Double-labeling Procedure Research Articles

Subcellular localization suggests novel functions for prolyl endopeptidase in protein secretion

For a long time, prolyl endopeptidase (PEP) was believed to inactivate neuropeptides in the extracellular space. However, reports on the intracellular activity of PEP suggest additional, as yet unidentified, physiological functions for this enzyme. Here, we demonstrate using biochemical methods of subcellular fractionation, immunocytochemical double-labelling procedures and localization of PEP-enhanced green fluorescent protein fusion proteins that PEP is mainly localized to the perinuclear space, and is associated with the microtubulin cytoskeleton in human neuroblastoma and glioma cell lines. Disassembly of the microtubules by nocodazole treatment disrupts both the fibrillar tubulin and PEP labelling. Furthermore, in a two-hybrid screen, PEP was identified as binding partner of tubulin. These findings indicate novel functions for PEP in axonal transport and/or protein secretion. Indeed, a metabolic labelling approach revealed that both PEP inhibition and PEP antisense mRNA expression result in enhanced peptide/protein secretion from human U-343 glioma cells. Because disturbances in intracellular transport and protein secretion mechanisms are associated with a number of ageing-associated neurodegenerative diseases, cell-permeable PEP inhibitors may be useful for the application in a variety of related clinical conditions.

HER2 expression in disseminated tumor cells (DTC) and corresponding primary tumors of breast cancer patients

9580 Background: The presence of disseminated tumor cells (DTC) in bone marrow (BM) of breast cancer patients is associated with poor prognosis. Several studies have indicated that these patients may benefit from secondary adjuvant immunotherapy. HER2 protein is suggested as one of the promising targets. The phenotype of the primary tumor is the current gold standard to make antibody based treatment decisions. However, the antigenic profile of primary tumors and DTC may be different. The aims of this study were (1) to determine the HER2 status of DTC in BM of breast cancer patients (2) and to compare the HER2 status of DTC and corresponding primary tumors. Method: Cytospins from bone marrow aspirates of 124 primary breast cancer patients (stage I-III) were analyzed for this study. A double labeling procedure was used for the identification of cytokeratin-positive (CK+)/HER2 positive cells. Slides were incubated with the pan-anti-cytokeratin antibody C11 conjugated to FITC and the polyclonal rabbit antibody CB11 against the HER2 protein. CB11 was detected by a secondary antibody labeled with Texas Red. HER2 status of the primary tumor was immunohistochemically assessed by the HERCEP-test (Dako Co., CA). Results: Disseminated tumor cells (DTC) were detected in the bone marrow of 47 patients. The number of DTC ranged from 1 to 70 per slide. 24 of these patients had HER2 positive DTC. HER2 positivity (score: 2+/3+) could be demonstrated in 12 of 46 primary tumors. Concordance rate between primary tumor and DTC was 63%. In three patients with HER2 positive tumors HER2 negative DTC were detected. In contrast, 14 patients with HER2 negative tumors (score: 0/1+) had HER2 positive DTC. The HER2 expression in DTC was heterogeneous in 8 of 20 patients with more than one CK-positive cell. Conclusions: (1) HER2 positive DTC can be detected in patients with HER2 negative primary tumors. Therefore, the antigenic profile of DTC should be included in the treatment decision for antibody based strategies. (2) The DTC are heterogeneous with regard to HER2 positivity. The effect of heterogeneity on the patient’s response is yet to be determined. No significant financial relationships to disclose.

A comparison of the proteins of the amoebal and plasmodial phases of the slime mould, Physarum polycephalum.

1. The proteins of amoebae and plasmodia of strain CL of Physarum polycephalum have been compared by two-dimensional gel electrophoresis. Both forms of the organism were labelled by growth on formalin-killed bacteria labelled with [35S]sulphate, [3H]lysine or [14C]lysine. Plasmodia were also labelled from radioactive lysine in the medium. 2. Of 306 relatively abundant proteins examined, 26% were phase-specific, that is they were found only in amoebae or in plasmodia. About a quarter of these apparent differences in gene expression may be due to minor changes in charge and/or size. 3. Amongst the 74% of the proteins present in both amoebae and plasmodia, there are substantial differences in differential rates of synthesis and these have been measured for a representative set of proteins by a double-label procedure.

Fluorescence in situ hybridization analysis of human oocytes: Advantages of a double-labeling procedure

To improve the fluorescence in situ hybridization (FISH) chromosomal analysis of human oocytes and first polar bodies. In situ chromosomal identification on isolated cells, with combinations of centromeric (or locus-specific) probes and whole-chromosome painting probes for chromosomes 9, 13, 16, 18, 21, and X. Montpellier University Hospital. Women participating in an IVF program. Fifty-four in vitro unfertilized oocytes were fixed on slides, and simple or double FISH labeling procedures were performed on preparations. Simultaneous in situ visualization of specific domains and chromosome arms of each targeted chromosome. Eight chromosomal abnormalities were identified, including two hyperhaploidies, three cases of extra single chromatid, and three cases of balanced separation of sister chromatids. Also, the double-labeling procedure allowed the avoidance of five interpretation errors, owing to additional artefactual signals. By ensuring precise identification of both chromosomes and single chromatids, the FISH double-labeling procedure limits the risk of erroneous interpretation and allows a more accurate cytogenetic analysis of human oocytes.

Sequential multiple probe fluorescence in situ hybridization (FISH) analysis of human oocytes and polar bodies

Sequential multiple probe fluorescence in situ hybridization (FISH) analysis of human oocytes and polar bodies

Immunomagnetic detection and clinical significance of micrometastatic tumor cells in malignant melanoma patients.

Positive associations between the presence of micrometastatic tumor cells and disease aggressiveness have been reported in several tumor types, but the clinical implications are still not established. We wanted to test a new, sensitive immunomagnetic detection method on bone marrow (BM) and peripheral blood (PB) samples from patients with malignant melanoma and relate the findings to clinical outcome. Samples from 210 patients admitted for relapse of cutaneous melanoma were examined. Mononuclear cell fractions isolated from BM and PB were incubated with superparamagnetic particles coated with antimelanoma antibodies. Live tumor cells with bound beads were isolated with a magnet and identified in a microscope as cell-bead rosettes. Beads without antibody or with an irrelevant antibody were used as controls. The whole procedure was completed within 2-3 h. The identity of the cells was confirmed with a new double labeling procedure with fluorescent microparticles. Rosetted melanoma cells were found in BM aspirates of 35 of 186 (19%) patients, but in only 2 of 208 (1%) PB samples. The controls were all negative. After a median observation time of 1.1 year (range, 0-6.8 years), patients with tumor cells in BM showed a significantly shorter overall survival from time of BM aspiration (P = 0.009). In multiple regression analysis, a positive BM test was a strong indicator of overall survival (P = 0.021), associated with disease stage (American Joint Committee on Cancer) and with the number of metastatic sites, but not with the primary (Breslow) tumor depth and morphology. The results demonstrate the prognostic significance of detecting BM micrometastasis in melanoma patients. The results strengthen the validity of the immunobead technique. In contrast to other techniques, the method identifies intact, live tumor cells that can be further characterized, making the assay attractive for extended use.

Molecular anatomy of placode development in Xenopus laevis

We analyzed the spatiotemporal pattern of expression of 15 transcription factors ( Six1, Six4, Eya1, Sox3, Sox2, Pax6, Pax3, Pax2, Pax8, Dlx3, Msx1, FoxI1c, Tbx2, Tbx3, Xiro1) during placode development in Xenopus laevis from neural plate to late tail bud stages. Out of all genes investigated, only the expression of Eya1, Six1, and Six4 is maintained in all types of placode (except the lens) throughout embryonic development, suggesting that they may promote generic placodal properties and that their crescent-shaped expression domain surrounding the neural plate defines a panplacodal primordium from which all types of placode originate. Double-labeling procedures were employed to reveal the precise position of this panplacodal primordium relative to neural plate, neural crest, and other placodal markers. Already at neural plate stages, the panplacodal primordium is subdivided into several subregions defined by particular combinations of transcription factors allowing us to identify the approximate regions of origin of various types of placode. Whereas some types of placode were already prefigured by molecularly distinct areas at neural plate stages, the epibranchial, otic, and lateral line placodes arise from a common posterior placodal area (characterized by Pax8 and Pax2 expression) and acquire differential molecular signatures only after neural tube closure. Our findings argue for a multistep mechanism of placode induction, support a combinatorial model of placode specification, and suggest that different placodes evolved from a common placodal primordium by successive recruitment of new inducers and target genes.

Molecular anatomy of placode development in Xenopus laevis

We analyzed the spatiotemporal pattern of expression of 15 transcription factors ( Six1 , Six4 , Eya1 , Sox3 , Sox2 , Pax6 , Pax3 , Pax2 , Pax8 , Dlx3 , Msx1 , FoxI1c , Tbx2 , Tbx3 , Xiro1 ) during placode development in Xenopus laevis from neural plate to late tail bud stages. Out of all genes investigated, only the expression of Eya1 , Six1 , and Six4 is maintained in all types of placode (except the lens) throughout embryonic development, suggesting that they may promote generic placodal properties and that their crescent-shaped expression domain surrounding the neural plate defines a panplacodal primordium from which all types of placode originate. Double-labeling procedures were employed to reveal the precise position of this panplacodal primordium relative to neural plate, neural crest, and other placodal markers. Already at neural plate stages, the panplacodal primordium is subdivided into several subregions defined by particular combinations of transcription factors allowing us to identify the approximate regions of origin of various types of placode. Whereas some types of placode were already prefigured by molecularly distinct areas at neural plate stages, the epibranchial, otic, and lateral line placodes arise from a common posterior placodal area (characterized by Pax8 and Pax2 expression) and acquire differential molecular signatures only after neural tube closure. Our findings argue for a multistep mechanism of placode induction, support a combinatorial model of placode specification, and suggest that different placodes evolved from a common placodal primordium by successive recruitment of new inducers and target genes.

Quantitative Analysis of Tryptase- and Chymase-containing Mast Cells in Eosinophilic Conditions of Cats

The presence and density of tryptase-positive/chymase-positive mast cells (MCs) (MC(TC)), chymase-positive/tryptase-negative MCs (MCC), and tryptase-positive/chymase-negative MCs (MC(T)) in lesional skin from cats with eosinophilic conditions were investigated. Skin biopsy specimens from eight cats with eosinophilic plaque (three cats), eosinophilic granuloma (two cats), and eosinophilic dermatitis (three cats) were studied. Toluidine blue staining and a double-enzyme-immunohistochemical staining technique were performed to determine MC density and MC subtypes, respectively. MC density varied from 170.3 to 503 cells/mm2 (mean value of 314.9 cells/mm2). In the superficial dermis, 5.9% of the MC belonged to the MC(T), 12.8% to the MC(C), and 81.2% to the MC(TC) subtype. In the deep dermis, 12.8% belonged to the MC(T), 12.8% to the MC(C), and 73.8% to the MC(TC) subtype. It is the first time that MC(C) have been identified. The double-labeling procedure proved to be a reliable tool for identifying simultaneously the presence of MC subtypes in feline skin.

Brush cells of rodent gallbladder and stomach epithelia express neurofilaments.

It has been suggested that brush cells (BCs), a distinct type of cell occurring in various epithelia of the respiratory and gastrointestinal tracts, may function as receptor cells. The major characteristics of BCs are a prominent brush border and an unusually highly ordered arrangement of cytoskeletal elements (F-actin, microtubules, and intermediate filaments). In this study we aimed to characterize the nature of the intermediate filaments in BCs by light and electron microscopic immunostaining. Gallbladder and stomach specimens from mice and rats, respectively, were fixed in various solutions, embedded either in paraffin or epoxy resin, and processed for immunodetection. Commercially available, well-characterized antibodies against neurofilaments, peripherin, and cytokeratin peptide 18 were used. The polyclonal antiserum cocktail to neurofilaments was applied as a supplement in a double-labeling procedure with anti-actin and anti-cytokeratin 18 antibodies. The results demonstrate that the BCs of both organs express two types of intermediate filaments, i.e., neurofilaments and cytokeratin 18 filaments, and that these have a compartmentalized distribution in the cytoplasm. BCs do not express peripherin. The immunodetection of intermediate filaments distinctive for mature neurons in BCs supports their putative receptor function. The co-expression of neurofilaments and cytokeratins is shown for the first time in healthy tissues.

Flow Cytometric Analysis of Hydroxyurea Effects on Fetal Hemoglobin Production in Cultures of β‐Thalassemia Erythroid Precursors

An increase in fetal hemoglobin (Hb F) ameliorates the clinical symptoms of the underlying disease in the β hemoglobinopathies—sickle cell anemia and β‐thalassemia (thal). Hydroxyurea (HU) can elevate Hb F production in erythroid cells and is the agent currently in clinical use for patients with sickle cell anemia; it is presently being tested in clinical trials for thalassemia. We have developed a two‐phase liquid culture system that mimics the in vivo hematological changes that are observed in patients treated with HU. Adding HU during the second phase of the culture increases the proportion of Hb F, increases the levels of total hemoglobin (Hb) content per cell and increases cell size, but it decreases the numbers of cells and the total amount of Hb produced. In the present study we developed and utilized a double labeling procedure for flow cytometric analysis of the cellular distribution of Hb F. Cells exposed to various concentrations of HU on day 6 of the second phase of the culture were harvested on day 12, and stained simultaneously with fluorochrome‐conjugated monoclonal antibodies specific for human glycophorin A, an erythroid specific marker, and human Hb F. Both the percentage of the Hb F‐containing cells and their intensity of fluorescence were recorded. The latter value gives a semi‐quantitative estimation of the mean cellular Hb F content. The results indicated that cultures derived from different β‐thalassemic patients differ in their response to HU. In most patients, low doses of HU decreased the percentage of Hb F‐cells as well as their Hb F content. At high doses, some patients showed an increase in both parameters, while others showed an increase in the percentage of Hb F‐cells with minimal increase in their mean Hb F content, while still other patients showed little effect at all. In all patients, high doses of HU caused a decrease in cell numbers. These results suggest that HU has mixed effects on erythroid precursors. Both the two‐phase liquid culture and the flow cytometric analysis procedures described herein provide the experimental tools for screening of Hb F‐inducing drugs and for evaluating patients' cell response prior to treatment.

Specific visualization of precipitated cerium by energy-filtered transmission electron microscopy for detection of alkaline phosphatase in immunoenzymatic double labeling of tyrosine hydroxylase and serotonin in the rat olfactory bulb.

To understand in detail the functional morphology of neuronal circuits it is important to identify at the ultrastructural level the incoming axon, its target neuron, and members of the signaling cascades involved. This, however, represents a formidable task, requiring highly sophisticated electron microscopic multiple-labeling techniques. To extend available double-labeling procedures such as combinations of immunogold and peroxidase methods, an additional, gold- and peroxidase-independent procedure would represent a considerable advantage. The present investigation therefore aimed to use alkaline phosphatase as the immunoenzymatic label at the electron microscopic level via cerium phosphate precipitates. To our surprise we found that available techniques, which are well established for the visualization of endogenous enzymes in sections from various tissues, are not suitable for application to immunocytochemistry. Careful characterization of the individual reaction conditions, however, resulted in an optimized procedure with largely increased sensitivity. The novel technique yields cerium-containing precipitates which are massive enough to allow the detection of the immunoenzymatic reaction product in the electron microscope. Using the rat olfactory bulb as the model system we showed further that our technique allows the combination with the peroxidase/diaminobenzidine system for ultrastructural double labeling. For this purpose, the alkaline phosphatase product is identified by its cerium content via energy-filtered transmission electron microscopy and thereby differentiated from cerium-free peroxidase-derived precipitates. Doing so, we found that ascending serotoninergic fibers do not establish synapses with dopaminergic periglomerular cells in the rat olfactory bulb.

Chemical-modification rescue assessed by mass spectrometry demonstrates that gamma-thia-lysine yields the same activity as lysine in aldolase.

The role of active site residues in fructose 1,6-bisphosphate aldolase is investigated by chemical-modification rescue. An active-site mutation, K107C, is constructed in a background where the four solvent-accessible cysteine residues are converted to alanine. The resulting mutant, tetK107C, when reacted with bromoethylamine (BrEA), shows a 40-fold increase in activity (to 80% that of wild type). Determination of the sites and their degree of modification using electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) is developed, allowing correlation of activity after chemical modification rescue to the degree of modification. The stoichiometry of the reaction is 2.5 aminoethylations per subunit, as measured by ESI-FTMS. Protein modification with a double-labeled mix (1:1) of natural abundance isotope (d(0)-BrEA) and 2-bromoethyl-1,1,2,2-d4-amine hydrobromide (d(4)-BrEA), followed by dialysis and trypsin digestion, shows aminoethylated peptides as "twin peptides" separated by four mass units in ESI-FTMS analysis. Using this detection procedure under nondenaturing (native) conditions, C107 is aminoethylated, whereas the four buried thiols remain unlabeled. Aminoethylation of other residues is observed, and correlates with those peptides containing histidine, methionine, and/or the amino terminus. Quantification of the aminoethylation reaction is achieved by labeling with nondeuterated d(0)-BrEA under denaturing conditions following double labeling under native conditions. In addition to complete labeling all five thiols, the intensity of the d(0)-BrEA peak for C107 containing peptides increases, and the change in the d(0)/d(4) ratio between native and denaturing conditions shows 82 +/- 4.5% aminoethylation at C107. This correlation of modification with the recovered activity, indicates that gamma-thia-lysine replaces lysine in the catalytic mechanism. Kinetic constants measured for the rescued K107C mutant enzyme with the substrates fructose 1-phosphate and fructose 1,6-bisphosphate are consistent with the role of the positively charged lysine binding to the C6-phosphate. ESI-FTMS, combined with this double-labeling procedure, allows precise identification of sites and measurement of degree of protein modification.

A Double-Label Immunohistochemical Method for Assessment of Epithelial Cell Replication in Paraffin Sections of Toxicant-Damaged Lungs

Enhanced epithelial cell replication is a sensitive indicator of toxicant-induced lung damage. Cell identification in the deep lung can be difficult and limit the usefulness of conventional techniques for examining cell-cycle kinetics in toxicant-damaged lungs. The purpose of this study was to develop a technique to reliably differentiate epithelial from nonepithelial cells in the deep lung. Formalin fixed, paraffin sections of toxicant-exposed lungs were used to develop a double, sequential, peroxidase/alkaline phosphatase method to detect bromodeoxyuridine and cytokewtins. Use of lü-5 cytokeratin antibody, which binds to all cytokeratin polypeptides and is epithelial cell specific, and a biotinstreptavidin amplified detection system maximized the detection of cytokeratin immunoreactivity and allowed cells with a negative reaction to be classified as nonepithelial. The staining produced was distinct and readily detected by light microscopy. In foci of severe damage, the cytokeratin immunostaining of epithelial cells highlighted the general architecture of the parenchyma. Cell counts and labeling data from inflammatory foci in endotoxin-instilled rat lungs were compared using the double-label versus a conventional single-label immunohistochemical method. The proportion of cells that could be identified was significantly increased in the double-label slides. We conclude that in lungs with severe damage, where the usual morphologic features such as cell size, shape, vacuolization, and position are unreliable indicators of cell type, the double-label procedure would aid the microscopist in obtaining reliable cells counts. (The J Histotechnol 25:15, 2002)Submitted: September 10, 2001; Accepted with revisions: November 7, 2001

Expression of tumour necrosis factor-α is associated with apoptosis in lungs of pigs experimentally infected with porcine reproductive and respiratory syndrome virus

Expression of tumour necrosis factor (tnf)-α and its association with apoptosis was examined in lungs from pig experimentally inoculated with porcine reproductive and respiratory syndrome virus (prrsv). Thetnf -α nucleic acid was detected in prrsv -infected alveolar macrophages by reverse transcription–polymerase chain reaction (rt - pcr). The tnf -α antigen was detected in alveolar macrophages by immunohistochemisty. dna fragmentation in apoptotic cells was detected by in situ terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labelling (tunel). A double-labelling procedure using immunohistochemisty for the detection of prrsv and the in situ tunel assay for the detection of apoptosis demonstrated that a majority of labelled cells were infected with prrsv or apoptotic, but not both. Apoptotic cells were more abundant than prrsv -infected cells in all lungs examined. A double-labelling procedure using immunohistochemisty for the detection of tnf -α and the in situ tunel assay for the detection of apoptosis demonstrated that a majority of labelled cells were apoptotic or for tnf -α, but not both. These data suggested that tnf -α released from macrophages after infection by prrsv may induce apoptosis in uninfected bystander cells.

Analysis of binding of mannosides in relation to Langerin (CD207) in Langerhans cells of normal and transformed epithelia.

Tandem-repeat C-type lectins (pattern-recognition receptors) with specificity for mannosides are intimately involved in antigen recognition, uptake, routing and presentation in macrophages and dendritic cells. In Langerhans cells, Langerin (CD207), a type-II transmembrane protein with a single C-type carbohydrate recognition domain attached to a heptad repeat in the neck region, which is likely to establish oligomers with an alpha-coiled-coil stalk, has been implicated in endocytosis and the formation of Birbeck granules. The structure of Langerin harbours essential motifs for Ca2+-binding and sugar accommodation. Lectin activity has previously been inferred by diminished antibody binding to cells in the presence of the glycan ligand mannan. In view of the complexity of the C-type lectin/lectin-like network, it is unclear what role Langerin plays for Langerhans cells in binding mannosides. In order to reveal in frozen tissue sections to what extent mannose-binding activity co-localizes with Langerin, we have used a synthetic marker, i.e. a neoglycoprotein carrying mannose maxiclusters, as a histochemical ligand, and computer-assisted fluorescence monitoring in a double-labelling procedure. Mannoside-binding capacity was detected in normal epithelial cells. Double labelling ensured the unambiguous assessment of the binding of the neoglycoprotein in Langerhans cells. Light-microscopically, its localization profile resembled the pattern of immunohistochemical detection of Langerin. This result has implications for suggesting rigorous controls in histochemical analysis of this cell type, because binding of kit reagents, i.e. mannose-rich glycoproteins horseradish peroxidase or avidin, to Langerin (or a spatially closely associated lectin) could yield false-positive signals. To show that recognition of carbohydrate ligands in dendritic cells is not restricted to mannose clusters, we have also documented binding of carrier-immobilized histo-blood group A trisaccharide, a ligand of galectin-3, which was not affected by the presence of a blocking antibody to Langerin. Remarkably, access to the carbohydrate recognition domain of Langerin appeared to be impaired in proliferatively active environments (malignancies, hair follicles), indicating presence of an endogenous ligand with high affinity to saturate the C-type lectin under these conditions.

Postmitotic basal cells in squamous cell epithelia are identified with Dolichos biflorus agglutinin – functional consequencesNote

Dolichos biflorus agglutinin (DBA) is a plant lectin specifically recognizing alpha-N-acetylgalactosamine. Controversial reports regarding the binding of DBA to the epidermis have been published. Using a double labeling procedure at the single-cell level, we studied the expression of DBA-reactive binding sites in conjunction with markers of cell proliferation and differentiation in normal human epidermis, cornea, and malignant tumors as well as in cultured keratinocytes. The results characterize the cells recognized by DBA as postmitotic early differentiating cells, identifiable by their lack of expression of the proliferation marker (Ki-67). The Golgi complex of a limited number of cultured keratinocytes was recognized by DBA and some of these cells show the accumulation of beta1 integrin chain in the Golgi complex. This process seems to be important for the migration of postmitotic cells from the basal to the suprabasal layers.

Osteopontin expression in primary sarcomas of the pulmonary artery.

Primary tumors of the great vessels (aorta, pulmonal artery, and inferior vena cava) are rare and represent in most cases vascular leiomyosarcomas. Furthermore, there also exists a group of sarcomas arising from the intima, known as intimal sarcomas, associated with early metastasis and a very poor prognosis. Osteopontin (OPN) is an extracellular matrix protein that binds to alphav integrins, thereby promoting cell attachment, chemotaxis, and signal transduction. The reported association of OPN with malignancy and metastasis prompted us to examine the expression of this protein in seven sarcomas of the pulmonary artery. Strong OPN-specific staining could be detected in tumor cells and the adjacent extracellular matrix. Using a double labeling procedure, proliferating cells showed a strong positive reaction with antibodies against OPN. In addition, this protein could be demonstrated in the cytoplasm of macrophages. CD44, a putative receptor of OPN, was expressed on the cellular surface of tumor-associated lymphocytes. The expression of OPN in macrophages and tumor cells indicates that this molecule could possibly mediate cellular adhesion of both cell types in pulmonary sarcomas. The detection in the extracellular matrix shows that OPN is actively secreted and may interact with the corresponding receptor, CD44, on the surface of lymphocytes. Although the function of OPN is not yet fully understood, our data indicate that strong expression of this molecule in poorly differentiated sarcomas could play a role in the progression of malignancy and metastasis as described previously for carcinomas.

Distribution and co-localization of choline acetyltransferase and p75 neurotrophin receptors in the sheep basal forebrain: implications for the use of a specific cholinergic immunotoxin.

The basal forebrain cholinergic system is involved in different forms of memory. To study its role in social memory in sheep, an immunotoxin, ME20.4 immunoglobulin G (IgG)-saporin, was developed that is specific to basal forebrain cholinergic neurons bearing the p75 neurotrophin receptor. The distribution of sheep cholinergic neurons was mapped with an antibody against choline acetyltransferase. To assess the localization of the p75 receptor on basal forebrain cholinergic neurons, the distribution of p75 receptor-immunoreactive neurons with ME20.4 IgG was examined, and a double-labeling study with antibodies against choline acetyltransferase and p75 receptor was undertaken. The loss of basal forebrain cholinergic neurons and acetylcholinesterase fibers in basal forebrain projection areas was assessed in ewes that had received intracerebroventricular injections of the immunotoxin (50, 100 or 150 microg) alone, as well as, in some of the ewes treated with the highest dose, with bilateral immunotoxin injections in the nucleus basalis (11 microg/side). Results indicated that choline acetyltransferase- and p75 receptor-immunoreactive cells had similar distributions in the medial septum, the vertical and horizontal limbs of the band of Broca, and the nucleus basalis. The double-labeling procedure revealed that 100% of the cholinergic neurons are also p75 receptor positive in the medial septum and in the vertical and horizontal limbs of the band of Broca, and 82% in the nucleus basalis. Moreover, 100% of the p75 receptor-immunoreactive cells of these four nuclei were cholinergic. Combined immunotoxin injections into ventricles and the nucleus basalis produced a near complete loss (80-95%) of basal forebrain cholinergic neurons and acetylcholinesterase-positive fibers in the hippocampus, olfactory bulb and entorhinal cortex. This study provides the first anatomical data concerning the basal forebrain cholinergic system in ungulates. The availability of a selective cholinergic immunotoxin effective in sheep provides a new tool to probe the involvement of basal forebrain cholinergic neurons in cognitive processes in this species.

Subcellular distribution of 5-HT(1B) and 5-HT(7) receptors in the mouse suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN), a circadian oscillator, receives glutamatergic afferents from the retina and serotonergic (5-HT) afferents from the median raphe. 5-HT(1B) and 5-HT(7) receptor agonists inhibit the effects of light on SCN circadian activity. Electron microscopic (EM) immunocytochemical procedures were used to determine the subcellular localization of 5-HT(1B) and 5-HT(7) receptors in the SCN. 5-HT(1B) receptor immunostaining was associated with the plasma membrane of thin unmyelinated axons, preterminal axons, and terminals of optic and nonoptic origin. 5-HT(1B) receptor immunostaining in terminals was almost never observed at the synaptic active zone. To a much lesser extent, 5-HT(1B) immunoreaction product was noted in dendrites and somata of SCN neurons. 5-HT(7) receptor immunoreactivity in gamma-aminobutyric acid (GABA), vasoactive intestinal polypeptide (VIP), and vasopressin (VP) neuronal elements in the SCN was examined by using double-label procedures. 5-HT(7) receptor immunoreaction product was often observed in GABA-, VIP-, and VP-immunoreactive dendrites as postsynaptic receptors and in axonal terminals as presynaptic receptors. 5-HT(7) receptor immunoreactivity in terminals and dendrites was often associated with the plasma membrane but very seldom at the active zone. In GABA-, VIP-, and VP-immunoreactive perikarya, 5-HT(7) receptor immunoreaction product was distributed throughout the cytoplasm often in association with the endoplasmic reticulum and the Golgi complex. The distribution of 5-HT(1B) receptors in presynaptic afferent terminals and postsynaptic SCN processes, as well as the distribution of 5-HT(7) receptors in both pre- and postsynaptic GABA, VIP, and VP SCN processes, suggests that serotonin plays a significant role in the regulation of circadian rhythms by modulating SCN synaptic activity.