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R Takada

Publications and source records attributed to R Takada.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cytoskeletal reorganization by soluble Wnt-3a protein signalling.

BACKGROUND: Wnt-3a is an intercellular signalling molecule that is involved in a variety of morphogenetic events. However, the molecular mechanisms underlying Wnt-3a signalling are poorly understood. We have sought to establish in vitro systems to assay the activity of this protein and investigate its biological roles. RESULTS: We prepared mouse L cells transfected with Wnt-3a cDNA, and found that their beta-catenin protein level was up-regulated. When conditioned medium (CM) was collected from cultures of the transfectants and added to nontransfected L cells, the beta-catenin level of the latter was also increased. Approximately 50% of the Wnt-3a proteins synthesized by the transfectants were secreted into the CM in a soluble form. These secreted Wnt-3a proteins formed an activity gradient in the environment surrounding the transfectants. Then, we studied whether Wnt-3a had any effect on cellular behaviour in vitro. When the CM containing Wnt-3a (W3a-CM) was added to cultures of C57MG mammary epithelial cells, their morphology was altered to exhibit closer intercellular contacts. Immunostaining for various adhesion and cytoskeletal proteins showed that the actin-microfilamental system was re-organized by the W3a-CM treatment. It induced a directional alignment of actin stress fibres and other actin-associated proteins. Moreover, villin, localized only at the perinuclear regions in untreated C57MG cells, was re-distributed to the leading edges of the cells, co-localizing with F-actin, in the presence of Wnt-3a. CONCLUSION: Our findings suggest that Wnt-3a protein, in the soluble form, can act to re-organize cytoskeletal structures.

Actins↗
PubMed1998

[Detection of isotype-specific autoantibodies to calpastatin in sera from patients with rheumatic diseases using heat-treated HeLa cell extract as an antigen source for immunoblotting].

To detect immunoglobulin isotype-specific autoantibodies to native human calpastatin in patients with rheumatic diseases, we performed immunoblot analysis using the heated HeLa cell extracts to enrich heat-resistant calpastatin. The calpastatin molecule that was apparently migrated to 110 kD by SDS-PAGE was confirmed to react with monoclonal anti-human calpastatin antibody in immunoblotting. IgG antibodies to calpastatin were detected in 22 of 48 sera (46%) from patients with RA, whereas only 20% (5/25), 11% (2/19) and 13% (2/15) of sera from SLE, SSc and PM/DM had IgG anti-calpastatin antibodies, respectively. IgM antibodies were also found in 40% (19/48) of RA and 12% (3/25) of SLE patients but not detected in sera from patients with other rheumatic diseases. IgA antibodies were found in only one RA and one SLE serum. In RA, 7 of 48 sera (15%) had IgM antibodies alone, but all SLE sera with IgM antibodies had IgG antibodies. Thus, anti-calpastatin autoantibodies were detected by using the native human calpastatin. Although these autoantibodies were found in patients with various rheumatic diseases, they were present in RA patients at the highest frequency. In particular, the presence of IgM antibodies appeared to be more specific in RA patients.

Arthritis, Rheumatoid↗
PubMed1997

Specific interactions and potential functions of human TAFII100.

Human transcription initiation factor TFIID contains the TATA-binding protein (TBP) and several TBP-associated factors (TAFs). To investigate the structural organization and function of TFIID, we have cloned and expressed a DNA encoding the third largest human TFIID subunit, hTAFII100. Immunoprecipitation studies demonstrate that hTAFII100 is an integral subunit that is associated with all transcriptionally-competent forms of TFIID. They further suggest that at least part of the N-terminal region lies on the surface of TFIID, while a C-terminal region containing conserved WD-40 repeats appears inaccessible. Both in vivo and in vitro assays indicate that hTAFII100 interacts strongly with the histone H4-related hTAFII80 and the histone H3-related hTAFII31, as well as a stable complex comprised of both hTAFII80 and hTAFII31. Apparently weaker interactions of hTAFII100 with TBP, hTAFII250, hTAFII28, and hTAFII20, but not hTAFII55, also have been observed. These results suggest a role for hTAFII100 in stabilizing interactions of TAFs, especially the histone-like TAFs, in TFIID. In addition, functional studies show that anti-hTAFII100 antibodies selectively inhibit basal transcription from a TATA-less initiator-containing promoter, relative to a TATA-containing promoter, suggesting a possible core promoter-specific function for hTAFII100.

Amino Acid Sequence↗
PubMed1996

Traumatic cervical tracheal disruption: report of two cases.

We report herein the cases of two patients who suffered tracheal disruption, both of whom underwent successful surgical treatment. The first patient was a 48-year-old truck driver who suffered severe dyspnea after jamming his neck in a truck door. An endotracheal tube was unable to be inserted due to bleeding and thus, an emergency tracheostomy was performed. On admission massive subcutaneous emphysema was noted in the neck and anterior chest, and tracheal disruption was confirmed by a lateral neck X-ray, computed tomography (CT), and fiberscopy. An emergency end-to-end anastomosis of the trachea with insertion of a T-type silicon tube into the lower trachea was performed. The second patient was a 36-year-old man who suffered severe dyspnea after having his neck caught in a chain while driving a motorcycle. On admission, marked subcutaneous emphysema in the neck and paradoxical movement of the trachea were noted. Tracheal disruption was confirmed by a lateral neck X-ray and CT, and a similar operation to that of the first patient was performed. This type of injury is rare; however, lateral neck X-ray, CT, and fiberscopy proved extremely useful for making an accurate diagnosis following which successful emergency surgery was able to be performed, achieving good long-term results.

Adult↗
PubMed1995

Evolutionary conservation of human TATA-binding-polypeptide-associated factors TAFII31 and TAFII80 and interactions of TAFII80 with other TAFs and with general transcription factors.

Human transcription initiation factor TFIID is composed of the TATA-binding polypeptide (TBP) and at least 13 TBP-associated factors (TAFs) that collectively or individually are involved in activator-dependent transcription. To investigate protein-protein interactions involved in TFIID assembly and in TAF-mediated activator functions, we have cloned and expressed cDNAs encoding human TAFII80 and TAFII31. Coimmunoprecipitation assays showed that TAFII80 interacted with TAFII250, TAFII31, TAFII20, and TBP, but not with TAFII55. Similar assays showed that TAFII80 interacted with TFIIE alpha and with TFIIF alpha (RAP74) but not with TFIIB, TFIIE beta, or TFIIF beta (RAP30). Further studies with TAFII80 mutations revealed three distinct interaction domains which fall within regions conserved in human TAFII80, Drosophila TAFII60, and yeast TAFII60. The N terminus of TAFII80 (residues 1-100) interacts with both TAFII31 and TAFII20, while two C-terminal regions are involved, respectively, in interactions with TAFII250 and TFIIF alpha (RAP74) (residues 203-276) and with TBP and TFIIE alpha (residues 377-505). The interactions between TAFII80 and general factors TFIIE alpha and TFIIF alpha (RAP74) could be important for recruitment of GTFs during activator-dependent transcription. Because TAFs 80, 31, and 20 show sequence similarities to histones H4, H3, and H2B, as well as some parallel interactions, this subset of TAFs may form a related core structure within TFIID.

Amino Acid Sequence↗
PubMed1995

Requirement of 19K form of Sonic hedgehog for induction of distinct ventral cell types in CNS explants.

The identity and patterning of ventral cell types in the vertebrate central nervous system depends on cell interactions. For example, induction of a specialized population of ventral midline cells, the floor plate, appears to require contact-mediated signalling by the underlying notochord, whereas diffusible signals from the notochord and floor plate can induce ventrolaterally positioned motor neurons. Sonic hedgehog (Shh), a vertebrate hedgehog-family member, is processed to generate two peptides (M(r) 19K and 26/27K) which are secreted by both of these organizing centres. Moreover, experiments in a variety of vertebrate embryos, and in neural explants in vitro, indicate that Shh can mediate floor-plate induction. Here we have applied recombinant Shh peptides to neural explants in serum-free conditions. High concentrations of Shh bound to a matrix induce floor plate and motor neurons, and addition of Shh to the medium leads to dose-dependent induction of motor neurons. All inducing activity resides in a highly conserved amino-terminal peptide (M(r) 19K). Moreover, antibodies that specifically recognize this peptide block induction of motor neurons by the notochord. We propose that Shh acts as a morphogen to induce distinct ventral cell types in the vertebrate central nervous system.

Animals↗
PubMed1995

Consumption of carbohydrate or medium- or long-chain triglycerides by unfed rats exerts different protein-sparing effects.

Consumption of nonprotein energy is reported to promote nitrogen retention (the protein-sparing effect). The present study was conducted to determine whether consumption of medium-chain triglycerides (MCT), long-chain triglycerides (LCT) or carbohydrate by unfed rats would exert different protein-sparing effects over 48 h. The amounts of MCT, LCT and carbohydrate were adjusted to be isoenergetic. The control group received only water. The urinary excretion of urea in the groups fed carbohydrate or MCT was significantly lower than in the LCT and control groups within the first 24 h. Urinary excretion of urea in the group fed LCT was similar to that of the control group for the first 24 h but was significantly lower after 48 h. Liver serine dehydratase activities in the groups fed MCT or LCT were similar, although they were significantly lower than in the control group. Serine dehydratase activity in the carbohydrate-fed group was the lowest. Liver L-lysine-2-oxoglutarate reductase activity was significantly lower in the groups fed carbohydrate, MCT or LCT than in the control group. Rats fed MCT had the lowest plasma concentrations of lysine and branched-chain amino acids. However, the plasma concentration of glutamic acid in the rats fed MCT was the highest. These results indicate that rats fed MCT, LCT or carbohydrate exhibit different protein-sparing effects and that the difference between rats fed MCT and those fed LCT is not attributable to differences in amino acid-degrading enzymes. Consumption of MCT may conserve some amino acid-derived nitrogen as glutamic acid.

Amino Acids↗
PubMed1995

Proteolytic processing yields two secreted forms of sonic hedgehog.

Sonic hedgehog (Shh) is expressed in tissues with known signalling capacities, such as the notochord, the floor plate of the central nervous system, and the zone of polarizing activity in the limb. Several lines of evidence indicate that Shh is involved in floor plate induction, somite patterning, and regulation of anterior-posterior polarity in the vertebrate limb. In this report, we investigate the biochemical behavior of Shh in a variety of expression systems and embryonic tissues. Expression of mouse Shh in Xenopus oocytes, COS cells, and baculovirus-infected insect cells demonstrates that in addition to signal peptide cleavage and N-linked glycosylation, chicken and mouse Shh proteins undergo additional proteolytic processing to yield two peptides with molecular masses of approximately 19 kDa (amino terminus) and 27 kDa (carboxy terminus), both of which are secreted. In transfected COS cells, we show that the 19-kDa peptide does not accumulate significantly in the medium unless heparin or suramin is added, suggesting that this peptide associates with the cell surface or extracellular matrix. This retention appears to depend on sequences in the carboxy-terminal part of the peptide. Finally, detection of the 19-kDa product in a variety of mouse and chicken embryonic tissues demonstrates that the proteolytic processing observed in cell culture is a normal aspect of Shh processing in embryonic development. These results raise the possibility that amino- and carboxyl-terminal regions of Shh may have distinct functions in regulating cell-cell interactions in the vertebrate embryo.

Amino Acid Sequence↗
PubMed1995

Distribution of Sonic hedgehog peptides in the developing chick and mouse embryo.

Sonic hedgehog (Shh) encodes a signal that is implicated in both short- and long-range interactions that pattern the vertebrate central nervous system (CNS), somite and limb. Studies in vitro indicate that Shh protein undergoes an internal cleavage to generate two secreted peptides. We have investigated the distribution of Shh peptides with respect to these patterning events using peptide-specific antibodies. Immunostaining of chick and mouse embryos indicates that Shh peptides are expressed in the notochord, floor plate and posterior mesenchyme of the limb at the appropriate times for their postulated patterning functions. The amino peptide that is implicated in intercellular signaling is secreted but remains tightly associated with expressing cells. The distribution of peptides in the ventral CNS is polarized with the highest levels of protein accumulating towards the luminal surface. Interestingly, Shh expression extends beyond the floor plate, into ventrolateral regions from which some motor neuron precursors are emerging. In the limb bud, peptides are restricted to a small region of posterior-distal mesenchyme in close association with the apical ectodermal ridge; a region that extends 50-75 microns along the anterior-posterior axis. Temporal expression of Shh peptides is consistent with induction of sclerotome in somites and floor plate and motor neurons in the CNS, as well as the regulation of anterior-posterior polarity in the limb. However, we can find no direct evidence for long-range diffusion of the 19 x 10(3) Mr peptide which is thought to mediate both short- and long-range cell interactions. Thus, either long-range signaling is mediated indirectly by the activation of other signals, or alternatively the low levels of diffusing peptide are undetectable using available techniques.

Animals↗
PubMed1995

[Relationship between birch pollen allergy and oral and pharyngeal hypersensitivity to fruit].

We evaluated the relationship between birch pollen allergy and oral and pharyngeal hypersensitivities to certain fruits. 1. Of 171 birch pollen CAP positive (score > or = 2) patients, twenty two (13%) were revealed to be hypersensitive to apples, eleven (6%) to be hypersensitive to peaches, both rates being higher than those found in patients with other CAP positive reactions (orchard grass pollen CAP, mugwort pollen CAP or Dermatophagoides pteronyssius CAP positive). 2. Among the birch pollen CAP positive patients, the higher the CAP score for birch pollen, the higher the prevalences of hypersensitivity to apples and peaches were found to be. 3. Of 171 birch pollen CAP positive patients, six (3.5%) were revealed to be hypersensitive to kiwi fruit. Of 253 patients with other CAP positive reactions, three (1%) were revealed to be hypersensitive to kiwi fruit.

Adolescent↗
PubMed1994

Dietary gamma-linolenic acid-enriched oil reduces body fat content and induces liver enzyme activities relating to fatty acid beta-oxidation in rats.

The objectives of this study were to examine the effects of dietary gamma-linolenic acid-enriched oil extracted from fungi on rat body composition and on the various enzyme activities relating to fat metabolism in the liver. The oil contained 25.3 g gamma-linolenic acid/100 g fatty acids. The levels of gamma-linolenic acid-enriched oil in the diets were 0, 1.5 and 4%, to give 0, 2.88 and 7.68 g gamma-linolenic acid/kg diet. The control diet contained 8% soybean oil. The rats were given free access to these diets for 4 wk. Body weight gain was less in the gamma-linolenic acid oil-fed groups than in the control group, although food intake was similar among the three groups. Absolute and relative carcass fat weights were significantly lower in the gamma-linolenic acid oil-fed groups than in the control group. Carcass protein and water contents were not different among the three groups, although values were slightly greater than controls in gamma-linolenic acid-fed groups when expressed relative to body weight. Plasma total cholesterol and free fatty acid concentrations generally were lower in the gamma-linolenic acid oil-fed groups than in the control group. In the liver, there were no significant differences in activities of malic enzyme and citrate cleavage enzyme among the three groups. However, the activities of carnitine palmitoyl-transferase and peroxisomal beta-oxidation were significantly higher in the gamma-linolenic acid oil-fed groups than in the control group. These results clearly demonstrate that dietary gamma-linolenic acid oil reduces body fat content and facilitates fatty acid beta-oxidation in the liver.

Adipose Tissue↗
PubMed1993

Identification of TFIID components required for transcriptional activation by upstream stimulatory factor.

A TATA box-binding initiation factor, TFIID, plays a central role in the transcriptional regulation by activators. Using anti-TFIID tau (a TATA box-binding component of native TFIID) immunoaffinity chromatography, nine polypeptides (230, 110, 85, 62, 58, 42, 28, 22, and 21 kDa) were identified as native Drosophila TFIID components that are tightly associated with TFIID tau. To verify the functional activity of the purified TFIID complex, template DNA and other transcription factors were reconstituted with purified TFIID bound to the antibody-Sepharose matrix. Immobilized TFIID mediated not only basal transcription but transcriptional activation by upstream stimulatory factor (USF). On the other hand, recombinant TFIID tau immobilized on the same antibody-Sepharose matrix could not mediate activation by USF. These results suggest that one or more of these additional polypeptides are required as functional TFIID subunits for activator-dependent transcription in conjunction with TFIID tau. As further evidence of the relevance of the Drosophila TFIID components identified in this analysis, including the previously unrecognized p230 (Dynlacht, B. D., Hoey, T., and Tjian, R. (1991) Cell 66, 563-576), protein blot analysis showed that TFIID tau interacts specifically and exclusively with p230. This suggests that p230 is an integral subunit of TFIID and that it may play a major role in tethering other subunits to TFIID tau.

Amino Acid Sequence↗
PubMed1993

Molecular cloning of the cDNAs for the four subunits of mouse DNA polymerase alpha-primase complex and their gene expression during cell proliferation and the cell cycle.

The DNA polymerase alpha-primase complex purified from mouse FM3A cells is composed of four polypeptides with molecular masses of 180, 68, 54, and 46 kDa. The largest subunit has DNA polymerase activity, the two smallest subunits have DNA primase activity, and the function of the 68-kDa subunit is unknown. We have isolated the cDNAs of the four subunits by low stringency hybridization and reverse transcription polymerase chain reaction and determined their nucleotide sequences. The predicted amino acid sequence of the 180-kDa subunit shows 88, 38, 34, and 32% identity to those of the catalytic subunits of human, Drosophila melanogaster, Schizosaccharomyces pombe, and Saccharomyces cerevisiae DNA polymerase alpha, respectively, and contains seven regions whose orders and sequences are highly conserved among viral and other eukaryotic DNA polymerases. The deduced amino acid sequence of the 68-kDa subunit shows 25% identity to that of the 73-kDa subunit of D. melanogaster DNA polymerase alpha-primase, shows no significant sequence similarity to any other protein in the data bases, but contains a potential phosphorylation site(s) for cdc2 kinase. The amino acid sequence of the 54-kDa subunit shows 32% identity to that of the large subunit of S. cerevisiae DNA primase. During activation of quiescent Swiss mouse 3T3 cells to proliferate, the levels of mRNA of the four subunits of the DNA polymerase alpha-primase complex increased before DNA synthesis. In growing mouse FM3A cells, the transcripts of the four subunits are present throughout the cell cycle and increase slightly prior to the S phase.

Amino Acid Sequence↗
PubMed1993

The p250 subunit of native TATA box-binding factor TFIID is the cell-cycle regulatory protein CCG1.

The protein TFIID is a general transcription factor which initiates preinitiation complex assembly through direct interaction with the TATA promoter element. It is a multisubunit complex containing a small TATA-binding polypeptide (TBP) and other TBP-associated factors (TAFs) ranging in size from about 30-250K (refs 7-10). Although native TFIID can mediate both activator-independent (basal) and activator-dependent transcription in reconstituted systems, TBP itself can mediate only basal transcription, even in cases where TBP or the general factor TFIIB are known to interact directly with transcriptional activators. TFIID subunits other than TBP must therefore be essential cofactors, and thus potential targets for activators, consistent with earlier demonstrations that activators interact with TFIID (refs 3, 5, 16, 17). Here we show that the 250K subunit of TFIID is identical to a gene product previously implicated in progression through the late G1 phase of the cell cycle. Part of p250 may thus serve a specific function in the activation of a subset of genes important for cell cycle progression.

Amino Acid Sequence↗
PubMed1993

Molecular cloning, expression, and characterization of the Drosophila 85-kilodalton TFIID subunit.

Transcription initiation factor TFIID is a multimeric protein complex that plays a central role in mediating promoter responses to various activators and repressors. To further understand the role of the 85-kDa TFIID subunit (p85), we have cloned the corresponding cDNA with a probe based on an amino acid sequence of the purified protein. The recombinant p85 interacts directly with both the TATA box-binding subunit (TFIID tau or TBP) and the 110-kDa subunit (p110) of TFIID, suggesting that p85 may play a role in helping to anchor p110 within the TFIID complex and, with other studies, that TFIID assembly and function may involve a concerted series of subunit interactions. Interestingly, the carboxy terminus of p85 contains eight of the WD-40 repeats found originally in the beta subunit of G proteins and more recently in other transcriptional regulatory factors. However, truncated p85 lacking all the WD-40 repeats maintained interactions with both TFIID tau and p110. These observations leave open the possibility of a distinct function for the WD-40 repeats, possibly in transducing signals by interactions with transcriptional regulators and/or other components of the basic transcriptional machinery.

Amino Acid Sequence↗
PubMed1993

Effects of long- and medium-chain triglycerides on amino acid uptake in rat intestinal brush border membrane vesicles.

1. Uptake of L-leucine, L-phenylalanine, L-proline and L-lysine into brush border membrane vesicles from rats fed either a medium-chain triglyceride (MCT) or a long-chain triglyceride (LCT) diet was studied under conditions of the presence of absence of a Na+ gradient. 2. From the results of initial rate, Na(+)-dependent transport in LCT feeding were lower than in feeding MCT. The Na(+)-independent transport did not vary in either group except for L-lysine uptake. 3. For L-leucine, L-phenylalanine and L-proline in Na+ dependence, kinetic analysis revealed 4-6-fold smaller Vmax values in LCT group than in MCT group. L-Lysine in Na(+)-independent transport was 10-fold lower in LCT group than in MCT group. The Km values were not affected by feeding the LCT or MCT diet. 4. It is clear that amino acid transport is regulated by different types of dietary fat. We consider that the alteration of transport activity is attributable to the changes in number of membrane-bound transport carriers but not to their affinity.

Amino Acids↗
PubMed1992

Identification of human TFIID components and direct interaction between a 250-kDa polypeptide and the TATA box-binding protein (TFIID tau).

Previous studies have indicated that human transcription initiation factor TFIID is a large complex that contains a TATA-binding polypeptide (TFIID tau or TBP) and other components that qualitatively alter promoter interactions and are uniquely required for activator-dependent (versus basal) transcription. TFIID tau-specific antibody columns have been employed to identify a number of human TFIID polypeptides that are tightly associated with TFIID tau. These differ in size from polypeptides in known general initiation factors, including the initiator-binding factor (TFII-I) which shares some promoter binding characteristics with TFIID. The largest component (p250) identified in TFIID was shown to interact directly and tightly with TFIID tau, suggesting that it may play a major role in the assembly of the TFIID complex.

DNA-Binding Proteins↗