Dale D. Vandré

Alzheimer's Disease Neurofibrillary Tangles Contain Mitosis-Specific Phosphoepitopes

Abstract: Paired helical filaments (PHFs) are the major components of neurofibrillary lesions present in Alzheimers disease (AD). PHFs are composed of the microtubule‐associated protein (MAP) τ, which is abnormally phosphorylated in AD. Normal fetal τ is also phosphorylated and shares certain phosphoepitopes with PHF‐τ. The abnormal phosphorylation of PHF‐τ 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 τ and mitotic phosphoepitopes. Both the MPM2 mitotic phosphoepitope antibody and the AT8 PHF‐τ 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 τ in the isolated PHF fraction but not normal adult τ. In addition, MPM2 failed to react with normal fetal or adult τ 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‐τ. Because normal fetal or adult rat brain τ did not express the MPM2 epitope, it is likely that this phosphoepitope is specific for the disease state.

2-Methoxymethylestradiol: a new 2-methoxy estrogen analog that exhibits antiproliferative activity and alters tubulin dynamics.

An estradiol metabolite, 2-methoxyestradiol (2-MeOE(2)), has shown antiproliferative effects in both hormone-dependent and hormone-independent breast cancer cells. Previously, a series of 2-hydroxyalkyl estradiol analogs had been synthesized in our laboratories as potential probes for comparison of estrogen receptor (ER)-mediated versus non-ER-mediated effects in breast cancer cells. A methoxy derivative of 2-hydroxymethyl estradiol was prepared for biological evaluation and comparison with 2-MeOE(2). Estrogenic activity of the synthetic analogs was evaluated in two ways, one by examining affinity of the analogs for the estrogen receptor in MCF-7 cells and the other by examining the ability of the analogs to induce estrogen-responsive gene expression. The analog, 2-methoxymethyl estradiol (2-MeOMeE(2)), demonstrated weak affinity for the estrogen receptor (0.9% of estradiol) and weak ability to stimulate estrogen-induced expression of the pS2 gene (0.02% of estradiol). Antitumor activity was evaluated both in vitro and in vivo. The steroidal nucleus seems to be an attractive target for developing novel tubulin polymerization inhibitors. Additionally, such steroidal compounds may have low toxicity compared to the natural products known to interact with tubulin. Interestingly, 2-MeOMeE(2) inhibited tubulin polymerization in vitro at concentrations of 1 and 3 microM and was more effective than 2-MeOE(2). In cells, 2-MeOMeE(2) was effective in suppressing growth and inducing cytotoxicity in MCF-7 and MDA-MB-231 breast cancer cells. The cytotoxic effects of 2-MeOMeE(2) are associated with alterations in tubulin dynamics, with the frequent appearance of misaligned chromosomes, a significant mitotic delay, and the formation of multinucleated cells. In comparison, 2-MeOE(2) was more effective than 2-MeOMeE(2) in producing cytotoxicity and altering tubulin dynamics in intact cells. Assessment of in vivo antitumor activity was performed in athymic mice containing human breast tumor xenografts. Nude mice bearing MDA-MB-435 tumor xenografts were treated i.p. with 50 mg/kg per day of 2-MeOMeE(2) or vehicle control for 45 days. Treatment with 2-MeOMeE(2) resulted in an approximate 50% reduction in mean tumor volume at treatment day 45 when compared to control animals and had no effect on animal weight. Thus, 2-MeOMeE(2) is an estrogen analog with minimal estrogenic properties that demonstrates antiproliferative effects both in vitro and in the human xenograft animal model of human breast cancer.

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) and 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-[H]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 β-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 M MAPs remain to be determined.

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.

Inhibition of Heat Shock Protein 90, a Novel RET/PTC1-associated Protein, Increases Radioiodide Accumulation in Thyroid Cells

RET/PTC1 is a rearranged form of the RET tyrosine kinase commonly seen in papillary thyroid carcinomas. It has been shown that RET/PTC1 decreases expression of the sodium/iodide symporter (NIS), the molecule that mediates radioiodide therapy for thyroid cancer. Using proteomic analysis, we identify hsp90 and its co-chaperone p50cdc37 as novel proteins associated with RET/PTC1. Inhibition of hsp90 function with 17-allylamino-17-demothoxygeldanamycin (17-AAG) reduces RET/PTC1 protein levels. Furthermore, 17-AAG increases radioiodide accumulation in thyroid cells, mediated in part through a protein kinase A-independent mechanism. We show that 17-AAG does not increase the total amount of NIS protein or cell surface NIS localization. Instead, 17-AAG increases radioiodide accumulation by decreasing iodide efflux. Finally, the ability of 17-AAG to increase radioiodide accumulation is not restricted to thyroid cells expressing RET/PTC1. These findings suggest that 17-AAG may be useful as a chemotherapeutic agent, not only to inhibit proliferation but also to increase the efficacy of radioiodide therapy in patients with thyroid cancer.

Nuclear Factor-Kappa B Regulates Inducible Prostaglandin E Synthase Expression in Human Amnion Mesenchymal Cells

Abstract The human amnion is a major intrauterine source of prostaglandin (PG) E2, a potent mediator of uterine contractions and cervical ripening. During parturition, inflammatory cytokines promote PGE2 production through increased prostaglandin-endoperoxide synthase-2 (PTGS2, also known as cyclooxygenase-2) expression. This is mediated, in part, through activation of the transcription factor nuclear factor kappa B (NFkappaB). Prostaglandin E synthase (PTGES, also known as microsomal PGE synthase-1) acts downstream of PTGS2 and is inducibly expressed in most systems. We hypothesized that NFkappaB might regulate cytokine-induced PTGES expression in amnion cells. With amnion mesenchymal cells, we found that proinflammatory cytokines coordinately upregulated PTGS2 and PTGES mRNA expression. In parallel, increased expression of the PTGS2 and PTGES proteins was observed. In comparison, the expression of two other PGE synthases (PTGES2 and PTGES3) was unmodified. PTGES induction was blocked both in the presence of pharmacological NFkappaB inhibitors and following adenovirus-mediated overexpression of a dominant-negative NFkappaB pathway protein. In cells transiently transfected with a luciferase reporter bearing a portion (−597/+33) of the human PTGES gene promoter, interleukin-1beta (IL1B) produced a moderate increase in luciferase activity; this effect was abrogated in the presence of an indirect NFkappaB inhibitor (MG-132). Finally, a kappaB-like regulatory element was identified that, when mutated, markedly attenuated IL1B-responsive PTGES promoter activity. In conclusion, our results support a role for NFkappaB in cytokine-induced PTGES expression in amnion mesenchymal cells in vitro. By coordinately regulating PTGS2 and PTGES, NFkappaB may contribute to an inducible PGE2 biosynthesis pathway during human parturition.

Casein kinase-1 isoforms differentially associate with neurofibrillary and granulovacuolar degeneration lesions

Alzheimer’s Disease (AD) is characterized by the appearance of neurofibrillary and granulovacuolar lesions in the brains of affected individuals. The former is composed of hyperphosphorylated aggregates of the microtubule-associated protein tau. The latter is poorly characterized but reacts strongly with anti-phosphoepitope antibodies indicating that it too accumulates phosphoproteins. Both lesions react strongly with antibodies directed against members of the casein kinase-1 family of phosphotransferases, a group of closely related protein kinases that frequently function in tandem with the ubiquitin modification system. To determine whether individual members of the casein kinase-1 family differentially associate with AD lesions, hippocampal sections isolated from late stage cases of AD were subjected to double-label fluorescence immunohistochemistry using a panel of selective anti-casein kinase 1 antibodies and small-molecule fluorochromes thioflavin S and thiazin red. The resultant colocalization patterns revealed that the alpha CK1 isoform strongly correlated with thioflavin S and thiazin red fluorescence, indicating that it preferentially associated with neurofibrillary lesions. In contrast, the delta isoform staining pattern was dominated by colocalization with granulovacuolar degeneration bodies. These findings suggest that granulovacuolar and neurofibrillary lesions occupy separate populations of neurons, and implicate CK1 isoforms in the generation of lesion-associated phosphoepitopes. They also suggest a nexus between the phosphorylation and ubiquitination modifications found in both lesions.

Dysferlin Is Expressed in Human Placenta But Does Not Associate with Caveolin

Abstract A proteomics screen of human placental microvillous syncytiotrophoblasts (STBs) revealed the expression of dysferlin (DYSF), a plasma membrane repair protein associated with certain muscular dystrophies. This was unexpected given that previous studies of DYSF have been restricted to skeletal muscle. Within the placenta, DYSF localized to the STB and, with the exception of variable labeling in the fetal placental endothelium, none of the other cell types expressed detectable levels of DYSF. Such restricted expression was recapitulated using primary trophoblast cell cultures, because the syncytia expressed DYSF, but not the prefusion mononuclear cells. The apical plasma membrane of the STB contained ∼4-fold more DYSF than the basal membrane, suggesting polarized trafficking. Unlike skeletal muscle, DYSF in the STB is localized to the plasma membrane in the absence of caveolin. DYSF expression in the STB was developmentally regulated, because first-trimester placentas expressed ∼3-fold more DYSF than term placentas. As the current literature indicates that few cell types express DYSF, it is of interest that the two major syncytial structures in the human body, skeletal muscle and the STB, express this protein.

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.

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. Microsc. Res. Tech. 42:13–23, 1998.