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T Fukasawa

Publications and source records attributed to T Fukasawa.

At least 37 records · Page 2Linked to original sources
PubMed1999

Steroidal saponins from the bulbs of Allium karataviense.

Chemical examination of the bulbs of Allium karataviense led to the isolation of five new spirostanol saponins (7-11) and a new furostanol saponin (12), together with a known steroidal sapogenin (1) and five known saponins (2-6). The structures of the new saponins were determined by detailed analysis of their spectral data, including two-dimensional NMR spectroscopy. The steroidal saponins produced by A. karataviense, except for 5 and 6, were found to be based upon (25R)-5 alpha-spirostane-2 alpha,3 beta,5,6 beta-tetrol (alliogenin) and contain a beta-D-glucopyranosyl moiety with the formation of an O-glycoside linkage to C-2 of the polyhydroxylated steroidal skeleton as the common structural feature. The isolated compounds were evaluated for cytostatic activity against human promyelocytic leukemia HL-60 cells.

Allium↗
PubMed1999

Microsatellite instability and loss of heterozygosity in primary and secondary proliferative lesions of the parathyroid gland.

Clonality and genetic abnormalities were evaluated to characterize proliferative lesions of the parathyroid gland. Fourteen lesions from patients with single-gland proliferation (adenomas [PA]), 6 lesions from patients with multiple-gland proliferation (primary hyperparathyroidism [PHPT]), and 47 lesions from 16 patients with secondary hyperparathyroidism (SHPT) were examined. Based on the X chromatin inactivation pattern, which was revealed by a HUMARA assay of lesions from female patients (n = 34; 24 informative cases), monoclonality was demonstrated in 6 of 10 PA (60%), 2 of 5 PHPT (40%), and 6 of 9 SHPT lesions (14 of 27 lesions, 52%). By PCR analysis using 17 microsatellite markers on eight chromosomes (chromosomes 1, 2, 3, 5, 6, 11, 13, and 17), loss of heterozygosity was sporadically observed in 4 of 14 PA, 3 of 6 PHPT, and 7 of 47 SHPT lesions, in most cases on a single locus of chromosome 11. On the other hand, microsatellite instability was observed more frequently: ie, in six PA, five PHPT, and nine SHPT lesions. The profile of microsatellite instability depended on the type of proliferation: microsatellite instability (MI) seemed to cluster in the region of chromosome 11 in PA. Microsatellite instability on TP53 was observed in 3 of 6 PHPT lesions and in 2 of 47 SHPT lesions but in no PA lesions. Microsatellite instability on Mfd47 was observed in only some cases of SHPT. Although no significant correlation was identified among histologic features, clonality, and genetic abnormalities in cases of primary proliferation, genetic abnormalities were more frequently observed in SHPT lesions that lacked fat tissues. Thus, genetic instability might be important in proliferative disorders of the parathyroid gland, either with or without uremia. However, genetic instability seems to be induced by different mechanisms in the three types of proliferation studied. In SHPT, the absence of fat tissues may indicate that the proliferation is accompanied by genetic changes.

Adenoma↗
PubMed1998

Functional correlation among Gal11, transcription factor (TF) IIE, and TFIIH in Saccharomyces cerevisiae. Gal11 and TFIIE cooperatively enhance TFIIH-mediated phosphorylation of RNA polymerase II carboxyl-terminal domain sequences.

Saccharomyces cerevisiae Gal11, a component of the holoenzyme of RNA polymerase II, interacts through its functional domains A and B with the small (Tfa2) and large (Tfa1) subunits of the general transcription factor (TF) IIE, respectively. We have recently suggested that Gal11 functions through a common pathway with TFIIE in transcriptional regulation (Sakurai, H., and Fukasawa, T. (1997) J. Biol. Chem. 272, 32663-32669). Here, we report that the activity of the TFIIH-associated kinase, responsible for phosphorylation of the largest subunit of RNA polymerase II at the carboxyl-terminal domain (CTD), is enhanced cooperatively by Gal11 and TFIIE. The enhancement of CTD phosphorylation was observed in the holoenzyme of RNA polymerase II, but not in its core enzyme. The stimulatory effect was completely abolished in the absence of either domain B of Gal11 or the Tfa1 subunit of TFIIE, suggesting that the domain B-Tfa1 interaction is necessary, if not sufficient, for an extensive phosphorylation of the CTD by TFIIH. Stimulation of basal transcription by Gal11 was coupled with enhancement of TFIIH-catalyzed CTD phosphorylation in a cell-free transcription system, suggesting that Gal11 activates transcription by stimulating the CTD phosphorylation in the cell.

Cell Nucleus↗
PubMed1997

Yeast Gal11 and transcription factor IIE function through a common pathway in transcriptional regulation.

The global transcription regulator Gal11, a component of RNA polymerase II holoenzyme, is required for full expression of many genes in yeast. We previously reported that Gal11 binds the small (Tfa2) and large (Tfa1) subunits of the general transcription factor (TF) IIE through Gal11 functional domains A and B, respectively. Here we demonstrate that the C-terminal basic region in Tfa2 is responsible for binding to domain A, whereas both the N-terminal hydrophobic and internal glutamic acid-rich regions in Tfa1 are responsible for binding to domain B. Yeast cells bearing a C-terminal deletion encompassing the Gal11-interacting region in each of the two TFIIE subunits, being viable, exhibited no obvious phenotype. In contrast, combination of the two deletions (TFIIE-DeltaC) showed phenotypes similar to those of gal11 null mutations. The levels of mRNA from TATA-containing genes, but not from TATA-less genes, decreased in TFIIE-DeltaC to an extent comparable to that in the gal11 null mutant. Combination of TFIIE-DeltaC with a gal11 null mutation did not result in an enhanced effect, suggesting that both TFIIE and Gal11 act in a common regulatory pathway. In a reconstituted cell-free system, Gal11 protein stimulated basal transcription in the presence of wild-type TFIIE. Such a stimulation was not seen in the presence of TFIIE-DeltaC.

Cell-Free System↗
PubMed1997

Promoter structure-dependent functioning of the general transcription factor IIE in Saccharomyces cerevisiae.

General transcription factor (TF) IIE is an essential component of the basal transcription complex for protein-encoding genes, which is widely conserved in eukaryotes. Here we analyzed requirement for TFIIE for transcription in vivo by using yeast Saccharomyces cerevisiae cells harboring mutations in the TFA1 gene encoding the larger one of the two subunits of TFIIE. Deletion analysis indicated that the N-terminal half of Tfa1 protein has an essential function to support the cell growth. In a temperature-sensitive tfa1 mutant cell, the steady-state level of bulk poly(A)+ RNA decreased rapidly at the restrictive temperature. Surprisingly, levels of several mRNAs, whose transcription is directed by the promoters lacking the typical TATA sequence, were not affected in the mutant cells at that temperature. This promoter-specific functioning of TFIIE was reproduced in a cell-free system composed of TFIIE-depleted nuclear extracts. These results strongly suggest that requirement for TFIIE varies in each gene depending on the promoter structures in vivo.

Fungal Proteins↗
PubMed1997

Galactose-dependent reversible interaction of Gal3p with Gal80p in the induction pathway of Gal4p-activated genes of Saccharomyces cerevisiae.

When galactose is added to logarithmically growing culture of the yeast Saccharomyces cerevisiae, a set of genes encoding galactose-metabolizing enzymes (GAL genes) starts to be transcribed within a few minutes. This rapid induction involves a serial interplay of Gal3p, Gal80p, and Gal4p. Recent experiments have indicated that a direct interaction between Gal3p and Gal80p plays a pivotal role in an early step of GAL induction. Here we demonstrate that complex of Gal3p and Gal80p, otherwise unstable, is stabilized in the presence of 0.1 mM galactose and 0.5 mM ATP. The requirement for galactose and ATP for stable complex formation is also observed by using highly purified Gal3p and Gal80p from yeast. We further show that thus formed Gal3p/Gal80p complex can easily be dissociated when it is washed with buffer lacking galactose. Finally, we show that mutant proteins encoded by GAL80S or GAL80DE21, which confer galactose-uninducible phenotype, fail to interact with Gal3p. These results strongly suggest that Gal3p functions as the sensor and transducer of galactose signal in the induction pathway of Gal4p-activated genes.

Adenosine Triphosphate↗
PubMed1997

Establishment of internal-image anti-idiotype monoclonal antibodies to a human antibody to lung cancer.

Internal-image anti-idiotype antibodies are expected to enhance anticancer effector mechanisms in vivo. The objective of this study was to establish hybridomas producing anti-idiotype monoclonal antibodies against a human monoclonal antibody (hmAb) 4G12 that reacts strongly with lung squamous cell carcinomas. BALB/c female mice 6 weeks old were immunized with 4G12. Splenocytes were hybridized with P3U1 cells and hybrid cells secreting anti-4G12 hmAb were cloned. Two clones reacted with 4G12 hmAb but not with 3H12 IgM hmAb, human IgM, human serum or fetal calf serum. These two Ab2 antibodies (IgG1 kappa) 2B12 and 2H1 demonstrated 91.5% and 90.3% inhibition in their reactivity with radiolabelled 4G12 on PC10 cells, indicating that 2B12 and 2H1 antibodies were of the Ab2 beta type. In criss-cross inhibition assays, the binding of 2B12 or 2H1 to 4G12 was not inhibited by 2H1 or 2B12. Thus 2B12 and 2H1 were thought to recognize the different epitopes on the antigen-binding sites. Antisera against 2B12 and 2H1 demonstrated specific reactivity to PC10 cells. The two Ab2 beta antibodies, 2B12 and 2H1, express internal images of lung squamous cell carcinoma recognized by the 4G12 antibody and may be useful for cancer immunotherapy.

Animals↗
PubMed1996

Core promoter elements are essential as selective determinants for function of the yeast transcription factor GAL11.

The GAL11 gene product, which copurifies with RNA polymerase II holoenzyme, is necessary for full expression of many, but not all, genes in yeast. Here we shows that the GAL11 dependence of a gene for expression is determined by the core promoter structure. In the GAL80 gene, a gal11 null mutation caused reduction of TATA-dependent transcription, but exerted no effect on initiator-mediated transcription. GAL11 stimulated TATA-dependent transcription, but did not affect the TATA-independent transcription in HIS4. GAL11 was also required for transcription mediated by a canonical TATA sequence but not by a nonconsensus TATA sequence of HIS3. These results suggest that GAL11 is specifically involved in the transcription machinery formed on the TATA element.

Fungal Proteins↗
PubMed1996

The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo function.

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many genes in yeast. The GAL11-encoded protein (Gal11p) has recently been shown to copurify with the holoenzyme of RNA polymerase II. Here we report that Gal11p stimulates basal transcription in a reconstituted transcription system composed of recombinant or highly purified transcription factors, TFIIB, TFIIE, TFIIF, TFIIH, and TATA box-binding protein and core RNA polymerase II. We further demonstrate that each of the two domains of Gal11p essential for in vivo function respectively participates in the binding to the small and large subunits of TFIIE. The largest subunit of RNA polymerase II was coprecipitated by anti-hemagglutinin epitope antibody from crude extract of GAL11 wild type yeast expressing hemagglutinintagged small subunit of TFIIE. Such a coprecipitation of the RNA polymerase subunit was seen but in a greatly reduced amount, if extract was prepared from gal11 null yeast. In light of these findings, we suggest that Gal11p stimulates promoter activity by enhancing an association of TFIIE with the preinitiation complex in the cell.

Binding Sites↗
PubMed1996

Analysis of the galactose signal transduction pathway in Saccharomyces cerevisiae: interaction between Gal3p and Gal80p.

The GAL3 gene plays a critical role in galactose induction of the GAL genes that encode galactose- metabolizing enzymes in Saccharomyces cerevisiae. Defects in GAL3 result in a long delay in GAL gene induction, and overproduction of Gal3p causes constitutive expression of GAL. Here we demonstrate that concomitant overproduction of the negative regulator, Gal80p, and Gal3p suppresses this constitutive GAL expression. This interplay between Gal80p and Gal3p is direct, as tagged Gal3p coimmunoprecipitated with Gal80p. The amount of coprecipitated Gal80p increased when GAL80 yeast cells were grown in the presence of galactose. When both GAL80 and GAL3 were overexpressed, the amount of coprecipitated Gal80p was not affected by galactose. Tagged gal3 mutant proteins bound to purified Gal80p, but only poorly in comparison with the wild type, suggesting that formation of the Gal80p-Gal3p complex depends on the normal function of Gal3p. Gal3p appeared larger in Western blots (immunoblots) than predicted by the published nucleic acid sequence. Reexamination of the DNA sequence of GAL3 revealed several mistakes, including an extension at the 3' end of another predicted 97 amino acids.

Amino Acid Sequence↗
PubMed1994

Yeast GAL11 protein stimulates basal transcription in a gene-specific manner by a mechanism distinct from that by DNA-bound activators.

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many, if not all, genes of the yeast Saccharomyces cerevisiae. We have recently shown that GAL11-encoded protein (Gal11p) enhances basal transcription from the CYC1 promoter in a cell-free transcription system [(1993) Proc. Natl. Acad. Sci. USA 90, 8382-8386]. Here we indicate that Gal11p stimulates basal transcription in a gene-specific manner in vitro. We further suggest that the mechanism underlying the transcriptional stimulation by Gal11p is distinct from that by DNA-bound activators, since Gal11p stimulated transcription in a reaction system where activators were unable to enhance transcription due to the lack of intermediary factors.

Cell-Free System↗
PubMed1994

Purification of yeast histones competent for nucleosome assembly in vitro.

We have developed a procedure to purify nucleosomal assembly-competent histones as a mixture of H2A, H2B, H3 and H4 from isolated nuclei of the yeast Saccharomyces cerevisiae with a purity of 70-80%. The mixture contained each of the histone subunits approximately at the equi-molar ratio. Plasmid pBR322 DNA was assembled into nucleosomes with the purified yeast histones in the presence of nucleoplasmin from unfertilized eggs of the frog Xenopus laevis. The efficiency of assembly of yeast histones was comparable to that of core histones purified from HeLa cells. The length of DNA fragment wrapping around a core histone particle and the molar ratio of histone components in an assembled nucleosome particle were estimated to be 150 +/- 10 bp long and H2A:H2B:H3:H4 = 1.0:0.9:0:9:1.0, respectively.

Cell Nucleus↗
PubMed1994

Two alternative pathways of transcription initiation in the yeast negative regulatory gene GAL80.

The yeast GAL80 gene, encoding a negative regulatory protein of galactose-inducible genes, shows both constitutive and galactose-inducible expression. The inducible transcription is under the control of Gal4p, a common activator for the galactose-inducible genes, which binds to an upstream activation sequence, called UASG, spanning between -105 and -89 in the 5'-flanking region of GAL80. Here we demonstrate that the constitutive transcription started at +1, whereas the inducible transcription occurs from a set of downstream sites at +37, +47, +56, and +67. Both transcriptions were enhanced 10-fold by another UAS, whose 5' boundary is located between -195 and -185. Gal4p stimulated transcription, which depends on the TATA box located at -20, from all the downstream sites. By contrast, the constitutive transcription depended on a small region of less than 16 bp long encompassing the +1 site, which directed transcription even in the absence of both the TATA box and the UASs. When a fragment covering that region was inserted immediately upstream of the open reading frame of HIS3, the resulting gene fusion, if introduced into a his3 yeast strain, supported growth on histidine-lacking medium. We detected by gel retardation assay a protein specifically interacting with this fragment. All the transcriptions observed in the in vivo experiments were faithfully reproduced in a cell-free transcription system. From these results, we suggest that initiation of GAL80 transcription involves two alternative pathways; one is initiator dependent, and the other is Gal4p regulated and TATA dependent.

Base Sequence↗
PubMed1994

[Establishment of anti-idiotype monoclonal antibodies to human anti lung cancer monoclonal antibody (4G12)].

The object of this study is to establish anti-idiotype (anti-ID) monoclonal antibodies (MoAbs) against a human MoAb (4G12) that highly reacts with lung squamous cell carcinomas. Two murine anti-ID MoAbs (2H1 and 2B12) were established by hybridoma technology. They showed specific reactivity with 4G12 but not with 3H12 human MoAb, human IgM and human IgG. These two MoAbs demonstrated more than 90% of binding inhibition of 4G12 to lung squamous cell cancer cell line (PC10). Moreover, cross inhibition test showed that 2H1 and 2B12 detect different idiotypes of 4G12 each other. Furthermore, specific reactivity of anti 2H1 and anti 2B12 sera to PC10 were observed by cell binding ELISA. These two anti-ID MoAbs had internal image of the original antigen.

Animals↗
PubMed1993

Yeast GAL11 protein is a distinctive type transcription factor that enhances basal transcription in vitro.

The yeast auxiliary transcription factor GAL11, a candidate for the coactivator, was partially purified from yeast cells, and its function was characterized in a cell-free transcription system. The partially purified GAL11 protein stimulated basal transcription from the CYC1 core promoter by a factor of 4-5 at the step of preinitiation complex formation. GAL11 protein also enhanced transcription activated by general regulatory factor 1, GAL4-AH, or GAL4-VP16 to the same extent as the basal transcription. Therefore, the apparent potentiation of the activators by GAL11 was attributable to the stimulation of basal transcription. The wild-type GAL11 protein (but not a mutant-type protein) produced in bacteria stimulated transcription as effectively as GAL11 from yeast. These results suggest that GAL11 functions as a positive cofactor of basal and activator-induced transcription in a cell-free transcription system.

Cell Nucleus↗