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P Dráber

Publications and source records attributed to P Dráber.

At least 19 recordsLinked to original sources
PubMed2006

Multiple tubulin forms in ciliated protozoan Tetrahymena and Paramecium species.

Tetrahymena and Paramecium species are widely used representatives of the phylum Ciliata. Ciliates are particularly suitable model organisms for studying the functional heterogeneity of tubulins, since they provide a wide range of different microtubular structures in a single cell. Sequencing projects of the genomes of members of these two genera are in progress. Nearly all members of the tubulin superfamily (alpha-, beta-, gamma-, delta-, epsilon-, eta-, theta-, iota-, and kappa-tubulins) have been identified in Paramecium tetraurelia. In Tetrahymena spp., the functional consequences of different posttranslational tubulin modifications (acetylation, tyrosination and detyrosination, phosphorylation, glutamylation, and glycylation) have been studied by different approaches. These model organisms provide the opportunity to determine the function of tubulins found in ciliates, as well as in humans, but absent in some other model organisms. They also give us an opportunity to explore the mechanisms underlying microtubule diversity. Here we review current knowledge concerning the diversity of microtubular structures, tubulin genes, and posttranslational modifications in Tetrahymena and Paramecium species.

Animals↗
PubMed2005

Distinct localization of a beta-tubulin epitope in the Tetrahymena thermophila and Paramecium caudatum cortex.

Many of the highly organized microtubular arrangements in ciliates are located in the cortical area containing membrane vesicles and vacuoles. In Tetrahymena thermophila and Paramecium caudatum, immunofluorescence microscopy with the monoclonal antibody TU-06, directed against beta-tubulin, revealed distinct staining of this cortical region alone, while the cilia and other microtubular structures were unstained. The specificity of the antibody was confirmed by immunoblotting and by preabsorption of the antibody with purified tubulin. Double-label immunofluorescence with antibodies against gamma-tubulin, detyrosinated alpha-tubulin, and centrin showed that the TU-06 epitope is localized outside the basal body region. This was also confirmed by immunogold electron microscopy of thin sections. Proteolytic digestion of porcine brain beta-tubulin combined with a peptide scan of immobilized, overlapping peptides disclosed that the epitope was in the beta-tubulin region beta81-95, a region which is phylogenetically highly conserved. As known posttranslational modifications of beta-tubulin are located outside this area, the observed staining pattern cannot be interpreted as evidence of subcellular sequestration of modified tubulin. The limited distribution of the epitope could rather reflect the dependence of TU-06 epitope exposition on conformations of tubulin molecules in microtubule arrangements or on differential masking by interacting proteins.

3T3 Cells↗
PubMed2005

Regulation of cys-based protein tyrosine phosphatases via reactive oxygen and nitrogen species in mast cells and basophils.

Activation of mast cells and basophils is accompanied by the production of reactive oxygen and nitrogen species that regulate diverse signaling pathways leading to the release of inflammatory mediators and production of a variety of cytokines. Although the functional pathways of reactive oxygen and nitrogen species in vivo are not completely understood, some novel metabolic pathways can be envisioned based on recent findings that protein tyrosine phosphatases can be regulated by reversible oxidation. In this review, we describe major sources and targets of reactive oxide and nitrogen species in mast cells and basophils. Direct and indirect regulations of class I and II Cys-based protein tyrosine phosphatases (LMW-PTP, PTEN, PTP-PEST, SHP-2, PTP1B, PTPalpha, PTPepsilon, DEP-1, TC45, SHP-1, HePTP and LAR) are discussed. The combined data highlight the role of redox-regulated protein tyrosine phosphatases as targets in the development of new ways of therapeutic intervention in allergies and inflammatory diseases.

Animals↗
PubMed2004

Preparation of human recombinant kinesin heavy chain and epitope mapping of its structural domains.

The isolation of the cDNA sequence encoding the human neuronal kinesin (a force-generating motor protein which transports various membrane organelles along microtubules in an ATP-dependent manner) heavy chain (nKHC) and the construction of expression vectors to produce the full-length nKHC and its domains in Escherichia coli is described. By tuning up the conditions for the expression of nKHC, a sufficient amount of the soluble protein intragenously tagged with 6xHis tag was obtained and purified by nickel chromatography. The recombinant structural domains of nKHC, including the motor domain (FKHC1--amino acids 1-330), the microtubule binding domain (FKHC2--amino acids 174-315) and the coiled-coil stalk domain (FKHC3--amino acids 331-906) were used to determine the epitope location for monoclonal antibodies KN-01, KN-02, and IB II raised against different kinesin heavy chains. The antibodies were shown to recognize epitopes located in the stalk domain of nKHC and represent thus useful probes for this domain.

Antibodies, Monoclonal↗
PubMed2004

A fast and simple dot-immunobinding assay for quantification of mouse immunoglobulins in hybridoma culture supernatants.

Mouse monoclonal antibodies of IgG subclasses and IgM class in hybridoma culture supernatants were quantified using a dot-immunobinding assay. Immunoglobulins were bound to nitrocellulose (NC) membrane and, after blocking, the membrane was incubated with anti-mouse antibody conjugated to horseradish peroxidase (HRP). Binding was revealed by incubation with a sensitive chemiluminiscence reagent. Quantitation was achieved by densitometric comparison with standard curves produced by purified monoclonal antibodies of the same subclass or purified antibodies of the same clone as the antibody to be quantified. These quantitative results were compared with those obtained using purified IgG from mouse serum or purified mouse myeloma IgM as standards. The dot-immunobinding assay requires 1 microl of hybridoma culture sample and takes about 1 h in total. Good linearity between the staining intensity and the amount of immobilized immunoglobulins was observed over the range of 0.05-5 ng/spot. The assay is simple, reproducible and can process simultaneously a large number of samples.

Animals↗
PubMed2001

Cell cycle-dependent changes in localization of a 210-kDa microtubule-interacting protein in Leishmania.

Using the monoclonal antibody MA-01, a new 210-kDa microtubule-interacting protein was identified in Leishmania promastigotes by immunoblotting and by immunoprecipitation. The protein was thermostable and was located on microtubules prepared by taxol-driven polymerization in vitro. On fixed cells the antibody gave specific staining of flagellum, flagellar pocket, and mitotic spindle. Subpellicular microtubules were basically not decorated but posterior poles of the cells were labeled in a cell-cycle-dependent manner. In anterior and posterior poles of cells the 210-kDa protein codistributed with the 57-kDa protein, immunodetected with anti-vimentin antibody, that was located only on cell poles. Immunolocalization of the 57-kDa protein was most prominent in dividing cells. The presented data suggest that the 210-kDa protein is a newly identified microtubule-interacting protein of Leishmania that could be involved in anchoring the microtubules in posterior poles of these cells. The striking codistribution of the microtubule-interacting protein and the 57-kDa protein in protozoa is described for the first time.

Animals↗
PubMed2001

Differential sensitivity to acute cholesterol lowering of activation mediated via the high-affinity IgE receptor and Thy-1 glycoprotein.

Lateral cross-linking of transmembrane high-affinity IgE receptors (FcepsilonRI) or glycosylphosphatidylinositol-anchored Thy-1 glycoproteins on the surface of rat mast cells and rat basophilic leukemia (RBL) cells triggers the signaling pathways that lead to the release of allergy mediators. Although both of these pathways are initiated by an increased activity of Lyn kinase, the exact mechanism by which Lyn kinase interacts with aggregated FcepsilonRI and Thy-1 is not completely understood. Here we demonstrate that pretreatment of RBL cells with methyl-beta-cyclodextrin (MBCD) resulted in a dose- and time-dependent decrease in cellular cholesterol, increased detergent solubilization of Thy-1 and Lyn kinase, and a transient increase in tyrosine phosphorylation of several proteins. Acute lowering of cholesterol suppressed the activation through Thy-1, as determined by tyrosine phosphorylation of Syk kinase and some other proteins, and modulation of free cytoplasmic calcium. In contrast, the FcepsilonRI-mediated activation events were more resistant. Thy-1 and FcepsilonRI in MBCD-pretreated cells also differed in the extent of aggregation after cross-linking: Thy-1 formed large caps, whereas FcepsilonRI accumulated in small patches. MBCD treatment induced an increased release of secretory components in both Thy-1- and FcepsilonRI-activated cells. The combined data indicate that cholesterol depletion does not merely block receptor signaling but has more complex consequences.

Animals↗
PubMed2001

Carcinoembryonic antigen-related cell adhesion molecule 1 is the 85-kilodalton pronase-resistant biliary glycoprotein in the cholesterol crystallization promoting low density protein-lipid complex.

A pronase resistant 85-kd glycoprotein in the Concanavalin A-binding fraction (CABF) of biliary glycoproteins has been reported to act as a promotor of cholesterol crystallization. De Bruijn et al. (Gastroenterology 1996;110:1936-1944) found this protein in a low-density protein-lipid complex (LDP) with potent cholesterol crystallization promoting activity. This study identifies and characterizes this protein. An LDP was prepared from CABF by discontinuous gradient ultracentrifugation. Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotting and immunochemical staining with anti-carcinoembryonic antigen, CEA-related adhesion molecule 1 (CEACAM1) cross-reacting antibodies. Biliary concentrations of CEA cross-reacting proteins were determined in patients with and without gallstones. Two isoforms of CEACAM1 (85- and 115-kd bands), CEA and 2 CEA cross-reacting protein bands of 40 and 50 kd were found in human bile. All bands were also present in CABF, but only a subfraction of the 85-kd band found in the LDP was resistant to digestion with pronase. CEACAM1-85 exhibited potent cholesterol crystallization promoting activity in vitro and accounted for most of the activity in CABF. Total CEA cross-reacting protein concentrations were the same in gallbladder biles from patients with cholesterol and pigment gallstones but only half of those in biles from nongallstone subjects. In conclusion, we have identified the protein component of the cholesterol crystallization promoting LDP to be CEACAM1-85.

Adenosine Triphosphatases↗
PubMed2001

Differential subcellular distribution of tubulin epitopes in boar spermatozoa: recognition of class III beta-tubulin epitope in sperm tail.

The exposure of tubulin epitopes was studied in ejaculated boar spermatozoa using a panel of four monoclonal antibodies specific to the N-terminal or C-terminal structural domains of tubulin and three monoclonal antibodies against class III beta-tubulin. The specificity of the antibodies was confirmed by immunoblotting. Immunocytochemical staining showed that antibodies discriminated between various parts of a spermatozoon, and that epitopes of class III beta-tubulin were present in the flagellum. A tubulin epitope from the C-terminal domain of beta-tubulin was detected in the triangular segment of the postacrosomal part of the sperm head. Its distribution changed after an A23187 ionophore-induced acrosome reaction, indicating that tubulin participates in the early stages of fertilization. Three monoclonal antibodies, TU-20, SDL.3D10, and TUJ1 directed against epitopes on the C-terminal end of neuron-specific class III beta-tubulin that is widely used as a neuronal marker, stained the flagella. The reactivity of TU-20 was further confirmed by absorbing the antibody with the immunizing peptide and by immunoelectron microscopy. Immunoblotting after two-dimensional electrophoresis revealed that the corresponding epitope was not present on all beta-tubulin isoforms. These results suggest that various tubulins are involved in the functional organization of the mammalian sperm flagellum and head.

Acrosome Reaction↗
PubMed2001

Structure-function analysis of Lyn kinase association with lipid rafts and initiation of early signaling events after Fcepsilon receptor I aggregation.

The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid- and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcepsilonRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, in cis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcepsilonRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca(2+). However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcepsilonRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcepsilonRI does not exhibit an increased association with lipid rafts, suggest that FcepsilonRI phosphorylation and early activation events can be initiated outside of lipid rafts.

Animals↗
PubMed2000

The specificity for the differentiation blocking activity of carcinoembryonic antigen resides in its glycophosphatidyl-inositol anchor.

Ectopic expression of various members of the human carcinoembryonic antigen (CEA) family of intercellular adhesion molecules in murine myoblasts either blocks (CEA, CEACAM6) or allows (CEACAM1) myogenic differentiation. These surface glycoproteins form a subset of the immunoglobulin (Ig) superfamily and are very closely related, but differ in the precise sequence of their external domains and in their mode of anchorage to the cell membrane. CEA and CEACAM6 are glycophosphatidyl-inositol (GPI) anchored, whereas CEACAM1 is transmembrane (TM) anchored. Overexpression of GPI-linked neural cell adhesion molecule (NCAM) p125, also an adhesion molecule of the Ig superfamily, accelerates myogenic differentiation. The molecular requirements for the myogenic differentiation block were investigated using chimeric constructs in which the COOH-terminal hydrophobic domains of CEA, CEACAM1, and NCAM p125 were exchanged. The presence of the GPI signal sequence specifically from CEA in the chimeras was sufficient to convert both CEACAM1 and NCAM into differentiation-blocking proteins. Conversely, CEA could be converted into a neutral protein by exchanging its GPI anchor for the TM anchor of CEACAM1. Since the external domains of CEA, CEACAM1, and NCAM can all undergo homophilic interactions, and mutations in the self-adhesive domains of CEA abrogate its differentiation-blocking activity, the structural requirements for differentiation-inhibition are any self-adhesive domains attached to the specific GPI anchor derived from CEA. We therefore suggest that biologically significant functional information resides in the processed extreme COOH terminus of CEA and in the GPI anchor that it determines.

Amino Acid Sequence↗
PubMed2000

New specificity and yield enhancer of polymerase chain reactions.

Tetramethylammonium (TMA) chloride, dimethyl sulfoxide and formamide are known to increase, under certain conditions, the specificity and efficiency of the polymerase chain reaction (PCR). We compared the ability of several TMA derivatives and some other reagents to increase the specificity of PCR and to improve the yield of amplification. A novel combination of the enhancer TMA and oxalate as anion is demonstrated to be a powerful enhancer of PCR. Addition of 2 mM TMA oxalate to the PCR mixture decreases the formation of non-specific DNA fragments and increases the yield of specific PCR products.

Animals↗
PubMed2000

Accumulation of 210 kDa microtubule-interacting protein in differentiating P19 embryonal carcinoma cells.

The MA-01 antigen, a thermolabile 210 kDa microtubule-interacting protein, is present in P19 embryonal carcinoma cells on microtubular structures as well as in cytosol. After aggregation of the cells and subsequent incubation with all-trans-retinoic acid (RA), the level of MA-01 expression increased approximately 10 times during 15 days. The increase started after 2 days of incubation with RA and preceded the appearance of neuron-specific tubulin betaIII, MAP2C and neurofilaments. Such elevated expression of MA-01 antigen was not detected in P19 cells treated with dimethylsulfoxide. These data indicate that enhanced expression of MA-01 antigen is one of the earliest events occurring in P19 cells during neuronal differentiation.

Animals↗