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A Takatsuki

Publications and source records attributed to A Takatsuki.

At least 55 records · Page 3Linked to original sources
PubMed1991

Brefeldin A inhibits the processing and secretion of envelope glycoproteins of human immunodeficiency virus type 1.

The processing and secretion of the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) were studied in chronically infected T cells and in primary macrophages treated with an antiviral antibiotic brefeldin A (BFA). BFA blocks the egress of proteins from the endoplasmic reticulum and has a profound effect on the structure of cis/medial Golgi. In MOLT-3 cells infected with the IIIB strain of HIV-1 (MOLT-3/IIIB), BFA inhibited the intracellular processing of gp160. The secretion of envelope proteins from these cells was significantly inhibited in the presence of BFA. The gag proteins, on the other hand, were processed and secreted normally. BFA also inhibited the proteolytic processing of gp160 in primary macrophages infected with HIV-1. The infectivity of virus pelleted from the medium of MOLT-3/IIIB cells treated with BFA was markedly lower than that obtained from untreated cells. These results demonstrate that the proteolytic processing of gp160 in HIV-1-infected cells takes place after the glycoprotein exists the endoplasmic reticulum and that the transport of glycoprotein to the cell surface is required for assembly of complete HIV-1 particles.

Antiviral Agents↗
PubMed1991

Brefeldin A blocks an early stage of protein transport in Candida albicans.

Brefeldin A (BFA) inhibited in a dose-dependent manner secretion of the cell-surface enzyme acid phosphatase (APase) into the periplasm of Candida albicans and caused intracellular accumulation of enzyme protein. Cells grown in the presence of BFA became more dense, implying that cell-surface growth was also blocked by BFA treatment. The APase that was accumulated intracellularly migrated faster on SDS-PAGE, suggesting less N-linked glycosylation compared with the mature, periplasmic APase produced in the absence of BFA. Pulse-chase experiments and gel-filtration of oligosaccharides released by Endo H treatment suggested that the core-glycosylated precursor form of APase accumulated in the presence of BFA. These results strongly suggested that endoplasmic reticulum (ER)-to-Golgi transport in C. albicans was inhibited by BFA. Aberrant membrane structures were observed in BFA-treated cells. Within 1 h of BFA removal these structures were replaced with rough ER membranes, suggesting that the accumulated membranes were derived from the ER.

Acid Phosphatase↗
PubMed1990

Degradation of newly synthesized apolipoprotein B-100 in a pre-Golgi compartment.

The synthesis and secretion of apolipoprotein (apo) B-100 have been studied in a human hepatoblastoma cell line, the Hep G2 cells. Pulse-chase analysis showed that apoB-100 was not quantitatively recovered in the culture medium. To reveal the intracellular degradation of apoB-100 prior to secretion, cells were incubated with 1 microgram/ml Brefeldin A (BFA) which impeded protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus and the fate of apoB-100 retained in the cells was traced at 37 degrees C. A significant amount of intracellular apoB-100 (40-60%/h) was degraded during the chase period, whereas apoA-1 remained intact. ApoB-100 degradation was temperature dependent, no degradation was observed below 20 degrees C. This degradation process was not inhibited by chloroquine, leupeptin, pepstatin, and chymostatin, suggesting that lysosomal proteases were not involved and that apoB-100 was degraded in a pre-Golgi compartment which is either part of, or closely related to, the ER. Preincubation of cells with low density lipoproteins (LDL) induced a 22-32% increase in the degradation of apoB-100. This result raised the possibility that secretion of apoB-100 might be regulated through the intracellular degradation of apoB-100. These results suggest the existence of the degradation pathway for apoB-100 in a pre-Golgi compartment and an unique regulatory mechanism for apoB-100 secretion.

Anti-Bacterial Agents↗
PubMed1990

Inhibitory effect of herpes simplex virus infection to target cells on recognition of minor histocompatibility antigens by cytotoxic T lymphocytes.

Target cell lysis by CTL specific for minor histocompatibility Ag (minor HA), which were generated in (C3H/He x BALB/c)F1 mice immunized with A/J mouse spleen cells, was dramatically reduced by infection of HSV to Neuro-2a (A/J mouse origin) cells as target. The reduction was apparent at 5 h after infection of HSV to target cells, when many viral proteins were produced in the cells. Conversely, MHC-restricted HSV-specific CTL-mediated cell lysis increased time dependently. Using an RNA virus, vesicular stomatitis virus, significant reduction of minor specific CTL-mediated target cell lysis was also found. During the time when this reduction of target cell lysis by HSV occurred, the surface expression of class I H-2Dd molecules was maintained, and anti-H-2a allo-MHC-specific CTL lysed HSV-infected Neuro-2a cells as strongly as uninfected Neuro-2a cells. When HSV-infected or uninfected Neuro-2a cells were treated with Brefeldin A that selectively blocks transportation of newly synthesized proteins out of endoplasmic reticulum, both HSV- and minor HA-specific CTL-mediated cell lyses were blocked. These observations demonstrated that minor HA are continuously synthesized and associated with class I molecules at pre-Golgi and transported via trans Golgi system with quick turnover, and that newly synthesized HSV Ag, which are also associated with class I molecules and transported via the same system, should take the place of intrinsic minor HA and be presented on the surface of the cells to be recognized by MHC-restricted CTL.

Animals↗
PubMed1990

Human urinary erythropoietin: preparation with high potency.

Human urinary erythropoietin has been purified to homogeneity. The seven-step procedure yielded a preparation with a potency of 225,000 U/mg protein. SDS-polyacrylamide gel electrophoretic analysis of the purified hormone revealed a single protein band with a molecular weight of about 35,000 that migrated with the biological activity. As to its stability, the purified hormone retained its activity in the presence of 0.001% Tween 20.

Anemia, Aplastic↗
PubMed1990

Morphological effects of brefeldin A on the intracellular transport of secretory materials in parotid acinar cells.

The morphological effects of Brefeldin A (BFA) on the parotid acinar cells of a rat were investigated at the stage of active resynthesis of secretory materials following administration of the secretogogue, isoproterenol. Incubation with BFA resulted in: a) marked dilation of the rough endoplasmic reticulum (RER), b) involution of the Golgi complex to rudimentary forms which disseminated throughout the cytoplasm, and c) agenesis of secretion granules. It appears that the primary action of BFA is inhibition of the export of secretory materials from the RER toward the Golgi complexes. Histochemical staining indicated the thiamine pyrophosphatase (TPPase) positive saccules of the Golgi stack to undergo degradation in autophagic vacuoles. In contrast, small vesicles showing the osmium reducing activity characteristic of cis elements, including osmium negative vesicles, continued to be present throughout a 4-h period of investigation, indicating the cis and, most likely, medial elements to be the components of the rudimentary Golgi complexes. On removal of the drug, a large number of transport vesicles appeared immediately from the RER and carried secretory materials to the rudimentary Golgi complex, so that the organelles were rapidly reconstructed within 30-60 min, followed by the reaccumulation of secretory granules by 90 min. It is thus indicated that the size and configuration of the Golgi complex is regulated by a dynamic equilibrium of the transport of secretory materials, and that the rudimentary Golgi complex containing cis and probably medial elements may function as the smallest units of the Golgi complex for full development as seen under normal conditions.

Animals↗
PubMed1990

Uncoupling of ganglioside biosynthesis by Brefeldin A.

We have studied the effect of Brefeldin A (BFA), an antiviral antibiotic, on glycosphingolipid metabolism in primary cultured cerebellar cells. Cells were labeled metabolically with [14C]galactose, or pulse-labeled with precursors of glycosphingolipid biosynthesis; i.e., [14]serine, [3H]palmitic acid or [3H]sphingosine. In all cases BFA (1 microgram/ml) strongly inhibited (75-95%) ganglioside biosynthesis beyond the stage of GM3 and GD3, that is the formation of GM1, GD1a, GT1b and GQ1b. Simultaneously an accumulation of GlcCer, LacCer, GM3 and GD3 was observed (up to 2000%). These effects could be reversed fully by removal of the BFA from the culture medium. These results indicate that the LacCer-, GM3- and GD3-synthases of murine cerebellar cells are localized together on the proximal site of the Golgi apparatus, probably in the cis-Golgi compartment. It is probable that sphingomyelin synthase and some of the other glycosyltransferases involved in ganglioside biosynthesis are localized in distinct compartments beyond the cis Golgi.

Animals↗
PubMed1988

Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum.

The antiviral antibiotic brefeldin A (BFA) strongly inhibits the protein secretion in cultured rat hepatocytes (Misumi, Y., Misumi, Y., Miki, K., Takatsuki, A., Tamura, G., and Ikehara, Y. (1986) J. Biol. Chem. 261, 11398-11403). We have further examined the inhibitory effect of the drug on intracellular transport of albumin by an immunocytochemical technique with peroxidase-conjugated Fab fragments of anti-rat albumin IgG. In hepatocytes treated with BFA (2.5 micrograms/ml) for 1 h at 37 degrees C, no characteristic structures of the Golgi complex could be observed, and albumin was diffusely distributed in the endoplasmic reticulum (ER), nuclear envelope, and small vesicles around, in contrast to its condensed localization in the Golgi complex in the control cells. Such an unusual distribution of the secretory protein, however, was rearranged to the normal localization in the Golgi complex after 4 h even in the presence of the drug, possibly due to a metabolism of the drug to an inert form. Exposure of the cells to BFA with constant renewals (2.5 micrograms/ml at 1-h intervals) or at a higher concentration (10 micrograms/ml) caused a prolonged accumulation of albumin in the ER, resulting in its dilation. These results indicate that BFA primarily blocks the protein transport from the ER to the Golgi complex, consistent with the biochemical data previously reported.

Animals↗
PubMed1987

Brefeldin A arrests the intracellular transport of a precursor of complement C3 before its conversion site in rat hepatocytes.

The effects of brefeldin A on intracellular transport and posttranslational modification of complement C3 (C3) were studied in primary culture of rat hepatocytes. In the control culture C3 was synthesized as a precursor (pro-C3), which was processed to the mature form with alpha- and beta-subunits before its discharge into the medium. In the presence of brefeldin A the secretion of C3 was strongly blocked, resulting in accumulation of pro-C3. However, after a prolonged interval the mature form of C3 was finally secreted. The results indicate that brefeldin A impedes translocation of pro-C3 to the Golgi complex where pro-C3 is converted to the mature form, but not its proteolytic processing, in contrast to the effects of monensin and weakly basic amines.

Animals↗
PubMed1986

Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes.

We examined the effect of brefeldin A, an antiviral antibiotic, on protein synthesis, intracellular processing, and secretion in primary culture of rat hepatocytes. The secretion was strongly blocked by the drug at 1 microgram/ml and higher concentrations, while the protein synthesis was maintained fairly well. Pulse-chase experiments with [35S]methionine demonstrated that brefeldin A completely blocked the proteolytic conversion of proalbumin to serum albumin up to 60 min of chase, although its conversion was observed as early as 20 min in the control cells. The drug also inhibited the terminal glycosylation of oligosaccharide chains of alpha 1-protease inhibitor and haptoglobin. These two modifications have been shown to occur at the trans region of the Golgi complex. The drug, however, had no effect on the proteolytic processing of the haptoglobin proform which takes place within the endoplasmic reticulum. Such an effect by brefeldin A is very similar with that induced by the carboxylic ionophore monensin. However, in contrast to evidence that monensin causes a delayed secretion of the unprocessed forms of these proteins, brefeldin A allowed the completely processed forms to be secreted after a prolonged accumulation of the unprocessed forms. Morphological observations demonstrated that the endoplasmic reticulum was markedly dilated by treatment with the drug at 10 micrograms/ml which continuously blocked the secretion. On the other hand, brefeldin A caused no inhibitory effect on the endocytic pathway as judged by cellular uptake and degradation of 125I-asialofetuin. These results indicate that brefeldin A is a unique agent which primarily impedes protein transport from the endoplasmic reticulum to the Golgi complex by a mechanism different from those considered for other secretion-blocking agents so far reported.

Adenosine Triphosphate↗
PubMed1986

Amplification of the amyE-tmrB region on the chromosome in tunicamycin-resistant cells of Bacillus subtilis.

In a class of tunicamycin-resistant mutants (tmrA7) of Bacillus subtilis, the production of extracellular alpha-amylase is increased by about five fold. The tmrA7 characteristics (tunicamycin resistance and hyperproduction of extracellular alpha-amylase) can be transferred to recipient cells by transformation. In the transformants and the original tmrA7 mutant, typical amplification of the region from 4 kb upstream of the amyE gene to the tmrB gene on the chromosome was detected. The repeating unit, 16 kb in size, repeats tandemly about five and ten times in the mutant and transformants, respectively, and the alpha-amylase production is proportional to the copy number of the amyE gene. Simultaneous amplification of the tmrB gene, which is responsible for tunicamycin resistance in the multicopy state, and the alpha-amylase structural gene (amyE) seems to be the cause of the pleiotropy of the tmrA7 mutation.

Bacillus subtilis↗
PubMed1982

Tunicamycin-resistant mutations in mouse FM3A cells.

Tunicamycin is an antibiotic that inhibits the oligosaccharide synthesis of glycoproteins. It greatly suppressed the growth of cultured mouse mammary carcinoma FM3A cells, when added to growth medium at concentrations of more than 0.1 microgram/ml. We have developed a single-step selection system for quantitatively detecting mutations resistant to the antibiotic in FM3A cells. Mutant colonies resistant to 1-1.2 micrograms tunicamycin per ml (the optimal concentration of the selecting agent) appeared at a frequency of 10(-4) to 10(-5) in an unmutagenized population, but they increased over 50-fold in the population mutagenized with 0.5 microgram N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) per ml for 2 h and selected under optimal conditions for the time of mutation expression and cell density in selective medium. Fluctuation analysis, by the method of Luria and Delbrück, revealed that tunicamycin-resistant mutations occurred at random during proliferation in normal medium at a rate of 1.2 x 10(-6) per cell per generation. So far 45 spontaneous and MNNG-induced mutant lines have been isolated and serially passaged in the absence of tunicamycin. These mutant lines all inherited their resistance for more than 60 generations. The mutants examined in detail were 12- to 26-fold more resistant than wild-type cells in terms of the D10 value, the concentration of tunicamycin reducing the plating efficiency to 10% of the control. In the hybrids between wild-type and mutant cells the tunicamycin resistance behaved in a co-dominant manner. Tunicamycin inhibited the incorporation of [3H]mannose into the acid-insoluble cell fraction; in this respect, mutant cells were over 30-fold more resistant than wild-type cells. Possible mechanisms of tunicamycin resistance are discussed.

Animals↗
PubMed1982

Effects of tunicamycin on cell adhesion and biosynthesis of glycoproteins in aggregation-competent cells of Dictyostelium discoideum.

The effects of tunicamycin (TM) on cell adhesion and on the biosynthesis of the membrane-associated glycoproteins in aggregation-competent cells of Dictyostelium discoideum were investigated. The cohesiveness due to the EDTA-stable contact site was completely inhibited in the presence of 1 microgram/ml of TM. In contrast, when cells were treated with high concentrations of TM, the EDTA-sensitive contact site in the early developmental stage remained functional. Scanning electron microscopy showed the surface of TM-treated cells to be smoother than that of untreated cells. The membrane proteins of the aggregation-competent cells and TM-treated cells were analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The results showed that several protein bands at 190 kd, 170 kd, 150 kd, 135 kd, 127 kd, 94 kd, 80 kd, 68 kd, 61 kd, and 52 kd which were observed in the untreated cells disappeared in the TM-treated cells, and protein bands at 105 kd and 70 kd were newly detected in the treated cells. Tunicamycin inhibited the incorporation of [3H]mannose or [14C]glucosamine into the trichloroacetic acid (TCA)-insoluble material of the aggregation-competent cells, while it did not affect [3H]leucine incorporation. These results suggest that an asparagine-linked carbohydrate moiety of membrane-associated glycoprotein(s) is involved in cell adhesion of aggregation-competent cells of D. discoideum.

Dictyostelium↗
PubMed1981

Isolation and characterization of Chinese hamster ovary cell mutants with altered sensitivity to high doses of tunicamycin.

A mutant, CTM422, resistant to low dose of tunicamycin (TM) was isolated from Chinese hamster ovary (CHO) cells, and it showed 7- to 10-fold higher resistance to TM than CHO. We further mutagenized CTM422, to isolate TM-high-resistant mutants which were resistant to about 100-fold higher dose of TM than CHO. The TM-high-resistant mutants (N101 and N102) acquired about 4-fold higher cross-resistance to 2-deoxy-d-glucose than CHO or CTM422, while both CHO and CTM422 showed similar sensitivity to 2-deoxy-d-glucose. TM-low-resistance appeared to be codominant, and TM-high-resistance was partially codominant against TM sensitivity, respectively. The transfer activity of N-acetylglucosamine from UDP-N-acetylglucosamine into the lipid fraction with CHO and CTM422 cell extracts was inhibited by TM to a similar extent, while the extract of N102 cells showed about 10-fold higher resistance to TM than CHO or CTM422.

Animals↗
PubMed1981

A colony-stimulating factor produced by mouse L . P3 cells in the presence of tunicamycin or 2-deoxy-D-glucose.

L . P3 cells grown in serum-free synthetic medium produced a colony-stimulating factor (CSF: a sialoglycoprotein stimulating proliferation of granulocyte-macrophage progenitor cells). Addition of tunicamycin (2 microgram/ml) or 2-deoxy-D-glucose (10 mM) to the culture neither decreased the yield of CSF relative to the cell number grown nor induced heterogeneity of the produced CSF. However, CSF produced in the presence of tunicamycin (CSF-tm) was about 23 percent smaller in its molecular weight than normally produced CSF (CSF-normal). Similarly, the addition of 2-deoxy-D-glucose resulted in the formation of a CSF (CSF-dGlc) which was about 16 percent smaller than CSF-normal. However, both CSF-tm and CSF-dGlc seemed to have retained sialic acid residues, because they were focused respectively at pH 4.2 and pH 3.7 upon isoelectric focusing, and both were converted to a pH 5.2 species by treatment with neuraminidase. In addition, CSF-tm was significantly less heat-stable than CSF-normal, whereas CSF-dGlc was only slightly less stable. These results suggest that complete glycosylation of the factor is not necessary for its production nor for its stimulatory action on the proliferation of myeloid stem cells, but is necessary for its maximal stabilization.

Animals↗