Differential interaction of 2,4,6-trinitrobenzenesulfonic (TNBS) and 2,4-dinitrobenzenesulfonic (DNBS) acids on mouse lymphocytes]

Modifications of lymphoid cells with dinitro-2,4 phenylsulfonic acid (DNBS) or trinitro-2,4,6 phenylsulfonic acid (TNBS) have been studied. TNBS action always produces an electrophoretic mobility increase in relation with the amount of amino-groups, according to the cell type. DNBS action produces an electrophoretic mobility increase for B cells of spleen and a decrease for T cells of spleen and thymic cells. The hydrophobic fluorescent probe 1-anilino-8-naphthalene sulphonate used to study conformational changes of cells revealed a slight fluorescence decrease for TNP-modified cells and an important fluorescence increase for DNP-modified cells.     

Demonstration of a tryptaminergic mechanism in the rat beta-cell.

Yellow 5-HT fluorescence has been histochemically demonstrated in rat pancreatic islet cells in animals injected with L-5-HTP with or without pretreatment with the monoaminoxidase inhibitor nialamide. This fluorescence was not observed after inhibition of the aromatic amino acid decarobxylase. The results strongly suggest the presence of a tryptaminergic mechanism in the rat islet cells.     

Chromatin fluorescence by pyronin staining

Human, chicken and mouse cells from different tissues show a bright red-orange fluorescence of the chromatin after staining with pyronin Y. The possibility that intercalation of the dye into double helical nucleic acids accounts for this fluorescence pattern is briefly discussed.     

Autonomous fluorescence of ascidian blood cells with special reference to identification of vanadocytes

Summary A tunichrome that has been suggested to be involved in the accumulation of vanadium ions in ascidian blood cells produces an autonomous fluorescence upon excitation with blue-violet light. However, we have found that signet ring cells, which contain large amounts of vanadium, do not fluoresce upon such excitation. The strongest fluorescence due to the tunichrome was observed in morula cells, which do not contain vanadium.     

Changes to cellular water and element content induced by nucleolar stress: investigation by a cryo-correlative nano-imaging approach

The cell is a crowded volume, with estimated mean mass percentage of macromolecules and of water ranging from 7.5 to 45 and 55 to 92.5 %, respectively. However, the concentrations of macromolecules and water at the nanoscale within the various cell compartments are unknown. We recently developed a new approach, correlative cryo-analytical scanning transmission electron microscopy, for mapping the quantity of water within compartments previously shown to display GFP-tagged protein fluorescence on the same ultrathin cryosection. Using energy-dispersive X-ray spectrometry (EDXS), we then identified various elements (C, N, O, P, S, K, Cl, Mg) in these compartments and quantified them in mmol/l. Here, we used this new approach to quantify water and elements in the cytosol, mitochondria, condensed chromatin, nucleoplasm, and nucleolar components of control and stressed cancerous cells. The water content of the control cells was between 60 and 83 % (in the mitochondria and nucleolar fibrillar centers, respectively). Potassium was present at concentrations of 128–462 mmol/l in nucleolar fibrillar centers and condensed chromatin, respectively. The induction of nucleolar stress by treatment with a low dose of actinomycin-D to inhibit rRNA synthesis resulted in both an increase in water content and a decrease in the elements content in all cell compartments. We generated a nanoscale map of water and elements within the cell compartments, providing insight into their changes induced by nucleolar stress.     

New histochemical contributions to the study of corticotropic cells in the adenohypophysis of the chick enbryo.]

Comparative analysis of results obtained after subsequent treatments of chick embryo adenohypophysis by anti-ACTH 17-39 Is, PAS and Bodian method demonstrates that ACTH cells are PAS positive, Bodian negative and are located in the cephalic lobe. With the histochemical fluorescence method of Falck, the cells marked by anti-ACTH 17-39 Is exhibit an induced fluorescence, after L-DOPA injection. However, after treatment by metopirone, numerous cells of the caudal part of the adenohypophysis take up L-DOPA but they are not corticotrophs.     

Demonstration of acidophilic neurosecretory cells in Crepidula fornicata Phil. (Mollusca, Gasteropoda, Prosobranchia) using the fluorescent dye, Geranine G]

The Fluorochrome Geranine G binds to acidophilic neurosecretory cells and produces red orange fluorescence. Chromolipids and pigments show green fluorescence. This method is easy and sensitive. Fluorescence fading during irradiation is low.     

Measurement of fluorescence decay of substances absorbed by an isolated living cell: an experimental device and early results]

A microfluorometric system allowing the mesurements of fluorescence decay (i.e. fluorescence lifetime) has been built. It will complete the microspectrofluorometric studies of absorbed compounds-polycyclic aromatic hydrocarbons, metabolites of benzo (a) pyrene-by single living cells and allows a better understanding of the intake and metabolisation of these compounds. The preliminary results with benzo (a) pyrene, dibenzocarbazole, pyrene are shown.     

Chromatin fluorescence by pyronin staining

Summary Human, chicken and mouse cells from different tissues show a bright red-orange fluorescence of the chromatin after staining with pyronin Y. The possibility that intercalation of the dye into double helical nucleic acids accounts for this fluorescence pattern is briefly discussed.This work was partially supported by a grant from the Comisión Asesora de Investigación Científica y Técnica, Spain.     

Surface display of a single-domain antibody library on Gram-positive bacteria

Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10⁷ camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.     

Fluorescein-concanavalin A conjugates distinguish between normal and malignant human cells: a preliminary report

A method is described for using a fluorescein isothiocyanate concanavalin A conjugate to stain human cell membranes in formalin fixed paraffin embedded tissue. 57 neoplastic and normal tissue sites were examined. In 54 malignant tumours, bright green fluorescence was confined to the cell membranes while in 23 benign tumours and normal tissue sites, the membranes were unstained or showed a diminished level of fluorescence. The distinction between malignant and hyperplastic or normal cells was clear cut and definite.     

Fluorescein-concanavalin A conjugates distinguish between normal and malignant human cells: A preliminary report

Summary A method is described for using a fluorescein isothiocyanate concanavalin A conjugate to stain human cell membranes in formalin fixed paraffin embedded tissue. 57 neoplastic and normal tissue sites were examined. In 34 malignant tumours, bright green fluorescence was confined to the cell membranes while in 23 benign tumours and normal tissue sites, the membranes were unstained or showed a diminished level of fluorescence. The distinction between malignant and hyperplastic or normal cells was clear cut and definite.     

Early detection of cell damage by supravital acridine orange staining

Summary Cell damage can be detected in living cells by acridine orange fluorescence earlier than with phase contrast microscopy or with conventional histological methods. The change in the acridine orange fluorescence from green to red indicates that the secondary structure of the DNA is altered very early during the cell death.Acknowledgments. We thank Miss Pirjo Vuori for excellent technical assistance, Engineer Charles Vane-Tempest for providing the necessary microscope equipment and the Finnish Academy for financial support.     

Rapid recording of the fluorescence spectrum emitted by an isolated living cell]

In order to use the selectivity of fluorescence emission for biomedical purposes, a microspectrofluorimeter has been built around an inverted microscope. The excitation is supplied by a 450 W Xenon lamp coupled with a monochromator allowing excitation from 230 to 600 nm. The fluorescence is analysed by a Grism (association prism-grating) and the spectrum focused on the 500 channels of the target of an Optical Multichannel Analyser. Related to good resolution and dispersion (0.44 nm/channel), improvement of signal to noise ratio (dark current suppression, and accumulation of spectra), this microspectrofluorimeter with high sensitivity is well adapted to the study of biological mechanism in single living cells (macrophages, monocytes, lumphocytes).     

Demonstration of catecholaminergic neurons in rat retina by fluorescence histochemistry on cryostat sections]

A fluorescence histochemical technique using glyoxylic acid and cryostat sections has been worked out to visualize the dopamine-containing neurons of the retina. It allows the demonstration of catecholaminergic junctional cells and alloganglion cells, classically described. Moreover, the observation of some fluorescent fibers in the external zone of the inner nuclear layer, in contact with the outer plexiform, suggests the presence of catecholamine-containing interplexiform cells, in the Rat retina.     

B Lymphocytes in obesity-related adipose tissue inflammation and insulin resistance

Obesity-related insulin resistance is a chronic inflammatory condition that often gives rise to type 2 diabetes (T2D). Much evidence supports a role for pro-inflammatory T cells and macrophages in promoting local inflammation in tissues such as visceral adipose tissue (VAT) leading to insulin resistance. More recently, B cells have emerged as an additional critical player in orchestrating these processes. B cells infiltrate VAT and display functional and phenotypic changes in response to diet-induced obesity. B cells contribute to insulin resistance by presenting antigens to T cells, secreting inflammatory cytokines, and producing pathogenic antibodies. B cell manipulation represents a novel approach to the treatment of obesity-related insulin resistance and potentially to the prevention of T2D. This review summarizes the roles of B cells in governing VAT inflammation and the mechanisms by which these cells contribute to altered glucose homeostasis in insulin resistance.     

On the self-association potential of transmembrane tight junction proteins

Tight junctions seal intercellular clefts via membrane-related strands, hence, maintaining important organ functions. We investigated the self-association of strand-forming transmembrane tight junction proteins. The regulatory tight junction protein occludin was differently tagged and cotransfected in eucaryotic cells. These occludins colocalized within the plasma membrane of the same cell, coprecipitated and exhibited fluorescence resonance energy transfer. Differently tagged strand-forming claudin-5 also colocalized in the plasma membrane of the same cell and showed fluorescence resonance energy transfer. This demonstrates self-association in intact cells both of occludin and claudin-5 in one plasma membrane. In search of dimerizing regions of occludin, dimerization of its cytosolic C-terminal coiledcoil domain was identified. In claudin-5, the second extracellular loop was detected as a dimer. Since the transmembrane junctional adhesion molecule also is known to dimerize, the assumption that homodimerization of transmembrane tight junction proteins may serve as a common structural feature in tight junction assembly is supported. Received 6 October 2005; received after revision 14 December 2005; accepted 27 December 2005 †These authors contributed equally to this work.