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D J Asai

Publications and source records attributed to D J Asai.

At least 19 recordsLinked to original sources
PubMed1998

Evidence for four cytoplasmic dynein heavy chain isoforms in rat testis.

Recent studies have revealed the expression of multiple putative cytoplasmic dynein heavy chain (DHC) genes in several organisms, with each gene encoding a separate protein isoform. This finding is consistent with the hypothesis that different isoforms do different things, as is the case for the axonemal dyneins. Furthermore, the large number of tasks ascribed to cytoplasmic dynein suggests that there may be additional isoforms not yet identified. Two of the mammalian cytoplasmic dynein heavy chains are DHC1a and DHC1b. DHC1a is conventional cytoplasmic dynein and is found in all organisms examined. DHC1b is expressed in organisms that have multiple dyneins, and has been implicated in the intracellular trafficking of molecules in unciliated and ciliated cells. In the present study, we examined the DHC1b protein from rat testis. Testis cytoplasmic dynein contains a large amount of dynein heavy chain reactive with an antibody raised against a peptide sequence of rat DHC1b. The testis anti-DHC1b immunoreactive protein is slightly smaller than testis DHC1a, as assessed by SDS-PAGE. In Northern blots, the DHC1b mRNA is smaller than the DHC1a mRNA. In sucrose gradients made in low ionic strength, DHC1a sedimented at approximately 20S, and the anti-1b immunoreactive heavy chains sedimented in a broad band centered at approximately 14S. The V1-photolysis reaction of individual sucrose gradient fractions revealed three distinct patterns of photolysis, suggesting that there are at least three separate 1b-like heavy chain isoforms in testis. Using a high-stringency Western blotting protocol, the anti-1b antibody and the anti-DHC2 antibody recognized the same heavy chain and specifically bound to one of the three 1b-like heavy chains. We conclude that rat testis contains three 1b-like dynein heavy chains, and one of these is the product of the DHC1b/DHC2 gene previously identified.

Animals↗
PubMed1996

Syk, activated by cross-linking the B-cell antigen receptor, localizes to the cytosol where it interacts with and phosphorylates alpha-tubulin on tyrosine.

Syk (p72syk) is a 72-kDa, nonreceptor, protein-tyrosine kinase that becomes tyrosine-phosphorylated and activated in B lymphocytes following aggregation of the B-cell antigen receptor. To explore the subcellular location of activated Syk, anti-IgM-activated B-cells were fractionated into soluble and particulate fractions by ultracentrifugation. Activated and tyrosine-phosphorylated Syk was found predominantly in the soluble fraction and was not associated with components of the antigen receptor. Similarly, the activated forms of Syk and its homolog, ZAP-70, were found in soluble fractions prepared from pervanadate-treated Jurkat T-cells. A 54-kDa protein that co-immunoprecipitated with Syk from the soluble fraction of activated B-cells was identified by peptide mapping as alpha-tubulin. alpha-Tubulin was an excellent in vitro substrate for Syk and was phosphorylated on a single tyrosine present within an acidic stretch of amino acids located near the carboxyl terminus. alpha-Tubulin was phosphorylated on tyrosine in intact cells following aggregation of the B-cell antigen receptor in a reaction that was inhibited by the Syk-selective inhibitor, piceatannol. Thus, once activated, Syk releases from the aggregated antigen receptor complex and is free to associate with and phosphorylate soluble proteins including alpha-tubulin.

Amino Acid Sequence↗
PubMed1996

Identification of seven rat axonemal dynein heavy chain genes: expression during ciliated cell differentiation.

Axonemal dyneins are molecular motors that drive the beating of cilia and flagella. We report here the identification and partial cloning of seven unique axonemal dynein heavy chains from rat tracheal epithelial (RTE) cells. Combinations of axonemal-specific and degenerate primers to conserved regions around the catalytic site of dynein heavy chains were used to obtain cDNA fragments of rat dynein heavy chains. Southern analysis indicates that these are single copy genes, with one possible exception, and Northern analysis of RNA from RTE cells shows a transcript of approximately 15 kb for each gene. Expression of these genes was restricted to tissues containing axonemes (trachea, testis, and brain). A time course analysis during ciliated cell differentiation of RTE cells in culture demonstrated that the expression of axonemal dynein heavy chains correlated with the development of ciliated cells, while cytoplasmic dynein heavy chain expression remained constant. In addition, factors that regulate the development of ciliated cells in culture regulated the expression of axonemal dynein heavy chains in a parallel fashion. These are the first mammalian dynein heavy chain genes shown to be expressed specifically in axonemal tissues. Identification of the mechanisms that regulate the cell-specific expression of these axonemal dynein heavy chains will further our understanding of the process of ciliated cell differentiation.

Amino Acid Sequence↗
PubMed1996

A novel cytoplasmic dynein heavy chain: expression of DHC1b in mammalian ciliated epithelial cells.

Organisms that have cilia or flagella express over a dozen dynein heavy chain genes. Of these heavy chain genes, most appear to encode axonemal dyneins, one encodes conventional cytoplasmic dynein (MAP1C or DHC1a), and one, here referred to as DHC1b, encodes an unclassified heavy chain. Previous analysis of sea urchin DHC1b (Gibbons et al. (1994) Mol. Biol. Cell 5, 57-70) indicated that this isoform is either an axonemal dynein with an unusual protein sequence or a cytoplasmic dynein whose expression increases during ciliogenesis. In the present study, we examined the expression of DHC1b in rat tissues. The DHC1b gene is expressed in all tissues examined, including unciliated liver and heart cells. In contrast, rat axonemal dyneins are only expressed in tissues that produce cilia or flagella. In cultured rat tracheal epithelial (RTE) cells, DHC1b is expressed in undifferentiated cells and increases in expression during ciliogenesis. In contrast, the expression of conventional cytoplasmic dynein, DHC1a, does not change during RTE differentiation and axonemal dynein is not expressed until after differentiation commences. In order to examine the expression of DHC1b protein, we produced an isoform-specific antibody to a synthetic peptide derived from the rat DHC1b sequence. The antibody demonstrated that DHC1b is a relatively minor component of partially purified cytoplasmic dynein. Indirect immunofluorescence microscopy revealed that DHC1b is not detected in cilia and remains in the cytoplasm of ciliated RTE cells, often accumulating at the apical ends of the cells. These results suggest that DHC1b is a cytoplasmic dynein that may participate in intracellular trafficking in polarized cells.

Amino Acid Sequence↗
PubMed1995

Multi-dynein hypothesis.

Axonemal dyneins and cytoplasmic dynein have evolved separate strategies to perform their tasks. The multi-dynein hypothesis accurately describes the highly specialized axonemal isoforms; each isoform is encoded by a separate gene, is located in a precise place, produces specific forces which contribute to the overall generation of propagated bending, and is not functionally interchangeable with other isoforms. In contrast, cytoplasmic dynein, although carrying many different cargoes, appears to be one isoform. An intriguing question is to determine whether there are additional cytoplasmic dyneins, heretofore uncharacterized, which, like their axonemal counterparts, are customized to perform specific tasks.

Animals↗
PubMed1995

Retrograde but not anterograde bead movement in intact axons requires dynein.

Dynein and kinesin have been implicated as the molecular motors that are responsible for the fast transport of axonal membranous organelles and vesicles. Experiments performed in vitro with partially reconstituted preparations have led to the hypothesis that kinesin moves organelles in the anterograde direction and dynein moves them in the retrograde direction. However, the molecular basis of transport directionality remains unclear. In the experiments described here, carboxylated fluorescent beads were injected into living Mauthner axons of lamprey and the beads were observed to move in both the anterograde and retrograde directions. The bead movement in both directions required intact microtubules, occurred at velocities approaching organelle fast transport in vivo, and was inhibited by vanadate at concentrations that inhibit organelle fast transport. When living axons were injected with micromolar concentrations of vanadate and irradiated at 365 nm prior to bead injections, a treatment that results in the V1 photolysis of dynein, the retrograde movement of the beads was specifically abolished. Neither the ultraviolet irradiation alone nor the vanadate alone produced the retrograde-specific inhibition. These results support the hypothesis that dynein is required for retrograde, but not anterograde, transport in vivo.

Animals↗
PubMed1995

The dynein genes of Paramecium tetraurelia: the structure and expression of the ciliary beta and cytoplasmic heavy chains.

The genes encoding two Paramecium dynein heavy chains, DHC-6 and DHC-8, have been cloned and sequenced. Sequence-specific antibodies demonstrate that DHC-6 encodes ciliary outer arm beta-chain and DHC-8 encodes a cytoplasmic dynein heavy chain. Therefore, this study is the first opportunity to compare the primary structures and expression of two heavy chains representing the two functional classes of dynein expressed in the same cell. Deciliation of paramecia results in the accumulation of mRNA from DHC-6, but not DHC-8. Nuclear run-on transcription experiments demonstrate that this increase in the steady state concentration of DHC-6 mRNA is a consequence of a rapid induction of transcription in response to deciliation. This is the first demonstration that dynein, like other axonemal components, is transcriptionally regulated during reciliation. Analyses of the sequences of the two Paramecium dyneins and the dynein heavy chains from other organisms indicate that the heavy chain can be divided into three regions: 1) the sequence of the central catalytic domain is conserved among all dyneins; 2) the tail domain sequence, consisting of the N-terminal 1200 residues, differentiates between axonemal and cytoplasmic dyneins; and 3) the N-terminal 200 residues are the most divergent and appear to classify the isoforms. The organization of the heavy chain predicts that the variable tail domain may be sufficient to target the dynein to the appropriate place in the cell.

Amino Acid Sequence↗
PubMed1994

Distribution of phosphorylated microtubule-associated protein 1B during neurite outgrowth in PC12 cells.

The functional significance of microtubule-associated protein 1B (MAP1B) phosphorylation during neuronal differentiation is unknown. In the present study we examined the hypothesis that the phosphorylation of MAP1B is required for neurite outgrowth. We reasoned that if MAP1B phosphorylation was important for neurite outgrowth then the intracellular distribution of phosphorylated MAP1B might exist as a discrete subset of the pattern for total MAP1B. We utilized a monoclonal antibody (mAb 7-1.1) that specifically recognizes a phosphorylated epitope on MAP1B and a polyclonal antiserum that recognizes all MAP1B protein to compare the distributions of phosphorylated and total MAP1B during neurite outgrowth. Phosphorylated MAP1B progressively accumulated in both the soluble and cytoskeletal fractions of differentiating cells. Similar proportions of total and phosphorylated MAP1B were associated with the cytoskeletons of differentiating PC12 cells. Within individual cells, phosphorylated MAP1B, in comparison with total MAP1B, was not limited to a particular intracellular domain. Phosphorylated MAP1B was present in both neurites and cell bodies. It was associated with fibrillar microtubules in neurites and growth cones, but it appeared nonfibrillar within cell bodies. In some cells that differentiated rapidly, there was little phosphorylated MAP1B in the early neurites despite the presence of extensive microtubules. In addition, although phosphorylated MAP1B increased in populations of mature PC12 cell cultures, increases in phosphorylated MAP1B did not always correlate with neurite outgrowth in individual cells. These results suggest that the phosphorylated isoform of MAP1B recognized by mAb 7-1.1 may not be required for neurite outgrowth.

Animals↗
PubMed1994

Phylogeny and expression of axonemal and cytoplasmic dynein genes in sea urchins.

Transcripts approximately 14.5 kilobases in length from 14 different genes that encode for dynein heavy chains have been identified in poly(A)+ RNA from sea urchin embryos. Analysis of the changes in level of these dynein transcripts in response to deciliation, together with their sequence relatedness, suggests that 11 or more of these genes encode dynein isoforms that participate in regeneration of external cilia on the embryo, whereas the single gene whose deduced sequence closely resembles that of cytoplasmic dynein in other organisms appears not to be involved in this regeneration. The four consensus motifs for phosphate binding found previously in the beta heavy chain of sea urchin dynein are present in all five additional isoforms for which extended sequences have been obtained, suggesting that these sites play a significant role in dynein function. Sequence analysis of a approximately 400 amino acid region encompassing the putative hydrolytic ATP-binding site shows that the dynein genes fall into at least six distinct classes. Most of these classes in sea urchin have a high degree of sequence identity with one of the dynein heavy chain genes identified in Drosophila, indicating that the radiation of the dynein gene family into the present classes occurred at an early stage in the evolution of eukaryotes. Evolutionary changes in cytoplasmic dynein have been more constrained than those in the axonemal dyneins.

Amino Acid Sequence↗
PubMed1994

The dynein genes of Paramecium tetraurelia. Sequences adjacent to the catalytic P-loop identify cytoplasmic and axonemal heavy chain isoforms.

Paramecium tetraurelia is a unicellular organism that utilizes both axonemal and cytoplasmic dyneins. The highly conserved region containing the catalytic P-loop of the dynein heavy chain was amplified by RNA-directed polymerase chain reaction. Eight different P-loop-containing cDNA fragments were cloned. Southern hybridization analysis indicated that each fragment corresponds to a separate dynein gene and that there are at least 12 dynein heavy chain genes expressed in Paramecium. Seven of the eight cloned contain sequence motif A, which is found in axonemal dyneins, and one contains sequence motif B, which is found in the dyneins from cell types that do not have cilia or flagella. Two of the Paramecium dynein genes were further investigated: DHC-6 which contains motif A, and DHC-8 which contains motif B. Additional sequencing of the central portions of these genes showed that DHC-6 most closely matches sea urchin ciliary beta heavy chain and DHC-8 is similar to the cytoplasmic dynein from Dictyostelium. Deciliation of the cells resulted in a substantial increase in the steady state concentration of DHC-6 mRNA but only a small change in DHC-8 mRNA. Antisera were produced against synthetic peptides derived from sequence motifs A and B. Competitive solid-phase binding assays demonstrated that each antiserum was peptide-specific. In western blots, the antiserum to motif A reacted with both ciliary and cytoplasmic dyneins. In contrast, the antiserum to motif B reacted with the cytoplasmic dyneins of Paramecium and bovine brain but did not react with ciliary dynein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
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↗
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↗