bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunocytochemical localization of mitochondrial carbonic anhydrase in rat tissues.
Authors: Väänänen H K , Carter N D , Dodgson S J
Published: April 2011

We used a monospecific polyclonal antiserum against mitochondrial carbonic anhydrase (CA V) from rat liver to study tissue localization of this new member of the carbonic anhydrase gene family. Strong granular immunostaining reaction of CA V was observed in hepatocytes, myocardium, and in certain populations of skeletal muscle fibers. This is the first time that mitochondrial carbonic anhydrase is described in cardiac tissue of rat or any other species. Different epithelial cells revealed very heterogeneous staining reaction, suggesting that mitochondria are a heterogeneous population with respect to their CA V content. Many cells in different glandular epithelia did not show any CA V, whereas some cells, such as gastric parietal cells, were intensely stained with CA V antibodies. No systematic co-expression of CA V with CA I, CA II, or CA III was observed, although the distribution of CA V in skeletal muscle was somewhat similar to that of CA III. Connective tissue cells such as fibroblasts, chondroblasts, and osteoblasts were negative.
Simultaneous immunohistochemical detection of IUdR and BrdU infused intravenously to cancer patients.
Authors: Miller M A , Mazewski C M , Yousuf N , Sheikh Y , White L M , Yanik G A , Hyams D M , Lampkin B C , Raza A
Published: April 2011

Cell cycle kinetics of solid tumors in the past have been restricted to an in vitro labeling index (LI) measurement. Two thymidine analogues, bromodeoxyuridine (BrdU) and iododeoxyuridine (IUdR), can be used to label S-phase cells in vivo because they can be detected in situ by use of monoclonal antibodies (MAb) against BrdU (Br-3) or IUdR (3D9). Patients with a variety of solid tumors (lymphoma, brain, colon cancers) received sequential intravenous IUdR and BrdU. Tumor tissue removed at the end of infusion was embedded in plastic and treated with MAb Br-3 and 3D9 sequentially, using a modification of a previously described method. Clearly single and double labeled cells were visible, which enabled us to determine the duration of S-phase (Ts) and the total cell cycle time (Tc), in addition to the LI in these tumors. Detailed control experiments using tissue culture cell lines as well as bone marrow cells from leukemic patients are described, including the comparison of this double label technique with our previously described BrdU-tritiated thymidine technique. We conclude that the two methods are comparable and that the IUdR/BrdU method permits rapid and reliable cell cycle measurements in solid tumors.
Thrombin stimulation of synthesis and secretion of fibronectin by human A549 epithelial cells and mouse LB fibroblasts.
Authors: Kang Y H , Kedar V P , Maheshwari R K
Published: April 2011

Thrombin, a serine protease generated at wound sites, takes part in multiple biological functions, including wound healing. The present report elucidates the effect of thrombin on fibronectin (FN) synthesis and secretion in fibroblasts and epithelial cells. Subconfluent cultures of mouse LB fibroblasts and human A549 epithelial cells were exposed to various concentrations of bovine plasma thrombin at 37 degrees C for 16 hr. After exposure, cells were processed for determination of cell-associated and secreted FN by metabolic labeling, immunoprecipitation, immunofluorescence, and peroxidase immunocytochemistry. The correlation of FN production with cell growth was studied by a combined procedure of peroxidase immunocytochemistry and light microscopic autoradiography. The amounts of cell-associated and secreted FN were significantly increased with dose increments of thrombin. The increases were most evident in secreted FN. The increase of cell-associated FN was also evidenced by results from immunofluorescence and immunocytochemical studies. Ultrastructurally, the intracellular FN was localized in rough endoplasmic reticulum, Golgi complexes, and secretory granules, whereas non-released extracellular FN was localized in the plasma membrane of cell-to-cell contacts and in the extracellular fibrils. More intense cytoplasmic FN staining was observed in cells that were not labeled with [3H]-thymidine, indicating that FN production may vary with different phases of cell growth. The results imply that thrombin may play an important role in the early phases of wound healing.
Malate content of picoliter samples of Raphanus sativus cytoplasm.
Authors: Bodson M J , Outlaw W H , Silvers S H
Published: April 2011

Malate, which plays many essential roles in plant metabolism, is a potent in vitro inhibitor of the cytosolic enzyme phosphoenolpyruvate carboxylase (PEPC). Because PEPC activity leads to malate biosynthesis, malate is assumed to attenuate its own synthesis in situ. To test this hypothesis, we measured directly the malate content of picoliter samples of Raphanus root-hair cytoplasm using quantitative histochemical techniques. We also obtained an estimate for malate accumulation in these cells. These values were compared with the PEPC activity of individual root hairs (less than 2 ng). The results indicate that high cytoplasmic malate concentration does not severely inhibit PEPC in situ. We suggest that the focus for studies on the regulation of organic anion accumulation be on the interactive effects of malate and other PEPC effectors.
Extraction of extendable beaded structures and their identification as fibrillin-containing extracellular matrix microfibrils.
Authors: Keene D R , Maddox B K , Kuo H J , Sakai L Y , Glanville R W
Published: April 2011

High molecular weight aggregates were extracted from human amnion using buffers containing 6 M guanidine hydrochloride. Rotary shadowed preparations and negatively stained samples examined by electron microscopy showed that each aggregate appeared to be a string of globular structures joined by fine filaments, giving the appearance of beads on a string. The periodicity of the beads was variable. A mouse monoclonal antibody directed against a previously characterized pepsin fragment of fibrillin was used with gold-conjugated secondary antibody and immunoelectron microscopy to show that the aggregates contained fibrillin. Similar structures were found in non-denaturing homogenates of skin, tongue, ligament, ciliary zonule, cartilage, and vitreous humor. When immunogold-labeled beaded structures were prepared for electron microscopy in the same manner as tissue, the beaded structures could no longer be seen. Instead, gold-labeled microfibrils were found which appeared to be the same as the fibrillin-containing matrix microfibrils observed in connective tissues and often associated with elastin. Thus, standard TEM protocols including fixation, dehydration, and embedding alter the ultrastructural appearance of microfibrils as compared with negative stain or rotary shadowing techniques. When skin was stretched and prepared for electron microscopy while still under tension, beaded filaments were seen in the tissue sections, but were not visible in non-stretched controls. In addition, when stretched ligament was immunolabeled with antibody directed against fibrillin while still under tension, the periodicity of antibodies along the microfibrils increased compared with non-stretched controls. We propose that microfibrils contain globular structures connected by fine filaments composed at lease in part of highly ordered, periodically distributed fibrillin molecules, whose periodicity is subject to change dependent on the tensional forces applied to the tissue in which they are contained.
Histochemical reactivity of the Geodia cydonium agglutinin (GCA) in human tissues.
Authors: Vierbuchen M , Uhlenbruck G , Hanisch F G , Müller W E , Ortmann M , Fischer R
Published: April 2011

We applied a peroxidase-antiperoxidase technique to study the distribution pattern and binding characteristics of the lectin from the marine sponge Geodia cydonium (Geodia cydonium agglutinin; GCA) in various human tissues. This lectin has been shown to possess a broad reactivity, but there was a distinct distribution of binding sites within the different organs. In the histochemical system GCA displayed no blood group specificity and labeled red blood cells, the vascular endothelium, and epithelial cells showing blood group antigen expression independent of the ABH blood group status. However, inhibition of GCA reactivity by simple sugars and complex carbohydrates demonstrated tissue-specific differences of lectin binding related to the ABH blood group status of the tissue and revealed information on the structural requirements of the histological lectin binding site. Tissues that totally lacked blood group antigens or that expressed only the H-antigen disclosed a GCA reactivity which was completely inhibited by lactose. In contrast, tissues that expressed blood group A- or blood group B-antigen exhibited a lactose-resistant lectin binding which was inhibited only by water-soluble blood group substance A from peptone A and by bovine glycophorin but not by other complex carbohydrates, including human glycophorin and human asialoglycophorin. Competitive inhibition studies in situ revealed that GCA binding was not inhibited by blood group type I/II carbohydrate sequence-specific lectins or by lectins with other sugar specificities. Inhibition by lactose of GCA binding to some histological sites indicates that the binding site consists of a beta-linked galactose-containing disaccharide. However, periodate oxidation of tissue sections had no effect on lectin binding, pointing to a subterminal location of the relevant sequence. The results obtained from inhibition studies with simple saccharides and complex carbohydrates in relation to the expression of ABH blood group antigens suggest a complex lectin combining site(s) in histological specimens. The lectin may possess either one binding site with a range of affinities for different carbohydrates (besides beta-linked disaccharides the GCA binding site accommodates to carbohydrate determinants carrying the blood group A or blood group B determinant), or may possess two different binding sites. Besides an acceptor site for beta-linked disaccharides, an additional binding site may exist accommodating to extended carbohydrate sequences related to A or B blood group structures. In conclusion, GCA represents a blood group-nonspecific lectin whose binding affinities are determined by the ABH blood group status of the tissue.
A double staining technique for simultaneous demonstration of astrocytes and microglia in brain sections and astroglial cell cultures.
Authors: Castellano B , González B , Jensen M B , Pedersen E B , Finsen B R , Zimmer J
Published: April 2011

We developed a double staining technique for simultaneous demonstration of astrocytes and microglial cells in histological brain sections and cell cultures. The procedure included a histochemical stain specific for microglial cells and an immunocytochemical stain specific for astroglial cells, with postponement of the final visualization of the staining products until both reactions had been performed. First, microglial cells were specifically but invisibly labeled by histochemical reaction for nucleoside diphosphatase (NDPase). Then the astroglial cells were labeled by performing the first parts of the immunocytochemical reaction for glial fibrillary acidic protein (GFAP). Finally, in a series of intervening steps, the NDPase reaction product was visualized and stabilized by treatment with ammonium sulfide and silver nitrate, while the 1-naphthol basic dye method was used to visualize the GFAP immunoreactive product. As an end product, the NDPase-positive microglial cells were brown and the GFAP-reactive astroglial cells blue. The two types of glial cells were clearly distinguishable in vibratome sections of rat brain tissue and in primary astroglial cell cultures, and we never observed cells that stained for both NDPase and GFAP. When the GFAP antibody was replaced by the OX-42 antibody, which recognizes microglial cells and macrophages, double staining of microglial cells was observed. The staining protocol has wide applications in studies of the functional interactions between microglial and astroglial cells in the normal brain and in different pathological states with neuronal or axonal degeneration, just as it can be used for experimental studies in cell cultures.
Immunolocalization of collagen types I and III, tenascin, and fibronectin in intramembranous bone.
Authors: Carter D H , Sloan P , Aaron J E
Published: April 2011

Structural components of the organic bone matrix were located by immunohistochemical techniques in fresh-frozen sections of normal and dysplastic bone. Fine and coarse birefringent fibers were identified as separate and distinctive features in the extracellular matrix by antibodies raised against human collagen Type III. The glycoprotein tenascin was located on a proportion of the fibers in a characteristic beaded pattern, which was absent in dysplastic bone. The fibers originated in the periosteum or in the fibrous stroma of the marrow cavity and were oriented with regard to both the spatial and the lamellar organization of the bone. The disposition and composition of the fibers suggests that they form a preliminary framework on which intramembranous bone modeling proceeds, and that the specific location of tenascin on the fibers in normal developing membrane bone may be important in determining the alignment of the bone tissue. Epitopes recognized by the collagen Type I and fibronectin antibodies were demonstrated throughout the mineralized matrix, but their incorporation into the collagen "Type III" fibers was evident only outside the mineralized matrix.
Simultaneous detection of highly phosphorylated proteins and viral major DNA binding protein distribution in nuclei of adenovirus type 5-infected HeLa cells.
Authors: Puvion-Dutilleul F
Published: April 2011

Highly phosphorylated proteins in situ in sections of Lowicryl-embedded cells are preferentially stained by bismuth, provided that the reactivity of the amino groups is blocked by glutaraldehyde fixation. This study showed that bismuth staining can be preceded by indirect immunocytochemistry using gold particles as markers. As a result, both immunostained and bismuth-stained proteins can be detected concomitantly on the same section. This was also carried out on sections of formaldehyde-fixed cells which were immunolabeled, then post-fixed with glutaraldehyde, and finally exposed to bismuth stain. These procedures were applied to sections of adenovirus Type 5-infected HeLa cells. Bismuth ions and viral anti-72 KD antibody bound concomitantly to intranuclear virus-induced single-stranded DNA (ssDNA) accumulation sites, structures in which viral replicative activity is intermittent, and also to the fibrillogranular peripheral replicative zones which surround the ssDNA accumulation sites and in which replication of viral genomes is continuous. The delicate fibrillar network enclosed within virus-induced compact rings of unknown function is slightly bismuth stained and binds few antibodies to viral 72 KD protein. Three intranuclear structures were stained exclusively with bismuth: the fibrillar component of the nucleolus, which is involved in ribosome formation; the interchromatin granules; and the virus-induced "fibrillar spots" of unknown significance. Thus, not all highly phosphorylated proteins in adenovirus-infected cells are viral 72 KD protein. In glutaraldehyde-fixed Miller spreads of nucleic acid molecules from adenovirus-infected cells, bismuth deposits occurred over unique thick filaments, the only portion of the viral deoxyribonucleoprotein molecules shown to be associated with viral 72 KD protein. In vitro studies revealed that the latter protein, known to be multiply phosphorylated, concomitantly binds anti-72 KD antibody and bismuth ions. These data have broadened the scope of the use of bismuth staining. Taken together, they indicate that in adenovirus infection highly phosphorylated proteins accumulate over intranuclear structures related to both replication of viral genomes and alteration of ribosomal metabolism.
Immobilization induces carbonic anhydrase III in type II fibers of rat skeletal muscle.
Authors: Laurila A L , Jeffery S , Savolainen J , Takala T E , Carter N D , Väänänen H K
Published: April 2011

The amount and fiber distribution of carbonic anhydrase III (CA III), a major soluble protein in Type I muscle fibers, were studied during cast immobilization of rat hindlimb with the ankle in plantar or dorsiflexion. The concentration of CA III increased two- (p less than 0.05) and three- (p less than 0.01) fold in the shortened and lengthened tibialis anterior muscle during a 3-weeks immobilization period, respectively. After 6 weeks of immobilization the increase was even greater (p less than 0.001). Concomitantly, the number of CA III positive fibers in the lengthened muscle increased so that almost all fibers were positive. In the soleus muscle no significant change in the CA III concentration was seen. On the basis of actomyosin ATPase staining, the transition of Type IIb fibers towards Type IIa occurred in the tibialis anterior muscle, whereas in the soleus muscle a transformation of Type I fibers towards Type IIa fibers occurred. Therefore, the increase in the muscle CA III concentration seems to be associated with a cell transformation of the muscle towards a more oxidative type.
Immunofluorescence analysis in flow cytometry: better selection of antibody-labeled cells after fluorescence overcompensation in the red channel.
Authors: Alberti S , Bucci C , Fornaro M , Robotti A , Stella M
Published: April 2011

Selection of cells labeled with fluorescein isothiocyanate-conjugated (FITC) antibodies can be difficult if large autofluorescent cells are used and if the cells bind only a few molecules of antibody. We have developed a simple flow cytometric procedure that allows better selection of stained cells. When an argon ion laser emitting at 488 nm is used, the green fluorescence detected is the sum of cell autofluorescence and of the signal generated by the FITC antibody. Thus, when we subtract green signal from the red by fluorescence compensation, the signal of stained cells is on average reduced more than for the unstained counterpart. In this scenario, positive selection of cells with low red signal allows more efficient selection of stained cells. We tested the overcompensation procedure on mixtures of cells unstained and stained with a relevant FITC antibody. Cell mixtures were analyzed using normal vs increased levels of compensation in the red channel. Increased levels of compensation resulted in easier gating and higher recovery of stained cells. The efficiency of the overcompensation procedure was particularly high when using red filters with low cutoff (i.e., 560 or 570 nm), possibly because of the significant emission of fluorescein in the red channel, which caused separation between stained and unstained cells also in the red dimension. This method is useful for sorting cells expressing low levels of surface markers and facilitates selection of rare cells transfected with surface antigen genes. This technique is compatible with the use of propidium iodide for live/dead cell discrimination and with the subtraction of the cellular background of autofluorescence.
Histochemical differences in expression of X-linked glucose-6-phosphate dehydrogenase between ectoderm- and endoderm-derived embryonic and extra-embryonic tissues.
Authors: Sobis H , Verstuyf A , Vandeputte M
Published: April 2011

We examined the activity of X-linked glucose-6-phosphate dehydrogenase (G6PD) in concepti of the enzyme-deficient mutant and wild-type C3H mice. By using different crosses between the G6PD-deficient homozygous, heterozygous, or wild-type females and hemizygous or wild-type males, we confirmed the inactivation of one of the two X chromosomes in female concepti by a histochemical method. With this technique, a dual (G6PD + or -) cell population could be observed in the tissue sections. We demonstrate that the paternal X chromosome is inactivated in the endoderm of parietal and visceral yolk sac and in the trophoblast, whereas in the embryo and in the yolk sac mesoderm this inactivation is random. Our results confirm biochemical observations showing that only the maternal X chromosome is expressed in all derivatives of trophectoderm and primitive endoderm, whereas derivatives of the primitive ectoderm show random X chromosome expression.
Ultrastructural localization of GP2 in acinar cells of pancreas: presence of GP2 in endocytic and exocytic compartments.
Authors: Beaudoin A R , St-Jean P , Grondin G
Published: April 2011

GP2 is a glycoprotein found in pancreatic acinar cells. Its subcellular distribution suggests that it may be involved both in exocytosis and endocytosis. Immunocytochemical studies have demonstrated GP2 to be present on the membrane and in the matrix of zymogen granules, on Golgi saccules, on the apical and basolateral surfaces of the plasma membrane, and in the lumina of acini. In addition, this protein was observed in small vacuoles and tubular structures previously identified as "basal lysosomes," "snake-like tubules," and in lysosomes. Because the latter group of structures are involved in endocytosis, it is possible that GP2 may be involved in this phenomenon. GP2 was readily detectable in pancreatic juice and was totally sedimentable by ultracentrifugation, as assessed by Western blot analysis. Induced lysis of isolated zymogen granules also caused release of GP2 in a sedimentable form which, by electron microscopy, appeared as a fibrillar structure. Immunocytochemical localization of amylase was studied in parallel with GP2 and was found in the secretory product to be associated with thread-like structures, presumably the pancreatic thread protein. The physiological significance of these observations is discussed.
Immunohistochemical study on arachidonate 5-lipoxygenase in porcine leukocytes and other tissues.
Authors: Komatsu N , Natsui K , Ueda N , Watanabe K , Yamamoto S
Published: April 2011

Arachidonate 5-lipoxygenase is an enzyme that catalyzes the oxygenation of arachidonic acid, producing 5-hydroperoxy acid. This enzymatic reaction initiates the biosynthesis of various bioactive leukotrienes. An antiserum was raised in a rabbit against the purified 5-lipoxygenase of porcine leukocytes, and various types of porcine leukocytes were immunostained by use of the antibody. As examined by light and electron microscopy, neutrophils and eosinophils were positively stained. The 5-lipoxygenase was localized in the cytoplasm but not in the plasma membrane and subcellular organelles of the positively stained cells. In contrast, lymphocytes were unstained. In porcine ileum, the majority of 5-lipoxygenase-positive cells were eosinophils and mast cells resident in the lamina propria mucosae, whereas parenchymal cells were not stained. In porcine lung, certain bronchiolar or bronchial epithelial cells were clearly immunostained, in addition to eosinophils and mast cells found in the interstitium.
Antibodies against neuroactive amino acids and neuropeptides. II. Simultaneous immunoenzymatic double staining with labeled primary antibodies of the same species and a combination of the ABC method and the hapten-anti-hapten bridge (HAB) technique.
Authors: Behringer D M , Meyer K H , Veh R W
Published: April 2011

In the present study we developed an immunoenzymatic double staining technique allowing the simultaneous detection of two neuroactive substances with primary antibodies of the same species and their simultaneous visualization in semithin sections of epoxy-embedded material. For this purpose, primary antibodies against glutamate, GABA, and serotonin were either biotinylated or labeled with the trinitrophenyl (TNP) group. The latter was visualized by a detection system here referred to as the hapten-anti-hapten bridge (HAB) technique. The HAB technique consists of anti-TNP antibodies, serving as bridges between the TNP-ylated primary antibody, and a TNP-ylated marker enzyme, such as alkaline phosphatase. The single components of the HAB technique were optimized by use of a dot-blot assay and an "artificial tissue" system. The optimal staining sequence consisted of TNP-ylated primary antibody with a molar TNP:antibody ratio of 12:1, followed by anti-TNP antibody and TNP-ylated alkaline phosphatase (molar TNP:enzyme ratio of 20:1). No further improvement of detection sensitivity could be obtained when soluble immunocomplexes between anti-TNP antibody and TNP-ylated alkaline phosphatase on the side of phosphatase excess were prepared and used instead of simple TNP-ylated alkaline phosphatase. When compared with other established procedures, such as avidin-conjugated alkaline phosphatase or the ABC method, the HAB technique revealed a similar detection sensitivity. The TNP-ylated primary antibody, however, had to be used at higher concentration than the corresponding unlabeled primary antibody. The suitability of the HAB technique in combination with a modified three-step ABC technique for the simultaneous demonstration of glutamate-like and GABA-like immunoreactivity in the rat brain was demonstrated. The advantages of the new technique in comparison with existing double staining methods are discussed.
Evaluation of human monoclonal antibody (2-139-1) in cutaneous melanocytic neoplasms in fixed tissue sections.
Authors: White W L , Tam S T , Mitchell M S , Yu M C , Kan-Mitchell J
Published: April 2011

Despite the growing list of xenogeneic monoclonal antibodies (MAb) that recognize malignant melanoma-associated antigens (MAA) in formalin-fixed, paraffin-embedded tissue, none has been able to detect epitopes found in malignant melanomas and not in melanocytic nevi. A human MAb, 2-139-1, that showed promise in this regard was evaluated against 85 melanocytic neoplasms, including malignant melanoma and histological simulators, particularly Spitz's nevus. MAb 2-139-1 stained 18 (53%) of 34 melanomas, eight (57%) of 14 dysplastic nevi, six (38%) of 16 Spitz's nevi, and three (14%) of 21 banal nevi, which included three small congenital nevi. We observed a significant increasing trend in reactivity (% positive cells x intensity) associated with the potential for malignancy (p for linear trend = 0.002). We conclude that human MAb 2-139-1 is applicable to the study of melanocytic neoplasms in routinely processed tissue. Although the ability of this MAb to separate benign from malignant cells is not absolute, our results suggest that the expression of the 2-139-1 epitope may be an early event in melanocytic tumor progression.
Use of anti-horseradish peroxidase antibody-gold complex in the ABC technique.
Authors: Gee B , Warhol M J , Roth J
Published: April 2011

We report a modification of the avidin-biotin-peroxidase complex (ABC) technique for the light and electron microscopic detection of antigens in tissue sections. An immunological approach was used instead of the DAB reaction to reveal ABC bound to antigen-antibody complexes. Affinity-purified polyclonal antibodies against horseradish peroxidase were complexed to particles of colloidal gold and applied for reaction with the horseradish peroxidase molecules of the ABC. For light microscopic immunolabeling, the signal produced by the anti-horseradish peroxidase antibody-gold complex required silver intensification. The ABC immunogold reaction as compared with the standard ABC technique, in particular with silver intensification of the DAB reaction product, provided superior resolution in paraffin sections. Furthermore, section pre-treatment to block endogenous peroxidase activity could be omitted and no potentially hazardous substrate was used. The ABC immunogold reaction was successfully applied for electron microscopic immunolabeling on Lowicryl K4M thin sections. We propose that the ABC immunogold reaction is a useful alternative to the standard ABC technique and can be equally well applied to light and electron microscopy.
Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in rat pituitary.
Authors: Tanaka S , Nomizu M , Kurosumi K
Published: April 2011

A synthetic peptide (ST-1) corresponding to the cleavage site between ACTH and beta-lipotropic hormone moieties of murine pro-opiomelanocortin (POMC) was constructed and its polyclonal antibody was generated. This antiserum immunoprecipitated only POMC from extracts of AtT-20 cells. Moreover, an antiserum raised against porcine ACTH immunoprecipitated both ACTH[1-39] and POMC. When ultra-thin frozen sections of melanotrophs in rat pars intermedia were immunolabeled with anti-ST-1 followed by protein A-gold, gold particles indicating the presence of POMC were selectively found in the electron-dense secretory granules in the Golgi area. In addition, the immunolabeling was also observed in the cisternae of the Golgi apparatus and rough endoplasmic reticulum. In contrast, with a polyclonal antibody specific for alpha-melanocyte-stimulating hormone the gold particles were found exclusively in the electron-lucent secretory granules, with none seen in the electron-dense secretory granules. With anti-ACTH serum, gold particles were observed in the electron-dense and -lucent secretory granules. In corticotrophs in the pars distalis, many gold particles indicating the presence of POMC were observed in the Golgi and peripheral secretory granules, but the percentage of immunolabeling in the peripheral secretory granules varied from cell to cell. On the other hand, ACTH immunolabeling was found in almost all the secretory granules. This finding suggests that the processing of POMC in corticotrophs might occur in the relatively peripheral granules. These results suggest that the intracellular sites of POMC processing are somewhat different between melanotrophs and corticotrophs in the pituitary.
Changes in rat pituitary POMC mRNA after exposure to cold or a novel environment, detected by in situ hybridization.
Authors: Wu P , Childs G V
Published: April 2011

Plasma adrenocorticotropin (ACTH) levels increase after acute cold exposure. The purpose of this study was to determine if there were parallel changes in pituitary proopiomelanocortin (POMC) mRNA. Male rats were exposed to cold (3-5 degrees C) or a novel environment for 15 or 30 min. Others were unstressed. POMC mRNAs in frozen sections or dissociated cells were hybridized with a photobiotinylated oligonucleotide probe which was detected in situ by streptavidin-alkaline phosphatase. The percentage of area labeled for POMC mRNA was quantified by the Cue-3 color image analysis system. In frozen sections, 24-fold increases in the percentage of area labeled for POMC mRNA were evident in intermediate lobes (IL) 30 min after stress. No change was seen in anterior lobes (AL). If the ALs were dissociated, a 66-99% increase in percentage of labeled cells was detected 2-3 hr after the cold exposure. Fifteen min of cold stress (CS) also caused a 117% increase in the area of label for POMC mRNA per corticotrope. No change was seen after 30 min. Exposure to a novel environment caused a 73% increase in the percentage of area labeled for POMC mRNA per AL corticotrope and an 11-fold increase in the IL. These results indicate that both AL corticotropes and IL melanotropes are stimulated by acute exposure to cold and novel environments.
Nuclear and cytoplasmic lectin binding sites in promastigotes of Leishmania.
Authors: Vannier-Santos M A , Saraiva E M , de Souza W
Published: April 2011

We demonstrated the presence of intracellular lectin binding sites in promastigotes of Leishmania mexicana amazonensis. Direct and indirect lectin-gold techniques were used on Lowicryl K4M-embedded cells. The nuclear compartment was labeled by most lectins. Nucleoli were mainly labeled by WFH (Wistaria floribunda hemagglutinin) and LFA (Limax flavus agglutinin) specific for D-galactose/N-acetyl-D-galactosamine (D-Gal/D-GalNAc) and sialic acid, respectively. Sections treated with the fetuin-gold complex without previous lectin incubation also exhibited labeled nucleoli, although less intensely, suggesting the presence not only of sialic acid but also of a sialic acid-specific endogenous carbohydrate binding molecule in Leishmania nuclei. Surprisingly, the Golgi complex was never labeled, whereas the endoplasmic reticulum was frequently labeled, especially by RCA (Ricinus communis agglutinin; D-GalNAc/D-Gal) and WGA (wheat germ agglutinin; D-GlcNAc). The kinetoplast, a DNA-containing structure located within the mitochondrion, was generally labeled towards its extremities, where previous studies have shown the presence of ribonucleoproteins. Some possible biological roles for these intracellular glycoconjugates are discussed.
In situ nick translation at the electron microscopic level: a tool for studying the location of DNAse I-sensitive regions within the cell.
Authors: Thiry M
Published: April 2011

The in situ nick translation method was adapted to the ultrastructural level, to study the location of DNAse I-sensitive sequences within the cell. Ultra-thin sections of Lowicryl-embedded cells were incubated in a medium containing DNAse I, DNA polymerase I, and all four deoxyribonucleotides, some being biotinylated. The nick-translated sites were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern is closely dependent on the DNAse I concentration in the nick-translation medium. The method reveals with great precision the specific DNAse I-sensitive regions within the nucleus. This technique can be used to discriminate between active and inactive regions of interphase chromatin.
Differential localization of annexins in ram germ cells: a biochemical and immunocytochemical study.
Authors: Feinberg J M , Rainteau D P , Kaetzel M A , Dacheux J L , Dedman J R , Weinman S J
Published: April 2011

We used antibodies that specifically bind annexins on Western blots to determine the distribution and abundance of these proteins in ram spermatids and sperm by immunogold electron microscopy. Annexins I and II were found essentially within the entire acrosome of spermatids. During epididymal maturation, they concentrated in the postacrosomal region or the acrosomal equatorial segment, respectively. They were also present in sperm flagellum, on the surface of the coarse fibers and fibrous sheath. These findings show that during ram germ cell maturation, annexins I and II are exported from the spermatid acrosome towards structurally and functionally defined parts of the sperm. Annexins III, IV, and V were not found in ram germ cells. Annexin VI was isolated from testis and sperm. In spermatids, it was found to be associated with endoplasmic reticulum and the mitochondria but was absent from the acrosome. In sperm, it was confined to the flagellum, the mitochondria, and on the coarse fibers and fibrous sheath. The presence of three annexins, in addition to calmodulin, in functional areas may indicate differential ways for sperm to control and regulate events that are known to be calcium dependent, such as flagellar motility, acrosome reaction, and fertilization.
Production and characterization of osteoclast-selective monoclonal antibodies that distinguish between multinucleated cells derived from different human tissues.
Authors: James I E , Walsh S , Dodds R A , Gowen M
Published: April 2011

Osteoclastoma-derived giant cells were used to produce 11 mouse monoclonal antibodies (MAb) reactive against human osteoclasts on undecalcified sections of adult human bone. All exhibited unique reactivities across a wide range of human tissues. Three in particular demonstrated distinctive reactivities; C35 was highly selective for bone osteoclasts, C27 showed selective reactivity for osteoclasts, tissue macrophages and blood-borne monocytes, and C22 showed selective membrane staining of osteoclasts. Consequently, C22 was used to coat Dynabeads to affinity-purify viable human osteoclasts from osteoclastoma-derived cell suspensions. Immunocytochemical staining of inflammatory osteoarthritic synovium/granulation tissue demonstrated positivity in the majority of giant cells with MAb C22 and C27. In contrast, C35 reacted with only very occasional giant cells. Furthermore, multinucleated cells formed in long-term human bone marrow cultures demonstrated similar selective staining. C27 stained all giant cells and the majority of mononuclear cells. C22 detected only a small proportion of giant cells. In contrast to its staining on bone osteoclasts, C22 demonstrated granular cytoplasmic staining in cultured giant cells. C35 stained no cells at all in these cultures. These MAb can therefore distinguish between giant cells of various origins and authentic mature osteoclasts. Alternatively, they can recognize antigens expressed at different stages of osteoclast differentiation and therefore provide an excellent tool for the study of the human osteoclast lineage.
R-phycoerythrin as a fluorescent label for immunolocalization of bound atrazine residues.
Authors: Sohn G , Sautter C
Published: April 2011

We established a highly sensitive immunofluorescence procedure for localizing bound atrazine in the aquatic macrophytes Elodea canadensis and E. densa. The technique included biotin-labeled anti-rabbit IgG as a first enhancement step and R-phycoerythrin (R-PE) coupled to streptavidin for fluorescent labeling as a second improvement on the procedure. A comparison with the conventional indirect immunofluorescence method confirmed the superior results of the R-PE approach. The use of atrazine-free plants (grown in charcoal-filtered water) and a variety of other controls excluded both contaminating atrazine and nonspecific incubation constituents as sources of tissue staining. Pre-incubations to block nonspecific binding sites proved to be unnecessary in this system. The highly sensitive procedure described here might be a useful tool for the localization of tissue-bound pesticides in general and possibly of other haptens as well.
Quantitative immunocytochemistry of rat submandibular secretory proteins during chronic isoproterenol administration and recovery.
Authors: Matsuura S , Hand A R
Published: April 2011

We utilized quantitative electron microscopic immunogold labeling procedures to follow changes in the intragranular content of five secretory proteins of the rat submandibular gland (SMG) during and after chronic treatment with the beta-adrenergic agonist isoproterenol (IPR). Labeling intensities (gold particles/microns2) of acinar cell secretory granules for mucin and glutamine/glutamic acid-rich proteins, major secretory proteins of the normal SMG, showed opposite responses to IPR. Labeling intensities increased for mucin and decreased for glutamine/glutamic acid-rich proteins immediately after IPR injections began, then rapidly returned to control levels after cessation of IPR treatment. SMG Protein C immunoreactivity, found in both acinar and intercalated duct granules, was less affected by IPR. However, opposite changes in labeling intensity were observed between acinar and intercalated duct granules. Labeling intensities for proline-rich proteins, IPR-inducible secretory proteins, increased only after 10 days of stimulation and maintained a high level even after cessation of drug treatment. Type 2 cystatin, another IPR-inducible protein, increased gradually with chronic IPR treatment and decreased slowly during the recovery phase. These results suggest that chronic beta-adrenergic stimulation affects the expression of genes for several rat SMG secretory proteins in a different manner.
Conjugate formation in urea: coupling of insoluble peptides to alkaline phosphatase for ELISA and in situ detection of antibody-forming cells.
Authors: Gerritse K , Fasbender M , Boersma W , Claassen E
Published: April 2011

We report here a new method to produce synthetic peptide/alkaline phosphatase (AP) conjugates in the presence of urea. The method allows the use of peptides that are not soluble to a sufficient degree in aqueous buffers. The presence of 8 M urea during the construction of the synthetic peptide/AP conjugates does not influence enzyme activity nor the affinity of the anti-peptide antibodies for the conjugated peptide. We demonstrate that these synthetic peptide/AP conjugates can be used for detection of specific antipeptide antibody-forming cells (AFC) in vivo. This method for constructing enzyme conjugates with insoluble proteins or peptides suggest not only new possibilities for detection of specific AFC in vivo but also for applications in receptor-ligand studies, ELISA (enzyme-linked immunosorbent assay), and spot ELISA for detection of antibody-secreting cells in vitro.
Detection of glomerular anionic sites in post-embedded ultra-thin sections using cationic colloidal gold.
Authors: Goode N P , Shires M , Crellin D M , Davison A M
Published: April 2011

We detected glomerular anionic sites in fixed, LR Gold-embedded ultra-thin tissue sections using cationic colloidal gold. Manual and computer-assisted quantitation were compared, and the influence of pH and glycosaminoglycan-degrading enzymes on site expression was examined. Both quantitation methods produced similar results. Alteration of pH within a narrow range (pH 2.5-3.0) markedly affected the staining pattern. At pH 2.5, epithelial and endothelial glycocalyx and regular sites restricted to the lamina rara externa were stained. At pH 3.0 and above, glycocalyx was unstained but intracellular and nuclear staining was present; glomerular basement membrane (GBM) and mesangial matrix sites were abundant. After chondroitinase ABC or hyaluronidase digestion, GBM staining was eliminated at pH 2.0 and reduced at pH 7.0 (p less than 0.001), suggesting that degraded sites are associated with chondroitin sulfate or hyaluronic acid. By contrast, prolonged heparitinase I digestion was ineffective at either pH. Digestion of purified substrates revealed crossreactivity of heparitinase towards chondroitin sulfate and of chondroitinase towards hyaluronic acid. Since tissue sites were reduced by chondroitinase but not heparitinase, we suggest that degradation is due to hyaluronidase activity of chondroitinase and the anionic sites are associated with hyaluronic acid. However, the influence of pH indicates that lamina rara externa sites are structurally distinct from other GBM anionic sites.
High glucose-6-phosphatase activity in non-pigmented epithelial cells of rabbit ciliary body.
Authors: Asaka Y , Watanabe J , Kanai K , Kanamura S
Published: April 2011

For study of the origin of glucose in the aqueous humor, glucose-6-phosphatase (G6Pase) and hexokinase activities, and glycogen, were cytochemically examined in the ciliary body (CB) of rabbit. G6Pase activity was also assayed biochemically. The staining reaction for G6Pase activity was strong in the non-pigmented epithelium (NPE) in the pars plana and tips of ciliary processes in the region containing large ciliary pockets within the pars plicata. NPE cells contained abundant reaction product for G6Pase activity in the endoplasmic reticulum (ER) and nuclear envelope. However, NPE in other regions of the CB and pigmented epithelium (PE) of CB, and other areas surrounding the anterior and (PE) of CB, and other areas surrounding the anterior and posterior chambers, showed weak or no G6Pase staining reaction. Biochemical G6Pase activity in the whole ciliary body was relatively high. Both NPE and PE in the pars plana and the tips showed strong staining reaction for hexokinase activity but no staining for glycogen. Furthermore, NPE cells in the tips bore large aggregates of smooth ER and many Golgi apparati. These suggest that the high G6Pase activity in NPE cells in the pars plana and the tips is related to glucose release into the aqueous humor.
Ultrastructural distribution of myosin heavy chain mRNA in cardiac tissue: a comparison of frozen and LR White embedment.
Authors: Wenderoth M P , Eisenberg B R
Published: April 2011

Electron microscopy (EM) in situ hybridization provides the higher resolution necessary to determine the spatial relationship between a specific mRNA and the organelle containing the protein encoded by that message. EM in situ hybridization was used to determine the subcellular myosin heavy chain (MHC) mRNA distribution with respect to the myofibril in normal cardiac tissue. Sections of frozen or acrylic-embedded tissue were compared for ultrastructural integrity and content of endogenous mRNA. Papillary muscles dissected from hearts of normal rabbits were aldehyde-fixed and either frozen or embedded in LR White. EM in situ hybridization with no riboprobe, vector sequence, same-sense, and anti-sense biotinylated riboprobes was detected by indirect immunocytochemistry. Labeling density using an antisense probe was highest over the intermyofibrillar space, with an average signal five times that of background. Background labeling by nonspecific sense probe was consistently low but not random, also having the highest density of gold clusters over the intermyofibrillar space. Ultracryomicrotomy yielded a higher absolute number of gold clusters, but sections were fragmented and disrupted striated muscle morphology. LR White embedment maintained ultrastructural integrity but gave a lower absolute signal. Fortunately, MHC mRNA is an abundant message and can tolerate the decreased sensitivity of LR White.
Differentiation-dependent expression of lectin binding sites on normal and neoplastic keratinocytes in vivo and in vitro.
Authors: Suter M M , Augustin-Voss H G , Pantano D M , Flanders J A , Varvayanis M
Published: April 2011

We used lectins as probes to demonstrate the composition of membrane carbohydrates of canine keratinocytes in various functional stages and various degrees of differentiation. Keratinocytes during normal epidermal turnover were compared by lectin immunohistochemistry to cells of hyperplastic epidermis and neoplastic keratinocytes. Three types of epidermal tumors and oral squamous cell carcinomas were examined. In addition, two in vitro tissue culture systems for keratinocytes were studied and compared with in vivo epithelium. In normal skin, PNA reacted only weakly with basal cells, whereas in hyperplastic skin basal cells bound this lectin strongly, demonstrating increasing expression of PNA binding sites with increasing thickness of the stratified squamous epithelium. ConA bound to basal cell tumors only. In oral squamous cell carcinomas, the expression of distinct lectin binding sites correlated with certain histological growth patterns, e.g., UEA-I reacted with highly invasive tumors but not with tumors showing a solid growth pattern. Using cell surface iodination and polyacrylamide gel electrophoresis, distinct differences in cell membrane protein expression were demonstrated between normal and neoplastic keratinocytes. SDS-polyacrylamide gel electrophoresis of cultured normal and neoplastic keratinocytes revealed several cell surface proteins that are specific for either cell type. Neoplastic cells specifically express a 140 KD lectin binding cell surface glycoprotein. The results of this study show that lectin binding patterns of keratinocytes are dependent on the functional state and the degree of differentiation of the cells and demonstrate correlation of some histological growth patterns with distinct lectin binding phenotypes, suggesting association of expression of cell membrane carbohydrate moieties with growth patterns. In addition, close similarities between "lifted cultures" grown at the air-liquid interface and native tissue demonstrate the value of this culture system as a model for differentiated stratified squamous epithelium.