Identification of a new vertebrate nucleoporin, Nup188, with the use of a novel organelle trap assay.
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The study of the nuclear pore in vertebrates would benefit from a strategy to directly identify new nucleoporins and interactions between those nucleoporins. We have developed a novel two-step "organelle trap" assay involving affinity selection and in vitro pore assembly. In the first step, soluble proteins derived from Xenopus egg extracts are applied to a column containing a ligand of interest. The bound proteins are then tagged by biotinylation and eluted. In the second step, potential nucleoporins are selected for by virtue of their ability to assemble into annulate lamellae, a cytoplasmic mimic of nuclear pores. The incorporated proteins are then recognized by their biotin tag. Here we use the lectin wheat germ agglutinin (WGA) as ligand; WGA inhibits nuclear transport and has been shown to directly bind three known nucleoporins from Xenopus extract, Nup62, Nup98, and Nup214, all of which contain N-acetylglucosamine residues. Under reduced-stringency conditions, three additional proteins bind to WGA-Sepharose and are revealed by the organelle trap assay. We identified all three as partner nucleoporins. Two were discovered to be Xenopus Nup93 and Nup205. The third is a novel vertebrate nucleoporin, Nup188. This new vertebrate protein, Xenopus Nup188, exists in a complex with xNup93 and xNup205. The Nup93-Nup188-Nup205 complex does not bind directly to WGA but binds indirectly via the N-acetylglucosamine-modified nucleoporins. A gene encoding human Nup188 was also identified. The discovery of vertebrate Nup188, related to a yeast nucleoporin, and its novel protein-protein interactions illustrates the power of the two-step organelle trap assay and identifies new building blocks for constructing the nuclear pore. (+info)
Identification of perivitelline N-linked glycans as mediators of sperm-egg interaction in chickens.
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This study demonstrates that carbohydrates play an essential role in sperm-egg interactions in birds. Sperm-egg interaction was measured in vitro as the ability of spermatozoa to hydrolyse a small hole in the inner perivitelline layer, the equivalent of the mammalian zona pellucida. Preincubation with Triticum vulgaris lectin (WGA) and succinyl-WGA (S-WGA) at 10 microgram ml(-1) resulted in complete inhibition of sperm-egg interaction, whereas at the same concentration a range of other lectins (Canavalia ensiformis (Con A), Arachis hypogea (PNA), Ulex europaeus II (UEA II), Solanum tuberosum (STA), Tetragonolobus purpureas (LTA) and Pisum sativum (PSA)) were unable to inhibit sperm egg interaction significantly, although fluorescein-labelled derivatives of these lectins were found to stain the inner perivitelline layer. Significant inhibition of sperm-egg interaction was achieved by the addition of N-acetyl-D-glucosamine and fucoidin to the assay mixture; however, D-glucose, D-galactose, D-fucose and L-fucose had no significant effect on sperm-egg interaction. Pretreatment of the inner perivitelline layer with N-glycanase significantly reduced sperm-egg interaction, whereas treatment with O-glycanase had no effect. These results demonstrate that N-linked glycans play an essential role in sperm-egg interaction in chickens. (+info)
Epithelial polarity: the ins and outs of the fly epidermis.
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Epithelial cells must polarize and establish apical and basolateral membrane domains during development. Recent experiments have shed light on how apical-basal polarity is generated during cellularization in Drosophila, when around 6000 epithelial cells are created synchronously from a syncytium. (+info)
Lectin histochemistry as a predictor of dysplasia grade in colorectal adenomas.
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Lectins are sugar-binding proteins that bind to specific cellular carbohydrates, commonly affecting cellular physiology. Phaseolus vulgaris leucoagglutinin (PHA), ulex europaeus isoagglutinin-I (UEA-I), wheat germ agglutinin (WGA) and peanut agglutinin (PNA) are among the most well studied lectins in various tissues. The purpose of this study was to detect the above lectins binding sites and so examine alterations in glycoconjugate expression in neoplastic cells of 52 colorectal adenomas with various clinicopathologic characteristics and proliferation rates. Lectin histochemistry was performed in paraffin sections with and without neuraminidase treatment. Proliferative fraction was determined by immunolabelling for Proliferating Cell Nuclear Antigen. PHA was the more frequently positive lectin in the examined specimens; however, it was simultaneously detected in normal colonic mucosa and so was WGA. The frequency of high grade dysplasia was significantly greater in older patients and in samples with UEA-I positivity without neuraminidase pretreatment. UEA-I-reactive adenomas were generally characterized by high cell proliferation rates. A statistical model based on patients age and UEA-I binding without neuraminidase treatment can generally predict grade of dysplasia in 83% of adenomas and particularly high grade dysplasia in up to 93% of adenomas; so, such a model may be potentially useful for the early detection of neoplasia, for instance in exfoliative cells from the large intestine. (+info)
Cleavage of SNAP-25 by botulinum toxin type A requires receptor-mediated endocytosis, pH-dependent translocation, and zinc.
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Previously we reported that SNAP-25, synaptobrevin II, and syntaxin I, the intracellular substrates of botulinum toxin originally identified in nontarget tissues, were present in a recognized mammalian target tissue, the mouse hemidiaphragm. Furthermore, we reported that SNAP-25, syntaxin I, and synaptobrevin II were cleaved by incubation of the intact hemidiaphragm in botulinum serotypes A, C, and D, respectively. The objective of the current study was to use the mouse phrenic nerve-hemidiaphragm preparation and botulinum serotype A to investigate 1) the relationship of substrate cleavage to toxin-induced paralysis, and 2) the relevance of substrate cleavage to the mechanism of toxin action. Immunoblot examination of tissues paralyzed by botulinum toxin type A (10(-8) M) revealed < or =10% loss of SNAP-25 immunoreactivity at 1 h postparalysis, and > or =75% loss at 5 h postparalysis. Triticum vulgaris lectin, an agent that competitively antagonizes toxin binding, antagonized toxin-induced paralysis as well as SNAP-25 cleavage. Methylamine hydrochloride, an agent that prevents pH-dependent translocation, also antagonized toxin-induced paralysis and SNAP-25 cleavage. Furthermore, zinc chelation antagonized toxin-induced paralysis and SNAP-25 cleavage. These results demonstrate that cleavage of SNAP-25 by botulinum serotype A fulfills the requirements of the multistep model of botulinum toxin action that includes receptor-mediated endocytosis, pH-dependent translocation, and zinc-dependent proteolysis. Furthermore, the minimal amount of SNAP-25 cleavage at 1 h postparalysis suggests that inactivation of only a small but functionally important pool of SNAP-25 is necessary for paralysis. (+info)
Carbohydrate moieties of the interstitial and glandular tissues of the amphibian Pleurodeles waltl testis shown by lectin histochemistry.
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The amphibian testis is a useful model because of its zonal organisation in lobules, distributed along the cephalocaudal axis, each containing a unique germ cell type. Sperm empty lobules form the so-called glandular tissue at the posterior region of the gonad. Androgen production is limited to the cells of the interstitial tissue surrounding lobules with spermatozoa bundles and to the cells of the glandular tissue. In this work, we have studied the distribution of terminal carbohydrate moieties of N- and O-linked oligosaccharides in the interstitial and glandular tissue of the Pleurodeles waltl testis, by means of 14 lectins combined with chemical and enzymatic deglycosylation pretreatment. Some differences in glycan composition between the interstitial and the glandular tissue have been detected. Thus in both tissues, N-linked oligosaccharides contained mannose, Gal(beta1,4)GlcNAc, and Neu5Ac(alpha2,3)Gal(beta1,4)GlcNAc, while O-linked oligosaccharides contained Con A-positive mannose, Gal(beta1,3)GalNAc, Gal(beta1,4)GlcNAc, Neu5Ac(alpha2,3)Gal(beta1,4)GlcNAc, and WGA-positive GlcNAc. Fucose was also detected in both tissues. However, GlcNAc on N-linked oligosaccharides and GalNAc and Neu5Ac(alpha2,6)Gal/GalNAc on both N- and O-linked oligosaccharides were found only in the interstitial tissue. As glandular tissue cells arise from the innermost cells of interstitial tissue that surround lobules, the differences in the glycan composition of interstitial and glandular tissue shown in this work may be related to the start of androgen synthesis when steroid hormone (SH)-secreting cells develop. (+info)
Imaging of the lectin-labeled cell surface of human lymphocytes by the use of atomic force microscope.
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The atomic force microscope (AFM) is a new useful tool to examine the surface structure of specimens with a higher resolution than the conventional scanning electron microscope. In the present study, we used the AFM to observe the surface of paraformaldehyde-fixed human lymphocytes processed for histochemistry using a biotinylated lectin, wheat germ agglutinin, followed by colloidal gold and silver-enhancement method. Before the treatment, no particles were attached to the cell surface. After treatment, many particles about 100 to 150 nm in diameter were visualized on it. Since we could observe the same cells on the slide glass before and after treatment, the AFM has the advantage to enable us the repeated imaging of samples treated with various kinds of histochemistries. (+info)
Streptococcus salivarius fimbriae are composed of a glycoprotein containing a repeated motif assembled into a filamentous nondissociable structure.
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Streptococcus salivarius, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of S. salivarius fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of S. salivarius ATCC 25975) with glass beads. Preliminary experiments showed that S. salivarius fimbriae did not dissociate when they were incubated at 100 degrees C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 x 10(6) to 40 x 10(6) Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 microm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that S. salivarius fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/phi, where X represents a modified amino acid residue and phi represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species, Streptococcus mitis and Streptococcus constellatus. (+info)