG-rhodamine secondary antibody. The sections were then "blocked" with a non-binding mouse MAb and unconjugated goat anti-mouse IgG F(ab) fragments. The actin-specific mAb was then applied and localized with a rabbit anti-mouse IgG-fluorescein secondary antibody. Laser scanning confocal microscopy and image analysis demonstrated that the I-bands, the A-bands, and the H-bands of each sarcomere were clearly identifiable by this approach. This protocol is not limited to use with mouse MAb but can be easily modified to permit indirect immunolocalization of two antigens in the same sample using any pair of same-species primary antibodies.
Ultrastructural detection of calcium and magnesium in the chromatoid body of mouse spermatids by electron spectroscopic imaging and electron energy loss spectroscopy.
Authors: Rouelle-Rossier V B , Biggiogera M , Fakan S
Published: April 2011

We studied at the ultrastructural level the presence of calcium and magnesium in the chromatoid body (CB) in mouse spermatids. In addition, the presence of these two cations was also examined in the nucleolus. By electron spectroscopic imaging (ESI) and electron energy loss spectroscopic (EELS) analyses on glutaraldehyde/pyroantimonate-fixed material, we showed the association of pyroantimonate precipitate granules containing calcium and/or magnesium with the CB. The granules in both active (primary spermatocytes) and inactive (spermatids) nucleoli contain mainly calcium. Our results confirm that although the pyroantimonate technique used alone suffers from a lack of specificity, it can be useful for in situ immobilization of different cations that are further resolved with methods of elemental analysis. Without pyroantimonate fixation, only very few spots containing calcium can be localized by ESI and EELS.
Quantitative comparison of intracellular concentration and volume of Clara cell 10 KD protein in rat bronchi and bronchioles based on laser scanning confocal microscopy.
Authors: Dodge D E , Rucker R B , Singh G , Plopper C G
Published: April 2011

We used an antibody to rat Clara cell 10 KD secretory protein (CC10) to compare the abundance of CC10 in non-ciliated epithelium of bronchioles and bronchi by immunohistochemistry and laser scanning confocal microscopy (LSCM) in the reflectance mode. Three zones of reflectance intensity (high, medium, low), directly related to CC10 content, were used to distinguish subcellular compartments of differing densities. Two major compartments contained CC10: granules and endoplasmic reticulum. Bronchiolar cell granules had relatively even reflectance, a high CC10 concentration (0.92 mg/ml), and occupied 7.5% of the cell volume. Bronchial cell granules were bi-zonal, lower in CC10 concentration (0.83 mg/ml), and occupied less cell volume (2.3%). Low-reflecting endoplasmic reticulum occupied a greater cell volume in bronchioles than in bronchi (32.8% and 22.1%, respectively). An intermediate-density zone consisted of endoplasmic reticulum close to granules. CC10 abundance, expressed as ng stored per unit surface area of epithelial basal lamina, was more than three times greater in bronchioles than in proximal bronchi (1.50 ng/mm2 vs 0.42 ng/mm2). These data demonstrate that the process of CC10 secretion differs markedly in non-ciliated epithelium of bronchi and bronchioles. Identifiable mucous-goblet cells did not contain CC10. The LSCM provides a method for quantifying intracellular CC10 pools in intact lung cells and has the potential for quantifying other proteins in subcellular compartments.
Immunohistochemical localization of taurine in rat renal tissue: studies in experimental disease states.
Authors: Trachtman H , Lu P , Sturman J A
Published: April 2011

Taurine, a sulfur amino acid, is present in abundant amounts in cells throughout the body. The kidney regulates taurine balance by modulating proximal tubule reabsorption in response to fluctuations in dietary intake of this nutrient. There is no information about the localization of taurine within the kidney in normal and diseased renal parenchyma. Therefore, using an antibody to a taurine-glutaraldehyde-BSA conjugate, we examined the distribution of taurine in renal tissue. Normal rats, those with streptozocin diabetes, puromycin aminonucleoside nephropathy, bilateral ureteral ligation, and 5/6 nephrectomy were studied. In normal animals, taurine was found primarily in medullary tubules, with minimal staining of proximal tubules and glomeruli. There was increased taurine staining of all structures, especially medullary tubules, in rats with streptozocin diabetes and puromycin aminonucleoside nephropathy. These changes were more pronounced in diabetic rats and were unrelated to renal medullary osmolality. The distribution of taurine within the kidney was unchanged in the models of acute and chronic renal failure. Alterations in the immunohistochemical localization of taurine correlated with the beneficial effect of this amino acid to preserve renal function in the rats with chronic diabetes and puromycin aminonucleoside nephropathy. These results suggest that taurine is preferentially localized in the medullary regions of the kidney, where it exerts a protective effect against renal injury in select disease states.
Establishment of a novel monoclonal antibody, SE-1, which specifically reacts with rat hepatic sinusoidal endothelial cells.
Authors: Ohmura T , Enomoto K , Satoh H , Sawada N , Mori M
Published: April 2011

A monoclonal antibody, SE-1, which specifically reacted with rat hepatic sinusoidal endothelial cells (HSE), was established with elutriator-separated HSE as immunogen. No crossreactivity was observed with other types of endothelial cells or non-endothelial cells. By immunoelectron microscopy, the antigen recognized by MAb SE-1 was localized to the membrane surface of HSE. Immunoblot analysis revealed that SE-1 recognized a single 45 KD band. Although the SE-1 antigen is not yet characterized, the above evidence strongly suggests that it is related to the specific function of HSE. Therefore, SE-1 may be a useful marker to study the role of HSE in various pathophysiological conditions of the liver.
Glycoproteins of mouse vaginal epithelium: differential expression related to estrous cyclicity.
Authors: Horvat B , Multhaupt H A , Damjanov I
Published: April 2011

We used lectin overlay blotting and SDS-PAGE to analyze the estrous cycle-specific expression of mouse vaginal epithelial glycoproteins. Seven lectins chosen for their differential carbohydrate-binding specificity revealed 15 glycoproteins that showed cycle-related expression. Each lectin had a unique binding pattern different from the patterns revealed by other lectins. However, several estrous cycle phase-specific glycoproteins reacted with more than one lectin. The most prominent of these glycoproteins (M(r) 92-95 KD) was weakly expressed in late diestrus and fully expressed only in proestrus, coincident with the transformation of two superficial layers of vaginal squamous epithelium into mucinous cuboidal cells. Electron microscopic lectin histochemistry revealed the glycoproteins in the mucinous granules of surface cuboidal cells and in the lumen of the vagina. Our results illustrate the complexity of glycoconjugate synthesis in mouse vagina and reveal the distinct cycle-specific patterns of individual glycoprotein expression. These cyclic glycoproteins could serve as vaginal biochemical markers for the specific phases of the estrous cycle.
Silver-enhanced diaminobenzidine-sulfide (SEDS): a technique for high-resolution immunoelectron microscopy demonstrated with monoamine immunoreactivity in monkey cerebral cortex and caudate.
Authors: Smiley J F , Goldman-Rakic P S
Published: April 2011

A common frustration of immunoelectron microscopy (IEM) is the density of the 3,3'-diaminobenzidine (DAB) label, which obscures intracellular details of labeled structures. To overcome this problem, a silver enhancement protocol was developed which leaves silver deposits on very low levels of DAB. The resulting label is composed of easily visualized punctate silver deposits, localized in processes with little or no detectable DAB. This technique incorporates several modifications into previously described methods for silver enhancement of DAB. The principal innovation is to pretreat the DAB label with sodium sulfide before silver enhancement, which substantially increases the sensitivity of the silver enhancement. In addition, cysteine was used in place of thioglycolic acid to suppress tissue argyrophilia, allowing use of both glutaraldehyde- and paraformaldehyde-fixed tissue without degradation of ultrastructure. We demonstrate this technique with dopamine, norepinephrine (NE), and serotonin (5HT) immunoreactivity in monkey prefrontal cerebral cortex and with dopamine immunoreactivity in the anterior caudate. The punctate label allows essentially unobscured visualization of the intracellular details and cell membranes of these monoamine axons. Whereas 5HT axons formed small asymmetric synapses, dopamine and NE axons typically formed small symmetric synapses with notably subtle membrane specializations. It is likely that these are often obscured by conventional DAB labeling. The use of several preparations indicates that this technique will be useful with a variety of antibodies. It might also provide an attractive alternative to colloidal gold, especially with glutaraldehyde-fixed tissue which is not easily penetrated by gold particles.
Histamine-storing cells in the oxyntic mucosa of the rat stomach: a transmission electron microscopic study employing fixation with carbodiimide.
Authors: Nissinen M J , Panula P
Published: April 2011

We studied the distribution of histamine (HA) immunoreactivity in endocrine cells of the acid-producing mucosa in rat stomach with pre-embedding immunoelectron microscopy (IEM) using an antiserum against HA. Four fixation modifications were compared to optimize the ultrastructural morphology and staining pattern with the antisera produced against carbodiimide-conjugated HA. Fixation with 4% 1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDCDI) combined with both 4% paraformaldehyde and 0.1% glutaraldehyde gave superior results compared with EDCDI alone. Enterochromaffin-like (ECL) cells were easily distinguished from other endocrine cells in optimally fixed samples. The peroxidase end-product was distributed within the cytoplasm surrounding the vesicles of the ECL cells. ECL cells comprised about 75% of all endocrine cells, and about 90% of them were HA immunoreactive (HA-IR). No other HA-IR cell types were identified by EM in the basal half of the oxyntic region of rat gastric mucosa. The results suggest that a combination of EDCDI and aldehydes is suitable for IM demonstration of HA in cells. ECL cells from a predominant portion of endocrine cells in the oxyntic glands and may constitute the only significant non-mast cell store of HA in rat gastric mucosa.
A novel hinge system and incubation chamber for emulsion-coated coverslip autoradiography.
Authors: Rhodes K J , Meiners K A , Rosene D L
Published: April 2011

We describe an emulsion-coated coverslip autoradiographic technique for large (50 x 50 mm) sections of monkey or human brain. The technique uses adhesive-backed, teflon-reinforced aluminum foil as a flexible hinge that allows the coverslip to swing away from the slide so that the emulsion and tissue can be processed independently. We also describe a plexiglas incubation chamber that allows two slides with coverslips folded away to be processed back-to-back in 5 ml of reagent solution. In general use, the chamber minimizes the volume of solution needed to cover large slide-mounted sections and is ideal for radioligand or immunohistochemical reactions that use expensive reagents. For autoradiography, the chamber greatly facilitates independent processing of the emulsion and tissue section. Together, the foil-hinge method and incubation chamber virtually eliminate the major technical pitfalls of previous emulsion-coated coverslip methods. Moreover, they facilitate combination of histochemical or immunocytochemical staining with autoradiographic localization of neurotransmitter receptors in the same tissue section. This is demonstrated by thioflavin S staining of neuritic plaques and tangles and autoradiographic localization of serotonin 1A receptors in sections of Alzheimer disease brain.
Chicken egg antibodies for immunohistochemical labeling of growth hormone and prolactin in bovine pituitary gland.
Authors: Schmidt P , Erhard M H , Schams D , Hafner A , Folger S , Lösch U
Published: April 2011

We describe the production of polyclonal chicken antibodies specific for bovine growth hormone (bGH) and prolactin (PRL). Antibodies were generated by immunization of laying hens with recombinant bGH (rbGH), pituitary derived bGH (pbGH), and ovine PRL (oPRL). After the lipoprotein fraction was removed by dextran sulfate precipitation the antibodies were isolated from the egg yolks by ammonium sulfate precipitation. Immunization with rbGH and oPRL generated large amounts of specific antibodies, as revealed by ELISA and Western blot analysis. Antibodies against pbGH showed pronounced crossreactions with oPRL. The antibodies against rbGH and oPRL were well suited for sensitive and specific labeling of the GH- and PRL-synthesizing cells in bovine pituitary glands by immunohistochemistry. In addition, a quick and sensitive procedure for demonstration of both bGH- and PRL-synthesizing cells in a single paraffin section by double immunohistochemistry is presented. The chicken anti-bGH antibodies showed excellent results in combination with rabbit anti-PRL antibodies. The main advantage of avian antibodies in double immunostaining methods is the lack of crossreactions between avian antibodies and mammalian immunoglobulins and receptors which bind to the crystalline fragment of mammalian immunoglobulins (Fc receptors).
Endogenous enzymes cause structural and chemical artifacts in methacrylate- and celloidin-embedded sections of unfixed freeze-dried tissues.
Authors: Goodwin A E , Grizzle J M
Published: April 2011

Bovine exocrine pancreas and fish (Rivulus ocellatus marmoratus) liver containing pancreatic acini were cryofixed, freeze-dried, and embedded in methacrylate or double-embedded in celloidin and paraffin. In chemically unfixed sections incubated in aqueous solutions, dissolution of zymogen granules was coincident with loss of tissue structure and antigenicity. Type II-S soybean protease inhibitor at 150 mg/liter during section flotation and in aqueous reagents used for immunohistochemistry prevented these artifacts and allowed the use of more dilute antibody solutions. Loss of glycogen from fish hepatocytes was most rapid in areas adjacent to pancreatic acini. Rapid loss of glycogen was attributed to amylase and was prevented by using poly-L-lysine instead of 3-aminopropyltriethoxysilane slide adhesive and by using alcoholic solutions during PAS staining. Inhibition of endogenous enzymes is an important consideration in the development of histological protocols with freeze-dried tissue sections.
A pre-embedding triple-label electron microscopic immunohistochemical method as applied to the study of multiple inputs to defined tegmental neurons.
Authors: Anderson K D , Karle E J , Reiner A
Published: April 2011

For many neural regions it is of interest to know the identity of the target structures of two different types of inputs to that neural region. Such studies require use of a triple-label immunohistochemical method to differentially label the class of target structure and the two types of input so that they can be visualized at the electron microscopic (EM) level. We describe here a procedure for combining three different markers (diaminobenzidine, benzidine dihydrochloride, and silver-intensified immunogold) for triple-label EM immunohistochemical pre-embedding labeling. All three markers are distinct at the LM and EM levels. An example of this approach as applied to studying striatal input to the ventral tegmental area is presented and the advantages of this approach are discussed.
A new assay for intracellular measurement of inosine monophosphate dehydrogenase activity: a guide for better selection of patients for enzyme-targeted chemotherapy.
Authors: Markovic N , Markovic O , Roberts J , Markovic S
Published: April 2011

We developed a new cytochemical assay for identification of cells containing inosine monophosphate dehydrogenase (IMPDH) and measurement of tumor cell sensitivity to the escalating dose/schedule of the IMPDH pattern-targeting drugs. The assay is based on cytochemical principles for development of DH activity markers inside morphologically classified cells, image analysis for measuring the amount of this marker, and computer assistance for data management. The assay was optimized on a human leukemia cell line (K562-NS) and reference values for enzyme activity were established. Assay specificity was determined with different substrates and enzyme inhibitors. Sensitivity depends on the measuring instrument (image analyzing system), and the precision of the biological model used for assessment of reference values was high. IMPDH-positive malignant cells were found in all specimens obtained from acute leukemia and solid tumor patients (13/13 and 29/29) and in four human tumor cell lines (K562, K562-NS, HL60, and HL60-M). Cells of the K562-NS line were exposed to tiazofurin and ribavirin in conventional assays for assessment of drug-induced acute and subacute toxicity/sensitivity. A reduction of IMPDH activity was recorded at drug concentrations below the range at which cell damage appeared.
Membrane glycoconjugates expressed on sheep airway epithelium.
Authors: Abdi K , Kobzik L , Li X , Mentzer S J
Published: April 2011

In mammalian lung, selective airway inflammatory reactions have been associated with viral infections, transplant rejection, and autoimmune diseases. Although the molecular basis for this selective reactivity is unknown, the importance of carbohydrates in immunologic processes suggests a potential role for membrane glycoconjugates in tissue-specific inflammatory reactions. In the present work we examined a panel of 39 lectins for their pattern of reactivity in the peripheral airways of the sheep lung. The size of the panel facilitated a comprehensive description of the glycoconjugate localization on the airway epithelium. Four lectins (agglutinins for Helix aspersa, Psophocarpus tetragonolobus, Trichosanthes kirilowii, and Griffonia simplifolia II) revealed selective reactivity with the small airway epithelium. On lectin Western blotting, these four lectins demonstrated a common low molecular weight banding profile that was distinct from control lectins. The histochemical staining patterns and Western blotting profiles provided evidence for the selective expression of membrane glycoconjugates in the peripheral airways of the sheep lung.
Detection of growth hormone-producing cells in human thymus by immunohistochemistry and non-radioactive in situ hybridization.
Authors: Maggiano N , Piantelli M , Ricci R , Larocca L M , Capelli A , Ranelletti F O
Published: April 2011

It is well recognized that growth hormone (GH) may act as a growth and differentiation factor for the thymus gland. Recently, it has been reported that Pit-1/GHF-1 transcription factor, which controls the expression of both GH and prolactin, is expressed in stromal (not lymphoid) cells of human thymus. Here, we demonstrated by immunohistochemistry and in situ hybridization the presence of distinct GH-producing epithelial cell subsets in human thymus. The cells positive for GH mRNA and GH-immunoreactive substance are both located in the same thymus compartments, i.e., along the thymus capsule, in the subcapsular cortex, and within the septa. Local concentration of GH higher than systemic ones, in combination with other factors, may be important in regulating the thymic microenvironment necessary for T-lymphocyte differentiation.
Transient expression of [D-Ala2] deltorphin I-like immunoreactivity in prenatal rat small intestine.
Authors: Matsui J , Fujimiya M , Matsui S , Amakata Y , Renda T , Kimura H , Maeda T
Published: April 2011

We studied the distribution of immunoreactive elements for [D-Ala2] deltorphin I (DADTI), a delta-opioid receptor ligand, in fetal and postnatal rat small intestine. DADTI-like immunoreactive cells were detected transiently on embryonic Days 20 and 21. Electron microscopic examination revealed that positive staining occurred in mucous epithelial cells, either mature goblet cells or undifferentiated cells containing only a few mucous granules. Positive immunoreaction products in mature goblet cells were confined in their apical cytoplasm to the luminal parts of mucous granule aggregates. The result suggests that a DADTI-like molecule(s) is synthesized in rat intestinal goblet cells and is secreted in a diacrine fashion into the intestinal lumen at a late fetal period. The molecule(s) thus secreted may be important for the intestine of rats just before birth, because DADTI-like immunopositive goblet cells are no longer seen at any postnatal period.
The DNA content of trophozoites and cysts of Giardia lamblia by microdensitometric quantitation of Feulgen staining and examination by laser scanning confocal microscopy.
Authors: Erlandsen S L , Rasch E M
Published: April 2011

We investigated direct measurement of the DNA content of the parasitic intestinal flagellate Giardia lamblia through quantitation by Feulgen microspectrophotometry and also by visualization of Feulgen-stained DNA chromosomes within dividing cells by laser scanning confocal microscopy. Individual trophozoites of Giardia (binucleate) contained 0.144 +/- 0.018 pg of DNA/cell or 0.072 pg DNA/nucleus. Giardia lamblia cysts (quadranucleate) contained 0.313 +/- 0.003 pg DNA or 0.078 pg DNA/nucleus. The genome size (C) value per nucleus ranged between 6.5-7.1 x 10(7) BP for trophozoites and cysts, respectively. Confocal microscopic examination of Giardia trophozoites undergoing binary fission revealed five chromosome-like bodies within each nucleus. Further information about genome size and DNA content within different Giardia species may help to clarify the pivotal role of these primitive eukaryotic cells in evolutionary development.
Detection of intestinal flora-derived bacterial antigen complexes in splenic macrophages of rats.
Authors: Kool J , De Visser H , Gerrits-Boeye M Y , Klasen I S , Melief M J , Van Helden-Meeuwsen C G , Van Lieshout L M , Ruseler-Van Embden J G , Van den Berg W B , Bahr G M
Published: April 2011

We studied the presence of bacterial antigens in rat tissues. We produced a monoclonal antibody (MAb 2E9) directed against intestinal flora-derived peptidoglycan-polysaccharide complexes from human and rat feces. With several immunological techniques, the specificity of 2E9 for this bacterial product was demonstrated. Using 2E9 in an immunohistological assay, we were able to show the presence of bacterial products in macrophages in the red pulp of spleens of conventional Lewis rats. However, we found no correlation between the development of the intestinal flora and positive spleen staining with MAb 2E9. The results were confirmed by immunohistology with a previously described MAb 2-4 directed to muramyl dipeptide. Other lymphoid organs did not stain positively with 2E9 and 2-4. Neonatal and young rats showed no staining of the spleen, but positivity could be induced by injecting peptidoglycan-polysaccharide complexes systemically. We conclude that bacterial fragments are present in splenic macrophages of conventional rats.
The Thomsen-Friedenreich antigen-related carbohydrate antigens in human gastric intestinal metaplasia and cancer.
Authors: Sotozono M A , Okada Y , Tsuji T
Published: April 2011

The Thomsen-Friedenreich antigen (T) is the core disaccharide of O-glycosylated complex carbohydrates and is presumed to be a cancer-associated carbohydrate antigen. However, we recently found that the expression of alpha-T and alpha 1-2 fucosyl alpha-T in the gastric surface epithelia was regulated allogeneically. In the present study we addressed their changes in gastric differentiation disorders. Expression of the T-related antigens was studied histochemically with monoclonal antibody MBrl and peanut agglutinin in 22 normal, 14 metaplastic, and 54 cancerous tissues. The sialylated antigens were detected after neuraminidase digestion. Gastric mucins were purified and examined to determine whether they were the carrier molecules of T. Expression of the normal antigens was decreased or not detected in about 80% of both disorders. Neosialylation of the T-related antigens was observed in the goblet cells of all metaplastic tissues. The absorptive cells did not express any T-related antigens. In gastric cancers, blocked synthesis, i.e., precursor accumulation or totally negative expression, was observed in approximately 66% and neosialylation in approximately 40%. The T-related antigens were carried by mucins. We conclude that blocked synthesis of the T-related antigens was found in a cancer-specific manner. Neosialylation was invariably associated with intestinal metaplasia and occasionally with cancer.
In situ localization and chromosomal mapping of the AG1 (Dmp1) gene.
Authors: George A , Gui J , Jenkins N A , Gilbert D J , Copeland N G , Veis A
Published: April 2011

Dentinogenesis is being used as a model for understanding the biomineralization process. The odontoblasts synthesize a structural matrix comprised of Type I collagen fibrils which define the basic architecture of the tissue. The odontoblasts also synthesize and deliver a number of dentin-specific acidic macromolecules into the extracellular compartment. These acidic macromolecules may be involved in regulating the ordered deposition of hydroxyapatite crystals within the matrix. AG1 is the first tooth-specific acidic macromolecule to have been cloned and sequenced. To identify which cells of the rat incisor pulp/odontoblast complex were responsible for synthesis of AG1, in situ hybridization was used. Digoxigenin labeled sense and anti-sense AG1 riboprobes were prepared. The AG1 mRNA was found to be expressed in the mature secretory odontoblasts. Neither pulp cells nor pre-odontoblasts showed any staining with the anti-sense probes. Chromosomal localization studies placed the AG1 gene on mouse chromosome 5q21, in tight linkage with Fgf5. AG1 has been renamed Dmp1 (dentin matrix protein 1) in accordance with present chromosomal nomenclature. Mouse 5q21 corresponds to the 4q21 locus in humans. This is the locus for the human tooth mineralization disorder dentinogenesis imperfecta Type II (DI-II). These data suggest that the Dmp1 gene is involved in mineralization and is a candidate gene for DI-II.
Localization of copper to afferent terminals in rat locus ceruleus, in contrast to mitochondrial copper in cerebellum.
Authors: Sato M , Ohtomo K , Daimon T , Sugiyama T , Iijima K
Published: April 2011

We examined copper localization in the locus ceruleus and cerebellum of rat brain by Timm's sulfide-silver staining, as modified by Danscher. Dense silver particles revealing copper localization were observed in sections of the locus ceruleus and cerebellum after pre-treatment with trichloroacetic acid. In the locus ceruleus, copper appeared to be distributed to neuropil rather than glial or neuronal cell bodies, and at the ultrastructural level copper was mainly localized on synaptic membranes of afferent terminals in contact with somatic spines or dendrites of locus ceruleus neurons, whereas copper was distributed to mitochondria in the granular layers of cerebellum and fine, sparse silver particles were observed throughout ependymal cells and epithelial cells of blood vessels. The specific localization of copper to afferent terminals in the locus ceruleus was confirmed by X-ray microanalysis, which showed a significant level of copper, but not zinc, in synaptic membranes. These results suggest a distinct role of copper depending on its regional distribution. Copper or copper protein may be involved in neurotransmission in the locus ceruleus but in mitochondrial activity in the cerebellum.
Liver regeneration: a comparison of in situ hybridization for histone mRNA with bromodeoxyuridine labeling for the detection of S-phase cells.
Authors: Alison M , Chaudry Z , Baker J , Lauder I , Pringle H
Published: April 2011

We developed an in situ hybridization technique for measurement of proliferative cell numbers through detection of histone mRNA in routinely fixed, paraffin-embedded tissue sections. Histone gene expression is coordinated with the cell cycle, and the increase in expression during S-phase permits unambiguous identification of cells undergoing DNA replication. Histone mRNAs were identified in routinely processed rat liver tissue by non-isotopic in situ hybridization with digoxigenin-labeled oligonucleotide probes. Specific hybrids were detected with alkaline phosphatase-labeled anti-digoxigenin antibody and visualized by BCIP-nitroblue tetrazolium indicator substrate. Unequivocal cytoplasmic labeling was observed in various cell types in the liver remnant during the first 72 hr after a two-thirds partial hepatectomy. The spatial and temporal patterns of histone labeling were almost identical to those obtained by staining with an antibody to bromodeoxyuridine. The identification of histone mRNA appears to be a reliable marker of the S-phase fraction, a technique with the further advantage that the tissue does not have to be first exposed to a nucleotide analogue. Hence, retrospective studies are possible. The probes can be applied to human and animal cells and tissues because the nucleotide sequences of histone genes are conserved.
Use of 1.4-nm immunogold particles for immunocytochemistry on ultra-thin cryosections.
Authors: Takizawa T , Robinson J M
Published: April 2011

We present a new application for the use of small immunogold particles (approximately 1.4-nm diameter) for ultrastructural immunocytochemistry. These small gold particles have been used on ultra-thin cryosections in conjunction with a silver enhancement procedure that does not degrade ultrastructural detail. We have used the human neutrophil as a model system, in which known protein markers of two different cytoplasmic granules were localized, in the development of this procedure. The 1.4-nm immunogold particles coupled with silver enhancement yield intense labeling for localization of lactoferrin, a marker for the specific granules, and myeloperoxidase, a marker for the azurophil granules. Double labeling in which one antigen was visualized with 1.4-nm gold and silver enhancement and a second antigen was detected with colloidal gold-IgG on the same ultra-thin cryosection was successfully achieved. We also show that 1.4-nm diameter immunogold particles penetrate into cryosectioned neutrophils to a greater extent than 5-nm or 10-nm immunogold probes. These results show that small immunogold particles, along with silver enhancement, are a useful addition to the immunolabeling methods available for use with ultra-thin cryosections.
Some ascites monoclonal antibody preparations contain contaminants that bind to selected Golgi zones or mast cells.
Authors: Spicer S S , Spivey M A , Ito M , Schulte B A
Published: April 2011

A small proportion of mouse ascites fluid induced by hybridomas producing monoclonal antibodies or myelomas secreting immunoglobulin yielded staining that was confined to the Golgi zone of certain epithelial cell types in rats and gerbils but not in mice. In addition, a commercial IgG fraction from mouse plasma similarly labeled the Golgi area, unlike IgG from mouse serum from another source. Culture supernatant from one hybridoma line contrasted with ascites fluid produced by the same hybridoma in failing to stain the Golgi region. The capacity of a fluid to react with the Golgi cisternae bore no relationship to the class of immunoglobulin secreted by the hybridoma or myeloma. Absorption of an ascites fluid with blood group A1 human erythrocytes eliminated its affinity for Golgi cisternae. Adsorption with blood group A2 or B or two type O cells used for screening for blood group antibodies had no effect on Golgi zone labeling by this ascites fluid. The positive cells included most serous secretory cells in rats, serous cells of sublingual and tracheal glands, and some endometrial and oviduct-lining cells in gerbils, and columnar lining cells of small intestine and cecum and all or part of the lining cells in some prostate lobes in both genera. Some of the tested ascites fluids stained mast cells. The agent accounting for mast cell labeling differed, however, from that reacting with Golgi cisternae in its distribution among the mouse ascites fluids examined, lack of relationship to the ABO blood group system, occurrence additionally in normal rat serum, and capacity to stain cells in mice as well as rats and gerbils.
A method for identifying peripheral connections of perivascular nerves based on sensitive acetylcholinesterase staining via perfusion.
Authors: Bleys R L , Groen G J , Matthijssen M A
Published: April 2011

To identify peripheral connections of perivascular nerves, which are usually hard to find and easily damaged, we developed a sensitive method for acetylcholinesterase (AChE) staining in rat. The procedure is based on primary staining via perfusion, which permits visualization of perivascular nerves before dissection and, after dissection, further staining via immersion for identification of peripheral connections of the earlier stained perivascular nerves. The AChE histochemistry is based on an intensification method originally described for sections. The procedure has been optimized for staining via vascular perfusion and can also be applied on whole-mount preparations via immersion. The results show intense nerve staining with minimal background. Although the procedure was initially developed for cerebrovascular nerves, the staining approach can also be applied in other regions, the main advantage being that perivascular neural structures can be identified without the damage that usually occurs during dissection.
Widespread expression of perlecan proteoglycan in basement membranes and extracellular matrices of human tissues as detected by a novel monoclonal antibody against domain III and by in situ hybridization.
Authors: Murdoch A D , Liu B , Schwarting R , Tuan R S , Iozzo R V
Published: April 2011

Perlecan, a multidomain heparan sulfate proteoglycan (PG), is an intrinsic component of basement membranes and extracellular matrices. We used a prokaryotic expression vector to generate fusion proteins encoding various domains of human perlecan protein core and these recombinant proteins were used as immunogens to produce mouse anti-human monoclonal antibodies (MAb). One MAb, designated 7B5, was characterized by Western blotting and ELISA and was shown to react specifically with the laminin-like region of perlecan (Domain III) but not with two other fusion proteins encoding Domain II or V. This perlecan epitope was detected by immunoenzymatic staining in the basement membranes of human tissues including pituitary gland, skin, breast, thymus, prostate, colon, liver, pancreas, spleen, heart, and lung. All vascular basement membranes tested contained this gene product. In addition, sinusoidal vessels of liver, spleen, lymph nodes, and pituitary gland expressed high levels of perlecan in the subendothelial region. In situ hybridization, using as probe the same human cDNA-encoding Domain III, localized perlecan mRNA to specific cell types within the tissues and demonstrated that in skin, perlecan appears to be synthesized exclusively by connective tissue cells in the dermal layer. The availability of MAb against precise regions of human perlecan will allow the investigation of this gene product in normal and diseased states.