Biomedical subjects

S Yamashiro

Publications and source records attributed to S Yamashiro.

At least 19 recordsLinked to original sources

PubMed1997

Identification of an actin binding region and a protein kinase C phosphorylation site on human fascin.

Fascin is a 55-58-kDa actin-bundling protein, the actin binding of which is regulated by phosphorylation (Yamakita, Y., Ono, S., Matsumura, F., and Yamashiro, S. (1996) J. Biol. Chem. 271, 12632-12638). To understand the mechanism of fascin-actin interactions, we dissected the actin binding region and its regulatory site by phosphorylation of human fascin. First, we found that the C-terminal half constitutes an actin binding domain. Partial digestion of human recombinant fascin with trypsin yielded the C-terminal fragment with molecular masses of 32, 30, and 27 kDa. The 32- and 27-kDa fragments purified as a mixture formed a dimer and bound to F-actin at a saturation ratio of 1 dimer:11 actin molecules with an affinity of 1.4 x 10(6) M-1. Second, we identified the phosphorylation site of fascin as Ser-39 by sequencing a tryptic phosphopeptide purified by chelating column chromatography followed by C-18 reverse phase high performance liquid chromatography. Peptide map analyses revealed that the purified peptide represented the major phosphorylation site of in vivo as well as in vitro phosphorylated fascin. The mutation replacing Ser-39 with Ala eliminated the phosphorylation-dependent regulation of actin binding of fascin, indicating that phosphorylation at this site regulates the actin binding ability of fascin.

Actins↗

PubMed1997

Low molecular-weight G-actin binding proteins involved in the regulation of actin assembly during myofibrillogenesis.

We previously demonstrated that small G-actin binding proteins, cofilin, ADF and profilin, are involved in the actin dynamics during myofibrillogenesis (OBINATA, T. (1993). Int. Rev. Cytol., 143: 153-189.). To better understand how they are responsible for the regulation of actin assembly, the amounts of the actin-binding proteins were quantified by means of quantitative immunoblotting and compared with that of G-actin pool. The sum of the amounts of cofilin, ADF and profilin was insufficient at early developmental stages but sufficient at later stages to account for the pool of G-actin in muscle cells. We detected expression of thymosin beta 4 at a considerable level in young embryonic but not in adult skeletal muscles. We, therefore, conclude that the G-actin pool in young embryonic skeletal muscle is mainly due to cofilin, ADF, profilin and thymosin beta 4. Switching from a non-muscle-type (NM-) cofilin to a muscle-type (M-) cofilin was observed during muscle development of mammals. In order to clarify cofilin-dependent regulation of actin assembly in muscle cells, cofilin tagged with fluorescence dyes was introduced into C2 myoblasts by a micro injection method. The exogeneous cofilin, but not ADF, caused quick disassembly of actin filaments and accumulated in furrow region of dividing cells. The analogs of the unphosphorylated form (A3-cofilin) and the phosphorylated form (D3-cofilin) were prepared by converting Ser3, a regulatory phosphorylation site, to Ala or Asp. When A3-cofilin and D3-cofilin were injected into living cells, the former was concentrated at the membrane ruffles and cleavage furrow, while the latter showed only diffuse distribution in the cytoplasm. These results suggest that the subcellular distribution of cofilin as well as its interaction with actin in vivo is regulated by its phosphorylation and dephosphorylation.

Actin Depolymerizing Factors↗

PubMed1997

Fascin, a sensitive new marker for Reed-Sternberg cells of hodgkin's disease. Evidence for a dendritic or B cell derivation?

Immunohistochemical localization of human fascin, a distinct 55-kd actin-bundling protein, was determined for a wide variety of lymphoid tissues (364 specimens total). In non-neoplastic tissues, reactivity was highly selective and localized predominantly in dendritic cells. In the thymus, this protein was distinctly localized to medullary dendritic cells. In reactive nodes, interdigitating reticulum cells of T zones, cells in subcapsular areas, and cells of the reticular network were reactive, with variable reactivity observed for follicular dendritic cells. Splenic dendritic cells of the white pulp and sinus-lining cells of the red pulp were reactive. Endothelial cells of all tissues exhibited variable reactivity. Lymphoid cells, myeloid cells, and plasma cells were uniformly nonreactive. In the peripheral blood, only dendritic (veiled) cells were reactive for fascin. A striking finding was observed for cases of Hodgkin's disease (total 187 cases). In all cases of nodular sclerosis (132), mixed cellularity (34), lymphocyte depletion (2), and unclassified types (5), all or nearly all Reed-Sternberg cells and variants were immunoreactive for fascin. Neoplastic cells exhibited strong diffuse cytoplasmic staining and frequently assumed dendritic shapes, particularly in the nodular sclerosis type, producing an interdigitating meshwork or syncytial network of cells. In cases of mixed cellularity type, neoplastic cells generally appeared more discrete. In all 14 cases of nodular lymphocyte predominance type, L&H variants were nonreactive. By contrast, neoplastic lymphoid cells of only 24 of 156 (15%) other lymphoid neoplasms (127 B cell, 27 T cell, and two null cell evaluated) were reactive for fascin. Fascin represents a highly effective marker for detection of certain dendritic cells in normal and neoplastic tissues, is an extremely consistent marker for Reed-Sternberg cells and variants of Hodgkin's disease (except L&H types), and may be helpful to distinguish between Hodgkin's disease and non-Hodgkin's lymphoma in difficult cases. The staining profile for fascin raises the possibility of a dendritic cell derivation, particularly an interdigitating reticulum cell, for the neoplastic cells of Hodgkin's disease, notably in nodular sclerosis type. However, as fascin expression may be induced by Epstein-Barr virus infection of B cells, the possibility that viral induction of fascin in lymphoid or other cell types must also be considered in Epstein-Barr virus-positive cases.

B-Lymphocytes↗

PubMed1996

Mice with disrupted GM2/GD2 synthase gene lack complex gangliosides but exhibit only subtle defects in their nervous system.

Gangliosides, sialic acid-containing glycosphingolipids, are abundant in the vertebrate (mammalian) nervous system. Their composition is spatially and developmentally regulated, and gangliosides have been widely believed to lay essential roles in establishment of the nervous system, especially in neuritogenesis and synaptogenesis. However, this has never been tested directly. Here we report the generation of mice with a disrupted beta 1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase; EC 2.4.1.92) gene. The mice lacked all complex gangliosides. Nevertheless, they did not show any major histological defects in their nervous systems or in gross behavior. Just a slight reduction in the neural conduction velocity from the tibial nerve to the somatosensory cortex, but not to the lumbar spine, was detected. These findings suggest that complex gangliosides are required in neuronal functions but not in the morphogenesis and organogenesis of the brain. The higher levels of GM3 and GD3 expressed in the brains of these mutant mice may be able to compensate for the lack of complex gangliosides.

Animals↗

PubMed1996

High expression of ganglioside alpha-2,8-sialyltransferase (GD3 synthase) gene in adult T-cell leukemia cells unrelated to the gene expression of human T-lymphotropic virus type I.

Expression of ganglioside alpha-2,8-sialyltransferase (GD3 synthetase; EC 2.4.99.8) gene and gangliosides in human leukemia cells were analyzed. Flow cytometric analysis of various types of leukemia cells using monoclonal antibodies revealed that GD3 was expressed at a moderate level on adult T-cell leukemia (ATL) cells and weakly on T-cell acute lymphocytic leukemia cells. Accordingly, high levels of GD3 synthase mRNA in ATL and low levels in T-cell acute lymphocytic leukemia were demonstrated by semiquantitative reverse transcription-PCR analysis. High-level expression of the GD3 synthase gene in ATL cells was detected in the uncultured state, and was not changed during short-term culture in vitro. This was in contrast with the rapid up-regulation of GM2/GD2 synthase gene, which was observed within 24 h after start of culture after the emergence of human T-lymphotropic virus type I (HTLV-I) gene expression in cultured normal T lymphocytes with or without HTLV-I p40tax protein, GD3 synthase gene was almost equally expressed, whereas GM2/GD2 synthase gene was much more strongly expressed in the p40tax-positive T cells. These findings indicated that the GD3 synthase gene was highly expressed in ATL cells unrelated to HTLV-I genome expression.

Acute Disease↗

PubMed1996

Phosphorylation of human fascin inhibits its actin binding and bundling activities.

Human fascin is an actin-bundling protein that is thought to be involved in the assembly of actin filament bundles present in microspikes as well as in membrane ruffles and stress fibers. We have found that human fascin is phosphorylated in vivo upon treatment with 12-O-tetradecanoylphorbol-13-acetate, a tumor promoter. The in vivo phosphorylation is gradually increased from 0.13 to 0.30 mol/mol during 2 h of treatment, concomitant with disappearance of human fascin from stress fibers, membrane ruffles, and microspikes. Human fascin can also be phosphorylated in vitro as judged by phosphopeptide mapping. The extent of phosphorylation depends on pH: the stoichiometries are 0.05, 0.38, and 0.6 alone does not affect fascin-actin binding. With the incorporation of 0.25 mol of phosphate/mol of protein, the actin binding affinity is reduced from 6.7 x 10(6) to 1.5 x 10(6) m(-1). The actin bundling activity is also decreased. These results suggest that phosphorylation of fascin plays a role in actin reorganization after treatment with 12-O-tetradecanoylphorbol-13-acetate.

Actins↗

PubMed1996

High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation.

A novel method for high-efficiency and region- controlled in vivo gene transfer was developed by combining in vivo electroporation and intraarterial plasmid DNA injection. A mammalian expression plasmid for the Escherichia coli lacZ gene (driven with a SV40 early promoter) was injected into the internal carotid artery of rats whose brain tumors (from prior inoculation) had been electroporated between two electrodes. The lacZ gene was efficiently transferred and expressed in the tumor cell 3 days after plasmid injection. However, neither any gene transfers nor any elevated lacZ activity was detected in tissues outside the electrodes. The plasmid was not transferred without electroporation. Human monocyte chemoattractant protein-1 cDNA was also transferred by this method, and its long-lasting (3 weeks) expression was confirmed by using the Epstein-Barr virus episomal replicon system. The expressed monocyte chemoattractant protein-1 protein was functional, as evident by the presence of a large number of monocytes in tumor tissue. This method, electrogene therapy, which does not require viral genes or particles, allows genes to be transferred and expressed in desired organs or tissues, and it may lead to the development of a new type of highly effective gene therapy.

Animals↗

PubMed1996

Site restricted and neuron dominant expression of alpha 2,8sialyltransferase gene in the adult mouse brain and retina.

Gene expression of the alpha 2,8sialyltransferase (alpha 2,8S-T) responsible for GD3 synthesis in the adult mouse brain and retina was analysed by reverse transcription-polymerase chain reaction/Southern blotting (RT-PCR/Southern) and in situ hybridization. Among various portions of the brain, high levels of 9.5 kb mRNA were observed in the retina and midbrain. Results of RT-PCR/Southern did not necessarily correlate with the enzyme activities in the individual sites. In situ hybridization analysis revealed that this gene was characteristically expressed in the inner segment of photoreceptor cells, some nuclei in the midbrain, cranial nerve nuclei in the pons-medulla, Purkinje cells in the cerebellum, pyramidal cells of the hippocampus and granular cells of the dentate gyrus. In the retina, the alpha 2,8S-T gene was broadly expressed over the layers during development, and retained high expression levels in the photoreceptor cells of adult mice consistent with high expression of GD3. Destruction of neurons in the hippocampus and dentate gyrus by injection of kainic acid and colchicine respectively resulted in the disappearance of the hybridization signal, suggesting that the alpha 2,8S-T gene was mainly expressed by neurons.

Aging↗

PubMed1996

A study on the relationship between parity and differentiation of granulated metrial gland cells.

Granulated metrial gland (GMG) cells are a feature consistently associated with rodent pregnancy. Little information is available, however, on the life history of GMG cells in pregnancies other than first pregnancy. To determine if the frequency of GMG cells in the mesometrial triangle of mice is altered by parity, a study of histological sections from pregnant uteri of several females of genotype C.B-17 scid/scid.bg/bg being retired from a breeding program was undertaken. Pregnancies in this study ranged from first to 13th; day 12.5 of gestation was used for all analyses. The frequency of GMG cells in a constant, measured area of the metrial gland on day 12.5 of pregnancy was not significantly altered between first and second pregnancies. A small but statistically significant decline in GMG cell frequency was observed in two females who had given birth to 10 or more litters. However, neither the size of individual day 12.5 GMG cells, nor their granularity changed with parity. The surface area of the placenta in sections was not altered by the decrease in GMG cells. These data suggest that the differentiation of GMG cells from their non-granulated progenitors is a tightly regulated process, that GMG cell progenitors may be fully mature in first pregnancy, and that depletion of GMG cell progenitors is not induced by advanced parity or age.

Animals↗

PubMed1996

Heterogeneity in the expression pattern of two ganglioside synthase genes during mouse brain development.

Gangliosides are synthesized by sequential catalytic reaction of multiple glycosyltransferases. GM2/GD2 synthase and GD3 synthase are key enzymes for ganglioside synthesis, because their relative activities regulate the main profiles of ganglioside expression. Mouse GD3 synthase (EC 2.4.99.8) cDNA was cloned by eukaryotic expression cloning, and its mRNA expression as well as that of GM2/GD2 synthase gene during the development of the mouse CNS was analyzed by using northern blotting, reverse transcription-polymerase chain reaction, and in situ hybridization. When brain tissue was analyzed as a whole mass, a typical pattern corresponding to the reported findings obtained by biochemical analyses was observed, i.e., high expression of GD3 synthase gene in the early stage and gradual increase of GM2/GD2 synthase gene expression in the late stage of the development. However, the results of in situ hybridization of these two genes revealed that the expression kinetics of these two genes were heterogeneous among various sites in the brain under development. These findings suggest that various expression patterns of the two genes reflect differences in the course of the development of individual sites, and also different ganglioside components are required in individual portions of the brain for development and maintenance of the function.

Amino Acid Sequence↗

PubMed1996

Obstructive jaundice caused by non-parasitic hepatic cyst treated with percutaneous drainage and instillation of minocycline hydrochloride as a sclerosing agent.

We report a case of obstructive jaundice caused by a large hepatic cyst which was successfully treated by percutaneous drainage and instillation of minocycline hydrochloride. An 88-year-old man presented with obstructive jaundice and upper abdominal fullness. Abdominal CT and ultrasonography revealed a large hepatic cyst and intrahepatic bile duct dilatation. After the percutaneous drainage of the cyst, minocycline hydrochloride was instilled as a sclerosant via a catheter into the cyst cavity. Liver enzyme levels returned to normal and the jaundice disappeared. Nine months after the treatment, the large cyst and dilatation of the intrahepatic bile duct had disappeared. The patient has remained healthy without symptoms.

Aged↗

PubMed1996

Accounting for the peripartum loss of granulated metrial gland cells, a natural killer cell population, from the pregnant mouse uterus.

Natural killer (NK) cells become a prominent cell population in the rodent uterus during pregnancy. The mature, heavily granulated form of these cells is rare in virgin or postpartum uteri. Death, migration, or dedifferentiation could account for the disappearance of these cells from late gestation uteri. We asked whether uterine NK cells, also known as granulated metrial gland (GMG) cells, die in situ and if expression of Fas antigen is essential for their death. Late in gestation, fragmentation of nuclear DNA was detected histologically by OH-end labeling, as were ultrastructural changes suggesting cell death. NK cells developed in and were lost from the uteri of pregnant Fas antigen-deficient lpr/lpr mice. Postpartum samples of retained placentas contained some residual NK cells that had moved from regions of uterine musculature toward the uterine lumen and were being expelled with the placenta. Thus, both cell death and placental separation remove NK cells from the peripartum uterus.

Animals↗

PubMed1995

The effects of the site-directed removal of N-glycosylation sites from beta-1,4-N-acetylgalactosaminyltransferase on its function.

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

Animals↗

PubMed1995

Substrate specificity of beta 1,4-N-acetylgalactosaminyltransferase in vitro and in cDNA-transfected cells. GM2/GD2 synthase efficiently generates asialo-GM2 in certain cells.

The substrate specificity of beta 1,4-N-acetylgalactosaminyltransferase has been analyzed using a fusion enzyme which consisted of the catalytic domain of the enzyme and the IgG binding domain of protein A, and also by extracts from cDNA transfectants. Both enzyme sources were capable of producing not only GM2 and GD2, but also asialo-GM2, GalNAc-sialylparagloboside, and Gal-NAc-GD1a from appropriate acceptors, although the efficiencies were at most 1-3% of those of GM2/GD2. The biological significance of these low specificities was studied with transient and stable transfectant cells. From the results of transient expression of the cDNA, asialo-GM2 expression appeared to inversely correlate with GM2 synthase levels in those lines. Consequently, GM2 seemed to be preferentially synthesized when both GM3 and lactosylceramide are available, and asialo-GM2 is synthesized in the absence of GM3 synthesis. However, the results of double immunostaining of CHO transfectants with anti-GM2 and anti-asialo-GM2 antibodies indicated that another factor may be involved in asialo-GM2 synthesis. From the in vitro assay using mixed acceptors, it was concluded that the presence of certain levels of GM2 might enhance the asialo-GM2 synthesis. These results suggest that even acceptors showing low efficiencies in vitro might be used in certain cells depending on the availability of precursors, expression levels of other gangliosides, as well as the kinetic properties of the enzyme, and the compartmentation of the glycosylation machineries in the cells.

Animals↗

PubMed1995

Characterization of the COOH terminus of non-muscle caldesmon mutants lacking mitosis-specific phosphorylation sites.

Phosphorylation of rat non-muscle caldesmon by cdc2 kinase causes reduction in most of caldesmon's properties, including caldesmon's binding to actin, myosin, and calmodulin, as well as its inhibition of actomyosin ATPase. We have generated and characterized the COOH terminus of caldesmon mutants lacking mitosis-specific phosphorylation sites, because the COOH-terminal half of caldesmon contains all 7 putative Ser or Thr sites for cdc2 kinase. Codons for the 7 putative Ser or Thr residues have been mutated to Ala, and resultant mutants were bacterially expressed. Analyses of the phosphopeptide maps of these mutants have identified 6 sites, including Ser-249, Ser-462, Thr-468, Ser-491, Ser-497, and Ser-527 as the mitosis-specific phosphorylation sites, whereas the phosphorylation of the remaining site, Thr-377, is not detected by this assay method. Actin binding experiments have suggested that 5 sites including Ser-249, Ser-462, Thr-468, Ser-491, and Ser-497 are important for the phosphorylation-dependent reduction in actin binding. Characterization of a mutant lacking all 7 Ser or Thr sites (7-fold mutant) has revealed that 7-fold mutation eliminates all phosphorylation sites by cdc2 kinase. While the in vitro properties of the 7-fold mutant, including actin, myosin, and calmodulin binding and inhibition of actomyosin ATPase, are very similar to those of nonmutated protein, such properties are not affected by the treatment with cdc2 kinase in contrast to nonmutated protein. This mutant should thus be useful to explore the functions of the mitosis-specific phosphorylation of caldesmon.

Amino Acid Sequence↗

PubMed1995

T cell receptor-mediated stimulation of mouse thymocytes induces up-regulation of the GM2/GD2 synthase gene.

cDNA clones of the mouse GM2/GD2 synthase (EC 2.4.1.92) gene were isolated, and their analyses revealed that the protein has a type II transmembrane structure with 533 amino acids, which was very similar to the human homolog except for the mRNA size. The mRNA level in thymocytes dramatically increased after treatment with anti-CD3 monoclonal antibody, whereas it was not elevated when treated with prostaglandin E2. In situ hybridization showed an elevation of mRNA levels in medullar thymocytes, suggesting that T cell receptor-mediated signaling induces up-regulation of the GM2/GD2 synthase gene in mature thymocytes.

Amino Acid Sequence↗

PubMed1995

Expression of alpha 2,8-sialyltransferase (GD3 synthase) gene in human cancer cell lines: high level expression in melanomas and up-regulation in activated T lymphocytes.

GD3 Synthase (alpha 2,8sialyltransferase) (EC 2.4.99.8) cDNA has been cloned by eukaryotic cell expression cloning. Using this cDNA as a probe, the expression level of the gene in human cancer cell lines was analysed by Northern blotting and RT-PCR, then correlated with the ganglioside expression and enzyme activity. Melanoma cell lines showed extremely strong bands in Northern blot and RT-PCR/Southern analysis. The enzyme activity was also very high in melanomas as expected. Neuroblastoma and astrocytoma lines showed relatively low levels of the gene expression, whereas they expressed high levels of GD2. Although the mRNA level of the GD3 synthase gene and enzyme activity in individual cell lines correlated positively, some cell lines showed much higher activity than expected from the mRNA level. Among leukaemia lines, adult T cell leukaemia-associated (HTLV-I+) lines showed fairly high levels of the mRNA. On the other hand, T-ALL lines showed very low levels. In addition, GD3 and GD2 expression and mRNA level of the gene during T lymphocyte activation were analysed. Only GD3 expression was induced by any of the stimulatory reagents used, and corresponding up-regulation of the GD3 synthase gene was shown in RT-PCR/Southern analysis.

Base Sequence↗

PubMed1995

Diverse expression of beta 1,4-N-acetylgalactosaminyltransferase gene in the adult mouse brain.

Among various tissues of mouse, beta 1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase) gene is expressed predominantly in the brain. Further analysis of the gene expression in the mouse CNS was performed by northern blotting and by enzyme assays using extracts from various parts of the CNS. In situ hybridization was also done to investigate the distribution of cells generating GM2/GD2 synthase. In northern blots, diverse levels of the gene expression were observed, depending on the regions examined. By in situ hybridization, pyramidal cells in the hippocampus, granular cells in dentate gyrus and cerebral cortex, Purkinje cells in cerebellum, and mitral cells in the olfactory bulb expressed high levels of the mRNA; these results corresponded to the results obtained by northern blot. Enzyme levels in these sites were accordingly high. However, enzyme levels in certain areas with low mRNA intensities, such as thalamus and pons medulla, were higher than expected from the results of northern blotting. The significance of the high gene expression in certain areas for brain function and the reason for the discrepancy between mRNA level and enzyme activity in some regions are discussed.

Animals↗