Biomedical subjects

D D Vandré

Publications and source records attributed to D D Vandré.

At least 19 recordsLinked to original sources

PubMed2001

Antigen retrieval in cells and tissues: enhancement with sodium dodecyl sulfate.

Immunocytochemistry provides important information on the localization of antigens in cells and tissues. However, the procedures used to prepare cells and tissues for immunocytochemical labeling may have deleterious effects on the results achieved. That is, the antigen of interest may be difficult or impossible to detect following labeling. These sorts of observations have led to the concept of antigen masking in which the antigen (or specific epitope) is hidden from antibodies specific for that antigen (or epitope). Various procedures to circumvent this problem have been developed. These different procedures generally fit under the term "antigen retrieval" (or epitope retrieval). The practice of antigen retrieval is widely employed with paraffin-embedded material. Antigen retrieval is less often applied to cells and tissues that are not embedded in paraffin. However, in the latter preparations there are situations in which the observed immunolabeling achieved falls short of expectations. This poor level of immunolabeling may, in some situations, be improved upon with antigen retrieval procedures. In this review, we describe experimental situations in which immunolabeling fell short of expectations. We also describe a procedure that has been useful in enhancing immunolabeling efficiency in these cases. The major feature of this procedure is the incorporation of a permeabilization/denaturation step using sodium dodecyl sulfate. This postfixation and prelabeling step dramatically improves immunolabeling for a number of antigens in both cells and cryosections of tissue.

Antigens↗

PubMed2000

Cell cycle-dependent phosphorylation of centrosomes: localization of phosphopeptide specific antibodies to the centrosome.

The microtubule nucleation capacity of the centrosome increases dramatically as cells progress from interphase into mitosis. The increase in nucleation capacity of the centrosome correlates with the cell cycle-dependent localization of the mitotic protein monoclonal-2 (MPM-2) phosphoepitope-specific antibody to the mitotic centrosome. Therefore, the phosphorylation state of centrosomal components may regulate the microtubule nucleation capacity of this organelle during mitosis. Neither the identity of the MPM-2 kinase(s) nor all of the MPM-2-reactive phosphoproteins associated with the centrosome have been fully elucidated. Only recently have the characteristics of the MPM-2 epitope site been defined, and we used this information to prepare polyclonal antibodies against synthetic phosphopeptides containing potential MPM-2 epitopes derived from the sequences of two MPM-2-reactive proteins, topoisomerase II, and microtubule associated protein 1B (MAP1B). We demonstrate that these phosphopeptide-specific antibodies also localize to the centrosome in a cell cycle-dependent fashion. Thus, polyclonal antibodies have been generated against defined phosphopeptides that reiterate many of the immunofluorescence staining properties exhibited by the MPM-2 antibody. These new phosphopeptide-specific antibodies will provide additional probes to examine the phosphorylation of centrosomal components and the functional consequences of their phosphorylation during mitosis.

Amino Acid Sequence↗

PubMed2000

Differential spindle assembly checkpoint response in human lung adenocarcinoma cells.

CI-980 is an antimicrotubule agent that binds the colchicine site on tubulin. We examined CI-980 cytotoxicity in two lung adenocarcinoma cell lines, A549 and A427. Depolymerization of microtubules following CI-980 treatment resulted in a mitotic arrest in the A549 population, but not in the A427 population. Similar responses were obtained following treatment with Taxol and nocodazole. Drug-treated A427 cells exited mitosis, generating a population dominated by multinucleated cells, while both multinucleated and apoptotic cells were present in the A549 population after extended drug treatment. CI-980-induced microtubule depolymerization was only partially reversible. However, regrowth of some microtubules in mitotic A549 cells following drug washout resulted in multinucleation of the population in the absence of apoptosis. These results show that A427 cells have a defective spindle assembly checkpoint. Levels of the MAD2 and BUB1 checkpoint proteins were similar in both A549 and A427 cells, suggesting that the checkpoint defect in the A427 cells is downstream of these proteins. In addition, induction of apoptosis in response to CI-980 correlates with the presence of a functional mitotic checkpoint and the extent of microtubule depolymerization.

Adenocarcinoma↗

PubMed2000

Applications of gold cluster compounds in immunocytochemistry and correlative microscopy: comparison with colloidal gold.

In this review, we discuss the immunocytochemical literature with respect to a comparison between conventional colloidal gold and gold cluster compounds as immunoprobes. The relative advantages and disadvantages of each of these types of particle for immunocytochemical applications are discussed. We present results from our own laboratories and those of others on the comparison of these immunoprobes in selected experimental situations. These results show the use of gold cluster compounds at both light and electron microscope levels. At the ultrastructural level, gold cluster compounds have been used in pre-embedding labelling of cultured cells, and for labelling of ultrathin cryosections and freeze-fracture preparations. Recently, fluorescently tagged gold cluster compounds have become available. Using ultrathin cryosections of human neutrophils as a model system, we demonstrate that a single immunoprobe (i.e. a fluorescently tagged gold cluster compound) is a robust probe for correlative fluorescence and electron microscopy.

Gold Colloid↗

PubMed2000

Enhanced labeling efficiency using ultrasmall immunogold probes: immunocytochemistry.

Detection of antigen-antibody interactions in immunocytochemistry relies on a reporter system. The most commonly employed reporter systems used are fluorochromes, enzymes, and particulate probes. This article considers the advantages and disadvantages associated with ultrasmall immunogold particles as the reporter system in immunocytochemical applications.

Gold↗

PubMed1998

Ultrasmall immunogold particles: important probes for immunocytochemistry.

In this article, we review the immunocytochemical literature with respect to a comparison between conventional colloidal gold and ultrasmall gold particles as immunoprobes. We discuss the relative advantages and disadvantages of each of these types of particles for immunocytochemical applications. We present results from our own laboratories, in which we compared these immunoprobes in selected experimental situations. In addition, we discuss our work on the use of a fluorescently labeled ultrasmall immunoprobe for correlative microscopy.

Fluorescent Dyes↗

PubMed1997

Partial characterization of the MPM-2 phosphoepitope.

The MPM-2 monoclonal antibody recognizes a distinctive group of proteins that are associated with structural components of the mitotic apparatus. These proteins become phosphorylated and MPM-2 reactive during M-phase and appear to be required for both the onset and completion of M-phase. Based upon the analysis of reported MPM-2 reactive sequences, we have developed a model for the essential elements that comprise the MPM-2 epitope. This model was tested by employing a series of synthetic phosphopeptides. We show here that a 14 amino acid synthetic phosphopeptide, derived from a potential MPM-2 site on human DNA topoisomerase II, is recognized by the MPM-2 antibody. This phosphopeptide was sufficient to compete for MPM-2 antibody recognition of (1) an isolated native mitotic MPM-2 antigen on dot blots, (2) proteins on immunoblots of mitotic cell lysates, and (3) specific immunostaining of mitotic cells. These results indicated that the topoisomerase peptide contained all of the essential elements of the MPM-2 epitope. By substituting selected amino acids with alanine, we were able to examine the contribution of different amino acids to the binding between the MPM-2 antibody and the epitope. Changing the amino acid that was adjacent to the phosphorylated threonine residue on the C-terminal side (the +1 position) had no effect on MPM-2 antibody binding. However, substitution of aromatic amino acids at either the -2 or +2 positions reduced antibody recognition. The aromatic amino acid at the -2 position appeared to be the most critical residue of those tested that influenced antibody binding. These results provide information required for the molecular definition of the MPM-2 epitope and should aid in the identification of potential MPM-2 reactive sites on other mitotic phosphoproteins.

Amino Acid Sequence↗

PubMed1997

Efficient immunocytochemical labeling of leukocyte microtubules with FluoroNanogold: an important tool for correlative microscopy.

We tested the immunoprobe FluoroNanogold (FNG) for its utility as an immunocytochemical labeling reagent. This immunoprobe consists of a 1.4-nm gold particle to which a specific Fab' fragment and a fluorochrome are conjugated. We employed the microtubules (MTs) of human phagocytic leukocytes as a model system for testing the usefulness of FNG as a secondary antibody for immunocytochemistry. We show that these fluorescently labeled ultrasmall immunogold particles are very efficient for labeling MTs in these cells. The signal from FNG can be detected directly by fluorescence microscopy or indirectly by other modes of optical microscopy and electron microscopy, after silver-enhancement of the gold. The spatial resolution of immunolabeled MTs obtained with FNG and silver enhancement was comparable to that of conventional immunofluorescence detection. Colloidal gold (5-nm and 10-nm in diameter), on the other hand, failed to label MTs in cells prepared in a similar manner. This difference in labeling was due in large part to greater penetration of 1.4-nm gold into aldehyde-fixed cells than either 5-nm or 10-nm gold particles. The fluorescent 1.4-nm immunoprobe was shown to be an important new tool for general use in correlative microscopy.

Animals↗

PubMed1996

High molecular weight microtubule-associated proteins contain O-linked-N-acetylglucosamine.

We have examined the post-translational modification of high molecular weight microtubule-associated proteins (MAPs) have shown that MAP1, MAP2, and MAP4 are glycosylated. The presence of carbohydrate residues on these proteins was indicated by labeling with biotin hydrazide following periodate oxidation, a specific and well established method for detecting saccharide moieties on proteins. Both MAP2 and MAP4 were also labeled in vitro by UDP-[3H]galactose in the presence of galactosyltransferase. Labeling by galactosyltransferase indicated that MAP2 and MAP4 contained terminal nonreducing GlcNAc residues, and they appeared to be O-linked to the proteins as shown by their sensitivity to beta-elimination. Chromatographic analysis showed that the GlcNAc residues were directly linked to the proteins as monosaccharides. Thus, we have added MAP2 and MAP4 to the list of intracellular O-GlcNAc-modified proteins, which includes other cytoskeletal proteins such as cytokeratins 8, 13, and 18 and neurofilament proteins NF-L and NF-M. We further characterized the O-GlcNAc modification of MAP2, and stoichiometric analysis indicated that nearly 10% of the MAP2 isolated from rat brain is modified by O-GlcNAc. However, this estimate is thought to reflect the minimal level of O-GlcNAc modification present on MAP2. We have also shown that both the O-GlcNAc and biotin hydrazide-reactive carbohydrate moieties are located on the projection domain of MAP2. Three O-GlcNAc-containing peaks were observed following fast protein liquid chromatography of a tryptic digest of MAP2, suggesting that multiple modification sites exist. The specific modification sites and functional significance of the O-GlcNAc glycosylation on the high Mr MAPs remain to be determined.

Acetylglucosamine↗

PubMed1996

Alzheimer's disease neurofibrillary tangles contain mitosis-specific phosphoepitopes.

Paired helical filaments (PHFs) are the major components of neurofibrillary lesions present in Alzheimer's disease (AD). PHFs are composed of the microtubule-associated protein (MAP) tau, which is abnormally phosphorylated in AD. Normal fetal tau is also phosphorylated and shares certain phosphoepitopes with PHF-tau. The abnormal phosphorylation of PHF-tau is considered to be involved in the formation of PHFs and subsequent degeneration of AD neurons. We have previously shown that other neuronal MAPs, such as MAP1B, contain mitosis-specific phosphoepitopes. In addition to mitotic cells, these epitopes are also expressed in fetal brain and PC12 cells during differentiation and neurite outgrowth. One hypothesis regarding the etiology of AD involves the reactivation of a fetal-like state and mitotic conditions in selected neurons. To determine if similar mitosis-associated phosphoepitopes appeared in AD, sections of hippocampal tissue were stained for immunoreactivity with antibodies recognizing both tau and mitotic phosphoepitopes. Both the MPM2 mitotic phosphoepitope antibody and the AT8 PHF-tau antibody stained neurofibrillary lesions and colocalized to pyramidal neurons in AD samples. In addition, PHFs isolated from an AD brain reacted with both antibodies. The MPM2 antibody specifically reacted with tau in the isolated PHF fraction but not normal adult tau. In addition, MPM2 failed to react with normal fetal or adult tau obtained from rat brains. The MPM2 antibody also recognized human MAP1B; however, MAP1B was not present in the PHF fraction. Our results indicate that MPM2 recognized a phosphoepitope present on PHF-tau. Because normal fetal or adult rat brain tau did not express the MPM2 epitope, it is likely that this phosphoepitope is specific for the disease state.

Adult↗

PubMed1995

Stimulus-dependent alterations in macrophage microtubules: increased tubulin polymerization and detyrosination.

Murine macrophage microtubules are very dynamic. The majority of the microtubules (approximately 80%) exist in a rapidly depolymerizing pool (t1/2 approximately 30 seconds). The remaining 20% of the microtubules are in a more slowly depolymerizing pool (t1/2 approximately 7 minutes). Macrophage microtubules are responsive to cell stimulation with phorbol esters; upon cell stimulation there is a rapid increase in total microtubule polymer and number. In addition there is stimulus-induced detyrosination of alpha-tubulin in macrophage microtubules that is rapid and essentially complete, occurring in all microtubules rather than a subset of microtubules. Detyrosination of the macrophage microtubules in response to phorbol esters does not confer increased stability to these microtubules, since treated cells have nocodazole-induced depolymerization kinetics similar to that in non-stimulated macrophages. Regrowth of microtubules following washout of nocodazole is also rapid. Interestingly the regrown microtubules are initially in the tyrosinated form even in the presence of phorbol ester. These experiments provide in vivo support for the model that detyrosination of alpha-tubulin occurs in polymeric tubulin while retyrosination occurs in dimeric tubulin. Macrophage microtubules also demonstrate an unusually rapid response to extracellular stimuli and thus provide a unique model system in which to examine signal transduction events and modulation of microtubules.

Animals↗

PubMed1995

The microtubule cytoskeleton in human phagocytic leukocytes is a highly dynamic structure.

The microtubule cytoskeleton of human leukocytes has been difficult to study, in part, due to the lack of a reliable protocol for the indirect immunofluorescence staining of microtubules in these cells. We report here the development of a simple and reliable immunocytochemical labeling protocol for the examination of microtubules in leukocytes including monocytes, neutrophils, and eosinophils. The dynamic properties of microtubules in both monocytes and neutrophils were examined by indirect immunofluorescence staining of cells following exposure to nocodazole. Nocodazole-induced depolymerization is extremely rapid in both cell types, as is the regrowth of microtubules following removal of the nocodazole. Rapid reorganization of the microtubule cytoskeleton was also observed in neutrophils undergoing chemotactic stimulation. Bundling of microtubules was observed in both monocytes and neutrophils isolated from patients undergoing taxol infusion chemotherapy. The taxol-induced bundles were transient in nature as they were absent from samples collected 48 h following the completion of the taxol infusion. These results demonstrate the unique dynamic properties of leukocyte microtubules and indicate that they can be altered in vivo. The development of this staining protocol should allow for the further analysis of leukocyte microtubules as related to the normal functional response of these cells and form the basis for correlating alterations in microtubule dynamics with the effects of taxol on leukocyte function.

Drug-Related Side Effects and Adverse Reactions↗

PubMed1992

Immunoelectron microscopic localization of phosphoproteins associated with the mitotic spindle.

We examined the immunogold staining of microtubules and microtubule organizing centers using an improved silver-enhancement reagent for small (1-1.4 nm) gold-conjugated secondary antibodies. First, the staining properties of different commercial preparations of gold-labeled antibodies were compared for sample penetration, label uniformity, and labeling density, and Nanogold 1.4-nm gold-conjugated F(ab') was found to be superior to the other probes examined. However, in samples examined for the localization of alpha- and beta-tubulin, gold staining did not extend through the pericentriolar material nor were the centrioles labeled. This apparent lack of centrosomal staining was not due to problems associated with penetration of the antibody probes, since staining adjacent to and within the centriolar cylinder was observed when phosphoprotein antigens recognized by the MPM-2 antibody were localized. The MPM-2 antibodies also localized to mitotic kinetochores, kinetochore fibers, and midbodies, in addition to mitotic centrosomes. The level of MPM-2 staining of the centrosome varied through the cell cycle. At interphase, this staining was restricted within the centriolar cylinder, whereas in mitotic cells extensive staining throughout the pericentriolar material was also observed. These results established the close relationship of MPM-2-reactive phosphoproteins with the centrosome, and suggest that this technique may be useful for ultrastructural localization of other cytoskeletal proteins.

Animals↗

PubMed1992

Silver enhancement of gold antibody probes in pre-embedding electron microscopic immunocytochemistry.

In pre-embedding EM immunocytochemistry with gold probes, the gold must be small enough to penetrate through cell membranes treated with mild detergents. Antibodies labeled with small gold probes (1-1.4 nm) are too small to be resolved in thin sections but can be seen if they are silver-enhanced after the gold has bound to the antigens in the cells. We investigated several aspects of gum arabic-silver lactate-hydroquinone enhancement solution (Danscher solution) by examining gold-conjugated antibodies embedded in agar, sectioned on a vibrotome, and enhanced with different solutions. The rate of silver enhancement was optimized in 50% gum arabic and 200 mM HEPES buffer, pH 5.8. We also examined chemicals used as developers and found that N-propyl gallate (NPG) gave a more uniform development than the routinely used hydroquinone (HQ). The diameter of the silver-enhanced particles after incubation in osmium tetratoxide (OSO4) decreased somewhat with longer incubation time and higher percentages, but the density (number per unit area) of silver-enhanced particles was little changed. The loss of silver-enhanced particle diameter was reduced by lowering the concentration of OSO4 to 0.1%. Comparison of commercial small gold probes showed that NPG enhancement of Nanogold gave more uniform particle size and a better correlation between enhancement time and particle density. When this procedure was applied to cell cultures with monoclonal antibodies, the silver-enhanced particles were similar to those in the agar sections. When free-floating tissue sections were used, longer silver enhancement times were needed to obtain similarly sized particles. This new NPG-silver-enhancement procedure offers a reliable and easy method to localize proteins in cultured cells and tissue sections by pre-embedding electron microscopic immunocytochemistry.

Animals↗

PubMed1992

Inhibition of mitosis by okadaic acid: possible involvement of a protein phosphatase 2A in the transition from metaphase to anaphase.

The effects of the protein phosphatase inhibitor okadaic acid were examined using the pig kidney cell line LLC-PK. At relatively low concentrations of the inhibitor (8-40 nM), cells became blocked in a metaphase-like mitotic state beginning 6-8 h after initial treatment. Spindle microtubules were present throughout the period of the mitotic block, but were not stabilized since they remained sensitive to nocodazole depolymerization. With increasing length of the mitotic block chromosome alignment at the metaphase plate was disrupted and multipolar spindles developed. Cells continued to accumulate in mitosis for at least 24 h, indicating that at these low concentrations okadaic acid was not cytotoxic, but rather acted as a cytostatic agent. Upon release of the okadaic acid block, mitotic LLC-PK cells recovered and completed anaphase. After extended periods of treatment some cells were able to escape the okadaic acid-induced mitotic block. These cells were multinucleate and had undergone cytokinesis in the absence of chromosome segregation. At higher concentrations of okadaic acid (0.5-1.0 microM), mitosis was blocked within 30-60 min of treatment. However, within 90-120 min treated cells rounded up and detached from the monolayer, regardless of whether they were in interphase or mitosis. Cytoplasmic microtubules were depolymerized in the detached cells, and these cells could not recover from the cytotoxic effects of such high concentrations of okadaic acid. Thus, differential effects of the phosphatase inhibitor could be demonstrated, depending upon the concentration of okadaic acid applied to the cultures. The okadaic acid-induced mitotic blockage was probably due to the inhibition of a type 2A protein phosphatase that is involved in the transition from metaphase to anaphase.

Anaphase↗

PubMed1992

Stimulus-dependent relocation of the microtubule organizing center in human polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNs) exhibit extensive directional migration (chemotaxis) and phagocytic activities. We have developed an in vitro model to evaluate the organization of the microtubule organizing center (MTOC) in PMNs as the latter interact with various substrata, including immobilized antigen-antibody complexes. PMNs were layered on poly-L-lysine substrata containing ferritin (PL+F) or ferritin-antiferritin complex (PL+F+AF) and the location of MTOCs was determined by indirect immunofluorescence of tubulin using conventional epifluorescence microscopy and confocal laser scanning microscopy. The MTOCs in the majority of the PMNs attached to PL+F occupied an apical location (81.29% +/- 3.34%), while in the majority of PMNs layered onto PL+F+AF, a basal location (79.37% +/- 5.26%) was observed. Following disruption of microtubules (MTs) by nocodazole before layering the cells on the substrata, the proportions of PMNs with apical MTOCs were 65.2% +/- 6.27% for PL+F and 47.2% +/- 4.1% for PL+F+AF substrata, while the proportions of PMNs with basal MTOCs were 26.11% +/- 8.89% for PL+F and 39.6% +/- 4.4 for PL+F+AF substrata. The results indicate that MTOCs in human PMNs in vitro (i) occupied a 'pre-defined' apical location; (ii) translocated to a 'newly defined' basal location upon stimulation with immobilized antigen-antibody complex; (iii) and depended on intact MTs for placement of MTOCs in both situations.

Antigen-Antibody Complex↗

PubMed1991

Proteins of the mammalian mitotic spindle: phosphorylation/dephosphorylation of MAP-4 during mitosis.

The phosphoprotein composition of isolated CHO spindles was analyzed using the MPM-1 and MPM-2 antibodies, which are reactive with a phosphorylated epitope enriched in mitotic cells and present on the centrosome, kinetochores, midbody and fibers of the mitotic spindle. Several high molecular weight phosphorylated spindle proteins were detected on immunoblots, including species of 410 x 10(3) Mr, 350 x 10(3) Mr, a 230-240 X 10(3) Mr doublet, 210 x 10(3) Mr and 120 x 10(3) Mr. The temporal and spatial distribution of the MPM-reactive phosphoproteins was determined by examining spindle structures isolated from cells at various stages of mitosis. The susceptibility of the staining pattern to extraction with salt, a procedure known to remove most microtubule-associated proteins (MAPs), was also examined. The phosphorylated 210 x 10(3) Mr species was identified as MAP-4 and localized to the spindle fibers using (1) a polyclonal antibody raised against this species, that reacted with known MAPs, and (2) established MAP-4 antibodies that reacted with the spindle 210 x 10(3) Mr MPM-reactive proteins. The comparative immunoblot and immunofluorescence analysis establishes a cycle of phosphorylation/dephosphorylation of MAP-4 upon entry and exit from mitosis. Regarding the other MPM-reactive proteins, comparative immunofluorescence staining and immunoblot analysis of isolated spindle samples before and after salt extraction indicate that they may be constituents of the centrosome, kinetochores or midbody, but their definitive identification awaits the production of monospecific antibodies.

Animals↗

PubMed1989

Anaphase onset and dephosphorylation of mitotic phosphoproteins occur concomitantly.

The cyclical phosphorylation and dephosphorylation of the centrosome during mitosis was analyzed by immunofluorescence methods using the MPM-2 antibody, which reacts with a subset of mitotic phosphoproteins. Quantification of MPM-reactivity indicated that centrosomal phosphorylation attained a maximal level just prior to anaphase onset. This level was maintained in metaphase cells blocked from further mitotic progression with the microtubule depolymerizing agent nocodazole. However, when nocodazole was added to cells that had just initiated anaphase, the level of centrosomal phosphorylation decreased rapidly as in untreated anaphase cells. We conclude that the onset of dephosphorylation of the centrosome coincided with the onset of anaphase and continued in the absence of chromosome movement. Dephosphorylation of MPM-2 reactive phosphoproteins may be taken as a biochemical indicator of anaphase onset.

Anaphase↗