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D J Asai

Publications and source records attributed to D J Asai.

At least 37 records · Page 2Linked to original sources
PubMed1993

Ciliary dynein of Paramecium tetraurelia: photolytic maps of the three heavy chains.

The ciliate Paramecium tetraurelia presents a powerful system to define the structural basis for dynein functional diversity within a single cell. This analysis will depend on the biochemical resolution of the dynein proteins. As an important first step, the three heavy chains of the ciliary outer arm dynein of paramecium were characterized. Sucrose density gradient centrifugation in a high salt buffer separated the dynein into a 22S species, which contained the alpha and beta heavy chains, and a 12S species, which contained the gamma chain as well as the inner arm dynein heavy chains. Both the 22S and 12S species retained enzymatic latency as indicated by stimulation of MgATPase activity by 0.1% Triton X-100. An unusual ATP-independent V1-like photolysis of only the beta chain provided the basis for estimating that the beta chain contributes almost half of the 22S MgATPase activity that is susceptible to V1 photolysis. The combination of the density gradient separation of the partially dissociated dynein and the ATP-independent V1-like photolysis of only the beta chain led to the unambiguous assignment of the V1 photolytic products to the appropriate parent heavy chains. An estimate of the molecular sizes of the three heavy chains was obtained. The photolytic peptide maps, which define the ATP-binding domains, were determined for the three heavy chains.

Amino Acid Sequence↗
PubMed1993

Dynein heavy chain isoforms and axonemal motility.

The translocation of dynein along microtubules is the basis for a wide variety of essential cellular movements. Dynein was first discovered in the ciliary axoneme, where it causes the directed sliding between outer doublet microtubules that underlies ciliary bending. The initiation and propagation of ciliary bends are produced by a precisely located array of different dyneins containing eight or more different dynein heavy chain isoforms. The detailed clarification of the structural and functional diversity of axonemal dynein heavy chains will not only provide the key to understanding how cilia function, but also give insights applicable to the study of non-axonemal microtubule motors.

Journal Article↗
PubMed1992

A cytoplasmic dynein heavy chain in sea urchin embryos.

By making the hypothesis that the pattern of conserved sequence residues in the vicinity of the hydrolytic ATP-binding site of dynein would resemble that in myosins from a broad variety of sources, we designed degenerate oligonucleotide primers capable of amplifying this region of multiple dynein isoforms from sea urchin embryo poly(A)+ RNA. Quantification of the expression of two of these dynein isoforms has shown that the level of mRNA encoding for the beta-heavy chain, like that of tubulin, increases 2-3-fold after deciliation of the embryos, whereas the expression of the second dynein isoform, like that of actin, is essentially unaffected. This second isoform is believed to be the cytoplasmic dynein of sea urchin embryos.

Amino Acid Sequence↗
PubMed1991

A PCR procedure to determine the sequence of large polypeptides by rapid walking through a cDNA library.

A procedure that uses the PCR to make rapid successive steps through a random-primed cDNA library has been developed to provide a method for sequencing very long genes that are difficult to obtain as a single clone. In each successive step, the portions of partial clones that extend out from the region of known DNA sequence are amplified by two stages of PCR with nested, outward-directed primers designed approximately 50 bases in from the end of the known sequence, together with a general primer based on the sequence of the vector. This procedure has been used to determine the coding sequence of the cDNA for the beta heavy chain of axonemal dynein from embryos of the sea urchin Tripneustes gratilla. By starting from a single parent clone, whose translated amino acid sequence overlapped the microsequence of a tryptic peptide of the beta heavy chain, and making 3 such walk steps downstream and 14 walk steps upstream, we obtained a sequence of 13,799 base pairs that had an open reading frame of 13,398 base pairs. This sequence encodes a polypeptide with 4466 residues of Mr 511,804 that is believed to correspond to the complete beta heavy chain of ciliary outer arm dynein.

Amino Acid Sequence↗
PubMed1991

Multiple nucleotide-binding sites in the sequence of dynein beta heavy chain.

Axonemal dyneins have two or three globular heads joined by flexible tails to a common base, with each head/tail unit consisting of a single heavy-chain polypeptide of relative molecular mass greater than 400,000. The sizes of the components have been deduced by electron microscopy. The isolated beta heavy chain of sea urchin sperm flagella, which is immunologically identical to that of the embryo cilia, is of particular interest as it retains the capability for microtubule translocation in vitro. Limited proteolysis of the beta heavy chain divides it into two fragments, A and B, which sediment separately at 12S and 6S, and possibly correspond to the head and tail domains of the molecule. Dynein ATPase is the energy-transducing enzyme that generates the sliding movement between tubules that underlies the beating of cilia and flagella of eukaryotes, and possibly also other large intracellular movements. Here we report that the deduced amino-acid sequence of the beta heavy chain of axonemal dynein from embryos of the sea urchin Tripneustes gratilla has 4,466 residues and contains the consensus motifs for five nucleotide-binding sites. The probable hydrolytic ATP-binding site can be identified by its location close to or at the V1 site of vanadate-mediated photo-cleavage. The general features of the map of photocleavage and proteolytic peptides reported earlier have been confirmed, except that the map's polarity is reversed. The predicted secondary structure of the beta heavy chain consists of an alpha/beta-type pattern along its whole length. The two longest regions of potential alpha helix, with unbroken heptad hydrophobic repeats 120 and 50 amino acids long, may be of functional importance. But dynein does not seem to contain an extended coiled-coil tail domain.

Amino Acid Sequence↗
PubMed1990

Molecular cloning and expression of sea urchin embryonic ciliary dynein beta heavy chain.

The determination of the structure and the expression of dynein during embryonic development are central to the understanding of dynein function. As an important first step toward these objectives, cDNAs encoding portions of sea urchin ciliary dynein were identified by antibody screening of a sea urchin cDNA expression library. Because of the complete lack of protein sequence data, it was first necessary to prove the identity of the dynein cDNAs. Of the five cDNA inserts initially cloned, one, designated P72A1, was characterized extensively. Four independent criteria demonstrated that P72A1 encoded a portion of a dynein heavy chain. (1) The beta-galactosidase-P72A1 fusion protein affinity-purified dynein-specific antibodies from crude antiserum. (2) Two other antisera to dynein, raised independently of the antiserum used to screen the cDNA library, reacted with the fusion protein. (3) A new antiserum raised against the fusion protein reacted with authentic dynein heavy chain on Western blots and stained embryonic cilia by indirect immunofluorescence microscopy. (4) Two new antisera, elicited against opposite ends of the P72A1 open reading frame, each reacted with authentic dynein heavy chain protein. Western blot analyses of dissociated dynein heavy chains revealed that P72A1 encoded a portion of the beta heavy chain. Epitope mapping experiments confirmed the identity of P72A1 as part of the beta heavy chain and also demonstrated that P72A1 encoded epitopes of the carboxyl-terminal fragment B domain of the dynein beta heavy chain. Northern blot analyses of poly(A)+ RNA revealed that P72A1 hybridized with a large RNA species ca. 12.5 kb in length. The dynein mRNA concentration increased during embryonic development. Dot blot analyses of RNA isolated at various times after embryo deciliation demonstrated that the dynein beta heavy chain mRNA accumulated rapidly in response to deciliation. The accumulation was similar to but not identical with the induction of tubulin mRNA in response to the same stimulus.

Adenosine Triphosphatases↗
PubMed1989

Tubulin isotype usage in vivo: a unique spatial distribution of the minor neuronal-specific beta-tubulin isotype in pheochromocytoma cells.

The neuronal cells of vertebrates express two beta-tubulin isotypes, called Class II and Class III, that are neuronal specific. In order to determine the distribution of the minor Class III isotype, site-directed antibodies were raised to synthetic peptides representing the carboxyl terminal, isotype-defining domains of the tubulins. These antibodies were applied to PC12 cells at various stages of differentiation. The Class III isotype was found to be expressed in undifferentiated PC12 cells as well as in cells at every stage of differentiation. The concentration of the Class III isotype, relative to the total beta-tubulin complement, did not change significantly. Indirect double immunofluorescence microscopy demonstrated that the Class III isotype was found in the soma and the neurites of differentiated PC12 cells; this spatial pattern of Class III expression paralleled the total beta-tubulin pattern. Although the anti-Class III antiserum could stain in vitro assembled neuronal microtubules in a filamentous pattern, a close examination of the Class III staining pattern in flattened PC12 cells revealed that this isotype was not incorporated into the nonaxoplasmic array of microtubules. Rather, the Class III isotype was localized in a nonfilamentous, granular pattern that was not readily extracted with nonionic detergent. Cells treated with taxol and then flattened and stained showed that the Class III isotype could be induced to assemble into microtubule bundles by taxol. Thus, the minor neuronal beta-tubulin isotype appears to be spatially specialized in its pattern of expression.

Adrenal Gland Neoplasms↗
PubMed1989

Physicochemical characterization and monoclonal and polyclonal antibody recognition of Baylisascaris procyonis larval excretory-secretory antigens.

Baylisascaris procyonis larval excretory-secretory (ES) antigens consisted of complex glycoproteins ranging from 10 kDa to over 200 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and lectin binding. Five monoclonal antibodies (Bapr1-Bapr5) produced against B. procyonis ES antigens were assayed by western blotting with larval ES antigens from B. procyonis, Baylisascaris melis, Baylisascaris transfuga, Ascaris suum, and Toxocara canis. Bapr1 and Bapr2 recognized periodate-sensitive epitopes on 14-kDa ES components of B. procyonis, B. melis, and B. transfuga, whereas Bapr4 and Bapr5 recognized periodate-resistant epitopes present on 55-kDa ES components of B. procyonis and B. melis. Bapr3 primarily recognized periodate-resistant epitopes on 33-45-kDa components of B. procyonis and B. melis ES. Heterologous rabbit antisera cross-reacted with many B. procyonis ES antigens on western blots, but recognition of the 33-45-kDa components was genus-specific. Normal human sera and T. canis-positive human sera also cross-reacted with many B. procyonis ES antigens, including those of 33-45 kDa. However, periodate oxidation markedly decreased cross-reactions and allowed for differential immunodiagnosis of B. procyonis versus T. canis. These studies demonstrated that antibody recognition of carbohydrate epitopes on ES components is an important cause of cross-reactions in antibody detection assays. Recognition of periodate-resistant (protein) epitopes on the 33-45-kDa B. procyonis ES components appears to be useful for genus-specific immunodiagnosis of larva migrans caused by Baylisascaris spp.

Animals↗
PubMed1989

Selective inhibition of cytokinesis in sea urchin embryos by low concentrations of stypoldione, a marine natural product that reacts with sulfhydryl groups.

Stypoldione is a marine natural product that inhibits cells division in marine embryos and in mammalian cell cultures. The mechanism responsible for the ability of the compound to inhibit cell division is not known. The compound was found in early studies to inhibit polymerization of tubulin into microtubules in vitro, which lead to the suggestion that inhibition of microtubule polymerization in cells might be responsible for the ability of the compound to inhibit cell division. More recently, stypoldione was found to react covalently with the sulfhydryl groups of a number of proteins including tubulin and with sulfhydryl groups of peptides and small molecules. Thus, stypoldione could potentially react with a large number of cellular targets. In the present study, we have examined the effects of stypoldione on the organization of microtubules and chromatin in cells, in relation to the ability of the compound to inhibit cell division. We used indirect immunofluorescence light microscopy of fixed and stained sea urchin embryos during the first and second divisions after fertilization, with stains specific for tubulin and DNA. We found that stypoldione exerted qualitatively different effects on cell division and microtubule organization and function at different concentrations. At the lowest effective concentrations, 5-10 microM, stypoldione selectively inhibited cytokinesis. Mitotic division occurred normally, usually with no discernible perturbation of microtubule organization or function, and cells became multinuclear. At somewhat higher concentrations, 20-40 microM, stypoldione blocked embryos before streak stage of the first division and, although microtubules were present, their organization was perturbed and they often formed unusual "spiral aster" arrays. At 80 microM and above, microtubules in blocked cells were largely absent. Thus, stypoldione uncouples cytokinesis from mitosis at the lowest effective concentrations and, although it can disrupt microtubules at relatively higher concentrations, it inhibits cell division at the lowest effective concentrations by a selective action on cytokinesis through a mechanism that does not appear to involve disassembly of microtubules.

Animals↗
PubMed1988

Ionic strength-dependent isoforms of sea urchin egg dynein.

Unfertilized sea urchin eggs provide a reservoir of molecules which later are involved in microtubule-mediated movements during embryonic development. Among these molecules is egg dynein, which has been isolated in two forms, 20 S and 12 S. Evidence obtained previously from our laboratory indicates that 20 S dynein is a latent activity precursor of ciliary dynein. In contrast, others have suggested that 12 S egg dynein functions in the mitotic apparatus. It is therefore important to determine the relationship between these egg dyneins. Here we demonstrate that the sedimentation velocity of the egg dynein is dependent on the ionic strength of the extraction conditions. The 20 S dynein is obtained with low ionic strength extraction, and the 12 S form is obtained in high salt (0.6 M KCl). The 20 S dynein, after collection from a sucrose gradient, can be converted quantitatively to the 12 S form by exposure to salt, and this conversion can be followed over time. Further, the 20 S dynein can be converted entirely to 12 S dynein and then partially reconstituted to a faster sedimenting species. During these conversions, the dynein high Mr heavy chains are always coincident with the MgATPase activity, and antibodies show that the dynein heavy chains of the 20 S, 12 S, and converted species are indistinguishable immunologically. These data suggest that 12 S dynein is an ionic strength-dependent isoform of 20 S dynein that results from a partial dissociation of the 20 S polypeptide complex, similar to the relationship between 12 and 21 S sperm flagellar dynein. If the 20 and 12 S enzymes are isoforms of the same dynein, then there is compelling evidence for only a single dynein in the unfertilized egg, and that dynein is probably a ciliary precursor.

Adenosine Triphosphatases↗
PubMed1988

Acetylated alpha-tubulin in microtubules during mouse fertilization and early development.

alpha-Tubulin in the microtubules of mouse oocytes and embryos is acetylated in a specific spatial and temporal sequence. In the unfertilized oocyte, a monoclonal antibody to the acetylated form of alpha-tubulin is bound predominantly at the poles of the arrested metaphase meiotic spindle. The labeling intensity of the spindle microtubules is weaker as observed by immunofluorescence using oocytes double-labeled for total tubulin and acetylated alpha-tubulin, and as measured by immuno high-voltage electron microscopy (immunoHVEM) with colloidal gold; cytasters are not acetylated. At meiotic anaphase, the spindle becomes labeled, and by telophase and during second polar body formation only the meiotic midbody is acetylated. The sperm axoneme retains its acetylation after incorporation though the interphase microtubules are not detected. First mitosis follows a pattern similar to that observed at the second meiosis and during interphase only the mitotic midbodies are acetylated. After treatment with cold, colcemid, or griseofulvin, the remaining stable microtubules are acetylated, but immunoHVEM observations suggest that these fibers might not have been acetylated prior to microtubule disruption. Taxol stabilization does not alter acetylation patterns. Acetylated microtubules are not necessarily old microtubules since acetylated fibers are observed at 30 sec after cold recovery. These results show the presence of acetylated microtubules during meiosis and mitosis and demonstrate a cell-cycle-specific pattern of acetylation, with acetylated microtubules found at the centrosomes at metaphase, an increase in spindle labeling at anaphase, and the selective deacetylation of all but midbody microtubules at telophase.

Acetylation↗
PubMed1987

Posttranslational modification and microtubule stability.

We have probed the relationship between tubulin posttranslational modification and microtubule stability, using a variation of the antibody-blocking technique. In human retinoblastoma cells we find that acetylated and detyrosinated microtubules represent congruent subsets of the cells' total microtubules. We also find that stable microtubules defined as those that had not undergone polymerization within 1 h after injection of biotin-tubulin were all posttranslationally modified; furthermore dynamic microtubules were all unmodified. We therefore conclude that in these cells the stable, acetylated, and detyrosinated microtubules represent the same subset of the cells' total network. Posttranslational modification, however, is not a prerequisite for microtubule stability and vice versa. Potorous tridactylis kidney cells have no detectable acetylated microtubules but do have a sizable subset of stable ones, and chick embryo fibroblast cells are extensively modified but have few stable microtubules. We conclude that different cell types can create specific microtubule subsets by modulating the relative rates of posttranslational modification and microtubule turnover.

Animals↗
PubMed1986

An antiserum to the sea urchin 20 S egg dynein reacts with embryonic ciliary dynein but it does not react with the mitotic apparatus.

Unfertilized sea urchin eggs contain one or more dynein-like enzymes which may be able to serve as microtubule translocators during embryonic development. There are at least two interesting possibilities for the function of the egg dynein: the enzyme may be involved in cytoplasmic microtubule movement such as mitotic spindle anaphase motion; or the enzyme may be a stored precursor for the dynein that functions in embryonic cilia, which are expressed and highly motile at the blastula stage of development. In order to determine directly the distribution and possible function of one of the previously described egg dyneins, the latent-activity 20 S egg dynein (Asai and Wilson, 1985), an antiserum was produced which was highly reactive with the important high Mr polypeptides of 20 S dynein. This antiserum reacted in "Western" immunoblots and in dot-blotting experiments with egg dynein and with embryonic ciliary dynein, but it did not react with any component of sperm flagella. Indirect double immunofluorescence microscopy demonstrated that the anti-20 S antiserum could brightly stain embryonic cilia but it did not stain the sperm flagella from the same sea urchin species. Under the same conditions that the antiserum stained cilia, anti-20 S did not stain the mitotic apparatus but it did appear to stain the cortical region of the dividing egg. In a time-course experiment, the antigen reactive with the anti-20 S antiserum gradually accumulated in the developing early sea urchin embryo. The most significant increase in the apparent concentration of the 20 S dynein occurred just prior to embryonic ciliation and during a period when the mitotic activity of the embryo was in decline. These results lead to two conclusions. First, ciliary dynein and sperm flagellar dynein, although derived from very similar organelles and from the same species of sea urchin, are immunologically distinct. Second, the 20 S egg dynein may be a stored precursor of embryonic ciliary dynein and does not appear to be a component of the mitotic apparatus.

Adenosine Triphosphatases↗
PubMed1986

Mechanism of action of the marine natural product stypoldione: evidence for reaction with sulfhydryl groups.

Stypoldione, a marine natural product that possesses an o-quinone functional group, has been shown to inhibit a variety of biological processes including cell division. We found that stypoldione binds covalently to sulfhydryl groups of thiol-containing compounds via addition of sulfur to the C-4' position of the quinone ring. We examined the ability of stypoldione to add to sulfhydryl groups of a number of thiol-containing substances, including glutathione, thiophenol, beta-mercaptoethanol, and the protein tubulin. We suggest that the biological actions of stypoldione may be caused by the addition of this compound to thiol groups of biological molecules.

Cell Division↗
PubMed1986

A radiolabeled monoclonal antibody binding assay for cytoskeletal tubulin in cultured cells.

To detect changes in the extent of tubulin polymerization in cultured cells, we have developed a radioactive antibody binding assay that can be used to quantitate total cytoskeletal tubulin or specific antigenic subsets of polymerized tubulin. Fibroblastic cells, grown to confluence in multiwell plates, were permeabilized and extracted with 0.5% Triton X-100 in a microtubule-stabilizing buffer. These extracted cytoskeletons were then fixed and incubated with translationally radiolabeled monoclonal antitubulin antibody (Ab 1-1.1), an IgM antibody specific for the beta subunit of tubulin. Specific binding of Ab 1-1.1 to the cytoskeletons was saturable and of a single apparent affinity. All specific binding was blocked by preincubation of the radiolabeled antibody with excess purified brain tubulin. Specific Ab 1-1.1 binding appeared to represent binding to cytoskeletal tubulin inasmuch as: pretreatment of cells with colchicine decreased Ab 1-1.1 binding in a dose-dependent manner which correlated with the amount of polymerized tubulin visualized in parallel cultures by indirect immunofluorescence, taxol pretreatment alone caused an increase in Ab 1-1.1 binding and prevented in a dose-dependent manner the colchicine-induced decrease in antibody binding, in cells pretreated with colcemid and returned to fresh medium, Ab 1-1.1 binding decreased and recovered in parallel with the depolymerization and regrowth of microtubules in these cells, and comparison of maximal antibody binding per cell between primary mouse embryo, 3T3, and human foreskin fibroblasts correlated with immunofluorescence visualization of microtubules in these cells. Thus, this assay can be used to measure relative changes in the level of polymerized cytoskeletal tubulin. Moreover, by Scatchard-type analysis of the binding data it is possible to estimate the total number of antibody binding sites per cell. Therefore, depending on the stoichiometry of antibody binding, this type of assay may be used for quantitating total cytoskeletal tubulin, specific antigenic subsets of cytoskeletal tubulin, or other cytoskeletal proteins.

Animals↗
PubMed1986

Dynein-like cytoplasmic microtubule translocators.

The eukaryotic flagellum presents an excellent predictive model of microtubule-mediated motility: movement is caused by microtubule translocators, called dyneins, which actively slide outer doublet microtubules against each other. Cytoplasmic movements, such as certain aspects of mitotic motion, may also be powered by dynein-like molecules. It is important, then, to carefully assess potential cytoplasmic dynein-like translocators by applying criteria defined by the properties of axonemal dynein. A cytoplasmic microtubule translocator may be only partially homologous to axonemal dynein; a modular construction may provide the translocator with domains that are shared with dynein, and with other domains that give it functional specificity. Finally, it is important to consider the possibility that a dynein-like enzyme that is found in the cytoplasm may not function in the cytoplasm but rather is awaiting incorporation into an axoneme.

Adenosine Triphosphatases↗