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Biomedical subjects

J De Mey

Publications and source records attributed to J De Mey.

At least 55 records · Page 3Linked to original sources
PubMed1985

Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus.

In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.

Anaphase↗
PubMed1985

Retention of immune complexes by Fc receptors on mouse follicular dendritic cells.

Follicular dendritic cells (FDC) are located inside lymph follicles and are mainly characterized by their capacity to retain antigens. We investigated this aspect in mice lymph nodes by using bovine serum albumin (BSA) labelled with 5-nm colloidal gold particles and homologous anti-BSA antibodies bound to 20-nm gold particles. Gold-labelled BSA injected alone in non-immunized mice was only rarely found in FDC cytoplasmic interdigitations. Injected in the form of immune complexes, it was retained by FDC. Antigen-free anti-BSA antibodies injected under similar conditions as immune complexes were always found in draining lymph nodes in the same locations as BSA-anti-BSA immune complexes. F(ab')2 from mouse immunoglobulins linked to colloidal gold particles were very rarely found between the FDC extensions, whereas it was intensely phagocytosed by macrophages. Our study permitted precise ultrastructural localization between FDC cytoplasmic extensions or inside macrophages and other cells of the lymph nodes, but it also pointed out that homologous antibodies linked to colloidal gold particles might be retained by FDC in the absence of antigens. These observations, carried out with colloidal gold, were checked by using 125I-labelled anti-BSA antibodies. Complement activation determinations of gold-labelled antibodies or immune complexes showed that antibodies or immune complexes fixed on colloidal gold particles do not activate the complement. This observation enabled us to conclude that Fc receptors play a significant part in the retention of gold-labelled antibodies or immune complexes by FDC of lymph nodes.

Animals↗
PubMed1985

Isolation of follicular dendritic cells from human tonsils and adenoids. III. Analysis of their Fc receptors.

Follicular dendritic cells (FDC), isolated from human tonsils or adenoids, were tested for their capacity to retain monomeric, aggregated or antigen-bound human antibodies in the absence of serum. FDC retain fluorescein-labelled heat-aggregated human immunoglobulins, but not monomeric ones nor fluorescein-labelled F(ab')2 in monomeric or aggregated form. Ultrastructural observations showed that colloidal gold-labelled monomeric, or antigen-bound, antibodies directed against tetanus toxoid are retained by dendrites and membrane infoldings of FDC but are never located in cytoplasmic vesicles. This retention was inhibited by incubating FDC with unlabelled aggregated or antigen-bound antibodies. When gold-labelled anti-tetanus toxoid antibodies were incubated in the presence of protein-A before the contact with FDC, a strong reduction of their retention occurred. This further suggested the presence of Fc receptors on isolated tonsillar FDC. Endocytosis was not observed in isolated FDC, even after prolonged incubation in presence of labelled immune complexes: their Fc receptors are, thus, not related to a phagocytic activity as they are in macrophages. Simultaneous ultrastructural labelling of Fc and C3b receptors with colloidal gold particles of different sizes did not reveal any clear relations between these two receptors on the surface of FDC.

Adenoids↗
PubMed1985

Probing microtubule-dependent intracellular motility with nanometre particle video ultramicroscopy (nanovid ultramicroscopy).

Colloidal gold particles of 20 to 40 nm diameter stabilized with polyethylene glycol (PEG) were microinjected in PTK2 cells. Aggregates and individual particles, which are smaller than the theoretical limit of resolution of the optical microscope and invisible to the eye are discernible from organelles by reflection of polarized light. They are optimally visualized using transmitted light and electronic subtraction of diffuse background light. The gold particles show saltatory motion. The direction, speed, median distance travelled and frequency of saltations are indiscernible from measurements made on cell organelles in the same preparations. Because microtubule treadmilling has been implicated as a potential motor for organelle motility, gold particles coupled to monoclonal antibodies, recognizing the alpha-subunit of tubulin (Kilmartin et al., 1982), were injected. These particles, often forming linear arrays, assumed entirely fixed positions in the cell. The results suggest that there is a transport system associated with microtubules which can carry synthetic particles through the cell without the need for them being covered with specific proteins. Microtubule treadmilling does not seem to be involved. The possibility of following 20-40 nm particles and probably even smaller ones, that can be coupled to most proteins, within living cells provides a tool of wide applicability to study the fate and behaviour of such proteins. It is suggested that this new method be called nanoparticle video ultramicroscopy or nanovid ultramicroscopy.

Animals↗
PubMed1984

Sensitive visualization of antigen-antibody reactions in dot and blot immune overlay assays with immunogold and immunogold/silver staining.

The use of colloidal gold and colloidal gold followed by silver enhancement as marker for dot or blot immune overlays is described. Colloidal gold probes concentrating at the sites of immune reaction gradually develop a pinkish colour during incubation that can be seen with the naked eye. With a physical developer, very high sensitivity and contrast is obtained by silver precipitation on the gold marker. Comparison of the immunogold and immunogold/silver staining methods with the indirect, PAP and ABC immunoperoxidase methods demonstrates that colloidal gold probes are excellent markers for immune overlay assays.

Animals↗
PubMed1984

Indirect immunofluorescence of microtubules in Dictyostelium discoideum. A study with polyclonal and monoclonal antibodies to tubulins.

Amebae of D. discoideum on coverslips were fixed in situ with glutaraldehyde and permeabilized with Triton X-100. Of six antibodies tested, only a monoclonal antibody to yeast tubulin consistently gave bright fluorescence. Counterstaining with DAPI facilitated the identification of interphase and mitotic stages. Most microtubules (MTs) in interphase amebae emanated from a nucleus-associated centre that had a non-fluorescent core. Amebae in early stages of mitosis lacked cytoplasmic MTs almost entirely. The nascent spindle in prophase appeared as a brightly fluorescent dot, whereas the prometaphase spindle was a short rod. Spindles in metaphase and anaphase nuclei were more elongate, some consisting of several fluorescent lines. Astral MTs were prominent on spindles in anaphase and telophase. Asters are obviously converted to the interphase complex of MTs in post-mitotic cells, while the shaft-like remnant of the central spindle disappears. The cyclical changes in the MT system related to cell division resemble those observed in higher eukaryotes and probably reflect changes in the locomotory behavior of the amebae rather than changes in cell shape.

Dictyostelium↗
PubMed1984

Intermediate filaments in monkey kidney TC7 cells: focal centers and interrelationship with other cytoskeletal systems.

Two-dimensional gel electrophoresis of intermediate-sized filament-enriched cytoskeletons of epithelial monkey kidney TC7 cells has shown that they are composed of at least two keratins (isoelectric focusing 36, Mr = 48,500; IEF 46, Mr = 43,500; HeLa protein catalogue number) and vimentin. Indirect immunofluorescence as well as immunoelectron microscopy using antibodies directed against specific polypeptides sometimes revealed a discontinuous staining of keratin-containing filaments. Indirect immunofluorescence analysis of cells stained with keratin or vimentin antibodies also revealed a bright perinuclear staining in 58% of the cells in interphase. Of particular interest were focal centers from which filaments radiated. Double-label immunofluorescence using tubulin and keratin antibodies showed that these centers codistributed with focal arrays of microtubules (most likely centrosomes) in interphase cells but were not colocalized with centrioles in mitosis or, in many cases, with the microtubule organizing centers seen after release from nocodazole treatment. Treatment of TC7 cells with demecolcine (10 micrograms/ml, 20 hr) resulted in a drastic rearrangement of the keratin and vimentin filaments. Likewise, treatment with cytochalasin B (10 micrograms/ml, 1 hr) produced a star-like arrangement of the keratin and vimentin filaments and, in most cases, these codistributed with patches of actin. The results provide evidence for the interaction of intermediate filaments (keratins and vimentin) with both microtubules and microfilaments.

Animals↗
PubMed1983

Intracellular topography of immunoreactive gastrin demonstrated using electron immunocytochemistry.

Gastrin (G)-producing cells from the mammalian gastric antrum have been investigated using computer-assisted morphometry and a novel double colloidal gold-labeled-immunoglobulin electron immunocytochemical procedure. Correlation analysis of human antral G-cells indicates (p less than 0.001) that a single population of granules exists with small (160 nm) electron-dense and large (240 nm) electron-lucent forms representing the extremes. Non-crossreacting region-specific antisera have been used to visualize G-17 and G-34 (progastrin) to the small electron-dense granules and G-17 to the other intermediate forms. From the results we propose a topographic segregation of immunoreactive gastrins within 2 apparently distinct granule subclasses and suggest that this may represent the pathway of granule maturation.

Adult↗
PubMed1983

Immunocytochemistry of serotonin-containing nerves in the human gut.

An immunocytochemical technique, using L-tryptophan enhancement and paraformaldehyde fixation, has been successfully applied for the demonstration of serotonin-containing neurons in the human enteric nervous system. Throughout the intestine, serotonin-immunoreactive nerves were mainly detected in the myenteric plexus and, to a smaller extent, in the submucous plexus, while hardly any nerve fibres were seen in the mucosa. This approach provides a useful tool for the investigation of neuropathological conditions of the gut.

Fluorescent Antibody Technique↗
PubMed1983

The interaction between microtubules and intermediate filaments in cultured cells treated with taxol and nocodazole.

Using double-label immunofluorescence and electron microscopy we studied the interaction between microtubules (MT) and intermediate filaments (IF) in MO cells treated with various combinations of taxol and nocodazole. With taxol, the organized MT of cultured cells are replaced by free MT and MT bundles. This rearrangement of MT is followed by a rearrangement of the IF. As in untreated cells a close association between these two filamentous systems is observed. In cells pretreated with nocodazole followed by addition of taxol, to induce the bundles of free MT, the preexisting IF coils disappear and IF associate with the MT. From these experiments we conclude that an interaction between MT and IF exists independent of the normal organisation of the MT system. The redistribution of IF always follows the redistribution of MT. The data show that MT determine the spatial distribution of IF which most probably involves some kind of physicochemical link.

Alkaloids↗
PubMed1983

Ultrastructural localization of tubulin and actin in polyethylene glycol-embedded rat seminiferous epithelium by immunogold staining.

The polyethylene glycol (PEG) method for immunofluorescence localization of cytoskeletal antigens has been extended to the ultrastructural level using glutaraldehyde-fixed tissues and immunogold staining. Semithin sections of fixed tissue embedded in polyethylene glycol are divested of the PEG, exposed to purified antibodies (e.g., antiactin, antitubulin) and anti-IgG-colloidal gold. The sections may be processed by dehydration and critical-point drying, or reembedment in hydrophilic substances. Tubulin is demonstrated in the mitotic spindles of dividing spermatogonia, manchettes, axonemes and centrioles of developing spermatids, and in the Sertoli cell cytoplasm; actin localization is demonstrated in the myoid cells of the tunica propria, and smooth muscle cells of arterioles in the interstitial tissue. The results demonstrate the applicability and versatility of PEG embedding for immunocytochemistry.

Actins↗
PubMed1983

Immunogold staining method for the light microscopic detection of leukocyte cell surface antigens with monoclonal antibodies: its application to the enumeration of lymphocyte subpopulations.

Colloidal gold was used as a marker for the light microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies. Suspensions of peripheral blood leukocytes were first incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. The cells were fixed and cytocentrifuge preparations or smears were made. Granulocytes and monocytes were then labeled by the cytochemical staining of their endogenous peroxidase activity. Lymphocytes reacting with the monoclonal antibody had numerous dark granules around the surface membrane. With electron microscopy, these granules appeared as patches of gold particles. This immunogold staining method proved to be a reliable tool for the enumeration of T-lymphocyte subpopulations in peripheral blood. The results were almost identical to those obtained with immunofluorescence microscopy. The procedure can also be applied on small volumes of capillary blood. This constitutes a good microtechnique for the determination of lymphocyte subsets in children.

Adult↗
PubMed1983

Cytochemical profile of immunoregulatory T-lymphocyte subsets defined by monoclonal antibodies.

Immunogold staining in combination with enzyme cytochemistry was used to determine the cytochemical profile of the immunoregulatory T-lymphocyte subpopulations defined by the monoclonal antibodies OKT3, OKT4, OKT8, and OKM1 in normal peripheral blood. Leukocyte suspensions were first incubated with the monoclonal mouse antibodies and then with colloidal gold-labeled goat antimouse antibodies. The cells were fixed and cytocentrifuge preparations were made. Cytochemical reactions for the detection of peroxidase, acid alpha-naphthyl acetate esterase, acid phosphatase, and beta-glucuronidase were performed on these preparations. Under light microscopy, lymphocytes reacting with the monoclonal antibodies had numerous dark granules around their surface membrane. In the cytoplasm the intracellular enzymatic activities were stained. The T-lymphocytes were characterized by a dot-like activity for the three enzymes. No significant difference could be found between the cytochemical profile of the T-helper (OKT4 positive) and T-cytotoxic suppressor cell populations (OKT8 positive). A few cells with lymphocyte morphology reacted with the OKM1 monoclonal antibody. Their cytochemical characteristics were different from those of mature T-cells (OKT3 population) or mature monocytes. From the comparison of their cytochemical characteristics, we can conclude that there is little correlation between the immunoregulatory T-lymphocyte subsets defined by these monoclonal antibodies and those defined by Fc receptors.

Acid Phosphatase↗
PubMed1983

Double immunogold staining method for the simultaneous ultrastructural localization of regulatory peptides.

Recent studies have suggested that the morphological characteristics of secretory granules contained within endocrine cells and nerves may be determined largely by their chemical composition. The use of the immunogold staining (IGS) method, which is based on the adsorption of colloidal gold to immunoglobulins, has been used in our laboratory to demonstrate a wide range of intracellular antigens at both the light and electron microscope levels. In this study we have applied a modification of the IGS method for the simultaneous detection of two separate antigens in a single tissue section, using a variety of region-specific antisera to different peptides. Peptide antisera were raised in rabbits or in guinea pigs and these were applied simultaneously or sequentially to grid-mounted ultrathin tissue sections. Antigenic sites were visualized at the electron microscope level using antisera raised in goats, adsorbed to gold particles of 12, 20, or 40 nm. Using this technique we have attempted to investigate the coexistence of multiple antigens in single tissue sections, in particular in single granules; the topographic distribution of molecular forms within one single granule or granule population; the heterogeneity of peptidergic neurons and also the heterogeneity of peptide content in morphologically similar granules. The double immunogold staining procedures described here have proved to be extremely effective for the simultaneous ultrastructural localization of two antigens (peptide-peptide; peptide-propeptide) on a single tissue section. The further development of this technique may provide useful information on neuroendocrine cell dynamics in normal and diseased states.

Animals↗
PubMed1983

A homologous radioimmunoassay for canine prolactin: plasma levels during the reproductive cycle.

A method is described for the purification of canine prolactin, involving preparative isoelectrofocusing. Canine prolactin has a molecular weight of 23 000 daltons, an isoelectric point of 5.7 and exhibits a high degree of homogeneity in polyacrylamide gels stained by means of a silver method. A specific, homologous radioimmunoassay is described using the Bolton-Hunter method for preparation of the labelled ligand, with a sensitivity of 0.1 ng/tube. Basal plasma prolactin levels of 2-4 ng/ml obtained through the oestrous cycle remained fairly constant but a rise of 9 ng/ml was found at the end of dioestrus in non-pregnant bitches. Level also rose 30 days after mating to reach a peak of about 50 ng/ml near parturition and during early lactation.

Animals↗
PubMed1983

Ultrastructural localization of four different neuropeptides within separate populations of p-type nerves in the guinea pig colon.

The ultrastructural localization of four neuropeptides, substance P, vasoactive intestinal polypeptide, Met-enkephalin, and somatostatin, in the guinea pig colon was investigated using electron immunocytochemistry. Each peptide was localized to the large granular vesicles in separate subpopulations of p-type nerves. These nerves could often be distinguished by the size and appearance of their immunostained granular vesicle cores. Thus, vasoactive intestinal polypeptide- and somatostatin-immunoreactive vesicles contained cores that were significantly larger (p less than 0.005) than those of substance P- or Met-enkephalin-positive vesicles. In addition, vasoactive intestinal polypeptide-immunoreactive vesicle cores were less well defined and more variable in shape than those of somatostatin-positive vesicles. Substance P- and Met-enkephalin-immunoreactive vesicle cores, however, were very similar in size (p greater than 0.05) and appearance and could only be differentiated using immunocytochemistry. This study demonstrates that the four neuropeptides under investigation are present within separate, often ultrastructurally distinct, neuronal systems in the gut. The distribution of these nerves within the colon is also described.

Animals↗