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Tadashi Wada

Publications and source records attributed to Tadashi Wada.

At least 37 records · Page 2Linked to original sources
PubMed2004

Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II.

Capping of the 5' ends of nascent RNA polymerase II transcripts is the first pre-mRNA processing event in all eukaryotic cells. Capping enzyme (CE) is recruited to transcription complexes soon after initiation by the phosphorylation of Ser-5 of the carboxyl-terminal domain of the largest subunit of RNA polymerase II. Here, we analyze the role of CE in promoter clearance and its functional interactions with different factors that are involved in promoter clearance. FCP1-mediated dephosphorylation of the carboxyl-terminal domain results in a drastic decrease in cotranscriptional capping efficiency but is reversed by the presence of DRB sensitivity-inducing factor (DSIF). These results suggest involvement of DSIF in CE recruitment. Importantly, CE relieves transcriptional repression by the negative elongation factor, indicating a critical role of CE in the elongation checkpoint control mechanism during promoter clearance. This functional interaction between CE and the negative elongation factor documents a dynamic role of CE in promoter clearance beyond its catalytic activities.

Nuclear Proteins↗
PubMed2004

Sialidase and malignancy: a minireview.

Aberrant sialylation in cancer cells is thought to be a characteristic feature associated with malignant properties including invasiveness and metastatic potential. Sialidase which catalyzes the removal of sialic acid residues from glycoproteins and glycolipids, has been suggested to play important roles in many biological processes through regulation of cellular sialic acid contents. The altered expression of sialidase observed in cancer would, therefore, suggest its involvement in the malignant process. In mammalian cells, three types of sialidase cloned and characterized to date were found to behave in different manners during carcinogenesis. Recent progress in molecular cloning of these sialidases has facilitated elucidation of the molecular mechanisms and significance of these alterations. Herein we briefly describe our own studies on sialidase changes associated with malignant transformation and summarize the topic from both a retrospective and a prospective viewpoint. Sialidases are indeed closely related to malignancy and are thus potential targets for cancer diagnosis and therapy.

Animals↗
PubMed2004

Further examination of infants referred from newborn hearing screening.

OBJECTIVES: Universal newborn hearing screening (UNHS) is considered beneficial and is accepted worldwide. However, some problems remain, and administrative systematization has yet to be established in many countries. This study assessed the hearing screening of referred newborn infants and discusses the problems that remain. MATERIALS AND METHODS: Over the two years from July 2001 to June 2003, 98 ears of 49 infants were judged as a "referral" from a newborn hearing screening program, and were subsequently referred to our hospital for further examination using conventional ABR and other audiological tests. The methodology used for hearing screening varied between practitioners and hospitals that utilized both different recording apparatus for AABR and/or automated DPOAEs and independent protocols. RESULTS: Conventional ABR identified 21 infants with bilateral normal hearing, 12 with unilateral hearing loss, and 16 with bilateral hearing loss, and a total correspondence rate of 40.8% (20 out of 49 infants). In a comparative analysis, 26 ears out of 98 (26.5%) were determined as false-positive, seven out of 98 as false-negative (7.1%), and there was a total correspondence rate of 66.3% (65 out of 98 ears). Five of the seven false-negative cases who were referred with unilateral hearing loss exhibited moderate to profound bilateral hearing loss (moderate; one infant, severe to profound; four infants). Of the 16 infants with bilateral hearing loss, nine with more than moderate loss had hearing aids fitted at our hospital or related educational institution before most were six months old. CONCLUSIONS: Our results suggest the accuracy of newborn hearing screening remains an issue, but may be improved by an experienced examiner and better protocols including a two-stage process and altered timing of screening. Other ongoing health care programs need to monitor for signs of hearing loss even in the "passed" infants because of possible false-negatives and delayed-onset hearing loss. Improvement is needed in both the intervention systems and diagnostic follow-up of hospitals. Early public support is also required for infants with either severe to profound or moderate hearing loss. From the viewpoint of test conditions and puerperal parental psychological problems, it is considered that the timing of screening needs further discussion. Here it is suggested that screening should be performed within the first three months of infant's life but not be limited to before hospital discharge, and incorporated into the routine health care program for one-month-old infants without reducing efficiency.

Acoustic Impedance Tests↗
PubMed2004

Bromodomain factor 1 (Bdf1) is phosphorylated by protein kinase CK2.

Bromodomain factor 1 (Bdf1) associates with Saccharomyces cerevisiae TFIID and corresponds to the C-terminal half of higher eukaryotic TAF1. It also associates with the SWR-C complex, which is important for Htz1 deposition. Bdf1 binds preferentially to acetylated histone H4. Bdf1 is phosphorylated, but the mechanism and significance of this modification have been unclear. Two distinct regions within Bdf1 are phosphorylated; one is just C terminal to the bromodomains and the other is near the C terminus. Mutational analysis shows that phosphorylation is necessary for Bdf1 function in vivo. Endogenous protein kinase CK2 purifies with Bdf1 and phosphorylates both domains. A similar mechanism may be responsible for phosphorylation of the C-terminal region of mammalian TAF1. These findings suggest that CK2 phosphorylation of Bdf1 may regulate RNA polymerase II transcription and/or chromatin structure.

Casein Kinase II↗
PubMed2004

Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro.

Recent studies have suggested that Spt6 participates in the regulation of transcription by RNA polymerase II (RNAPII). However, its underlying mechanism remains largely unknown. One possibility, which is supported by genetic and biochemical studies of Saccharomyces cerevisiae, is that Spt6 affects chromatin structure. Alternatively, Spt6 directly controls transcription by binding to the transcription machinery. In this study, we establish that human Spt6 (hSpt6) is a classic transcription elongation factor that enhances the rate of RNAPII elongation. hSpt6 is capable of stimulating transcription elongation both individually and in concert with DRB sensitivity-inducing factor (DSIF), comprising human Spt5 and human Spt4. We also provide evidence showing that hSpt6 interacts with RNAPII and DSIF in human cells. Thus, in vivo, hSpt6 may regulate multiple steps of mRNA synthesis through its interaction with histones, elongating RNAPII, and possibly other components of the transcription machinery.

Animals↗
PubMed2003

Overexpression of plasma membrane-associated sialidase attenuates insulin signaling in transgenic mice.

Plasma membrane-associated sialidase is a key enzyme for ganglioside hydrolysis, thereby playing crucial roles in regulation of cell surface functions. Here we demonstrate that mice overexpressing the human ortholog (NEU3) develop diabetic phenotype by 18-22 weeks associated with hyperinsulinemia, islet hyperplasia, and increased beta-cell mass. As compared with the wild type, insulin-stimulated phosphorylation of the insulin receptor (IR) and insulin receptor substrate I was significantly reduced, and activities of phosphatidylinositol 3-kinase and glycogen synthase were low in transgenic muscle. IR phosphorylation was already attenuated in the younger mice before manifestation of hyperglycemia. Transient transfection of NEU3 into 3T3-L1 adipocytes and L6 myocytes caused a significant decrease in IR signaling. In response to insulin, NEU3 was found to undergo tyrosine phosphorylation and subsequent association with the Grb2 protein, thus being activated and causing negative regulation of insulin signaling. In fact, accumulation of GM1 and GM2, the possible sialidase products in transgenic tissues, caused inhibition of IR phosphorylation in vitro, and blocking of association with Grb2 resulted in reversion of impaired insulin signaling in L6 cells. The data indicate that NEU3 indeed participates in the control of insulin signaling, probably via modulation of gangliosides and interaction with Grb2, and that the mice can serve as a valuable model for human insulin-resistant diabetes.

Adipocytes↗
PubMed2003

Human alpha2,3-sialyltransferase (ST3Gal II) is a stage-specific embryonic antigen-4 synthase.

Monosialosyl globopentaosylceramide (MSGb5), originally described as stage-specific embryonic antigen-4, is expressed in testicular germ cell tumors and in aggressive cases of human renal cell carcinoma (RCC). Clarification of the molecular mechanisms regulating synthesis of MSGb5 is very important to understand testicular carcinogenesis and the malignant progression of human RCC. For this purpose, we have investigated alpha2,3-sialyltransferase involved in the synthesis of MSGb5. We used the method of expression cloning combined with polymerase chain reaction targeted to sialylmotif to isolate a cDNA clone from RCC cell line ACHN library. The cloned cDNA was found to be identical to the previously cloned ST3Gal II in sequence. A soluble recombinant form of the protein in COS-1 cells showed an enzyme activity of alpha2,3-sialyltransferase toward globopentaosylceramide (Gb5) in addition to asialo-GM1 and GM1a. Transient transfection of COS-7 and ACHN cells with this cDNA induced an increase of MSGb5, whereas stable transfection of antisense ST3Gal II cDNA suppressed expression of MSGb5 in ACHN cells. The ST3Gal II mRNA level was increased in 7 of 8 RCC cell lines and in all six RCC tissues surgically obtained, although it was not necessarily consistent with the MSGb5 level in RCC cell lines. This study indicates that ST3Gal II is a MSGb5 (stage-specific embryonic antigen-4) synthase and that its increased expression level is closely related to renal carcinogenesis.

Animals↗
PubMed2003

Bromocriptine augmentation therapy in a patient with Cotard's syndrome.

A 72-year-old female with bipolar I disorder developed Cotard's syndrome, i.e., various delusions of negation accompanied by severe depressive symptoms. She responded neither to the combination of antipsychotic drug and antidepressant nor to the lithium augmentation therapy. However, the delusions and depressive symptoms improved dramatically after the addition of bromocriptine 2.5-5 mg/day to the combination of clomipramine and lithium. This report suggests that bromocriptine augmentation therapy might be effective at least for some patients with Cotard's syndrome in mood disorders.

Aged↗
PubMed2003

Mechanism of H-8 inhibition of cyclin-dependent kinase 9: study using inhibitor-immobilized matrices.

BACKGROUND: Positive transcription elongation factor b (P-TEFb), which phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII), is comprised of the catalytic subunit cyclin-dependent kinase 9 (CDK9) and the regulatory subunit cyclin T. The kinase activity and transcriptional activation potential of P-TEFb is sensitive to various compounds, including H-8, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), and flavopiridol. RESULTS: We investigated the molecular mechanism of the H-8 inhibition of CDK9 using matrices to which H-9, an amino derivative of H-8, was immobilized. CDK9 bound specifically to H-9, and this interaction was competitively inhibited by ATP and DRB, but not by flavopiridol. Mutational analyses demonstrated that the central region of CDK9, which encompasses the T-loop region, was important for its binding to H-9. CONCLUSIONS: H-9-immobilized latex beads are useful for trapping CDK9 and a subset of kinases from crude cell extracts. The flavopiridol-binding region of CDK9 is most likely different from its H-9-binding region. These biochemical data support previously reported observations which were based on crystallographic data.

Adenosine Triphosphate↗
PubMed2003

Structure-function analysis of human Spt4: evidence that hSpt4 and hSpt5 exert their roles in transcriptional elongation as parts of the DSIF complex.

BACKGROUND: The human Spt4/Spt5 complex, termed DRB-sensitivity inducing factor (DSIF) is a dual regulator of transcription that stimulates, or, when cooperating with negative elongation factor (NELF), represses RNA polymerase II (RNAPII) elongation. Spt4 and Spt5 are also thought to be involved in mRNA capping, homologous DNA recombination, and transcription-coupled DNA repair. As a first step to understanding how these proteins regulate diverse cellular processes, we investigated the structure and function of hSpt4 in vitro. RESULTS: Immunodepletion of hSpt5 from HeLa nuclear extracts resulted in the efficient co-depletion of hSpt4. Using DSIF-depleted nuclear extracts and a series of Spt4 mutants, we examined the amino acid sequence of hSpt4 which was important for hSpt5 binding and for transcriptional repression and activation by DSIF. Unexpectedly, the zinc finger of hSpt4, which is critical for the yeast counterpart to function in vivo, was dispensable for hSpt5 binding and for transcriptional regulation in vitro. CONCLUSION: These and other results suggest: (i) that the central region of hSpt4 is necessary and sufficient for its function in vitro and (ii) that there is no free hSpt4 or hSpt5 in cells. We propose that hSpt4 and hSpt5 exert their roles in transcriptional regulation, and possibly in other nuclear processes, as parts of the DSIF complex.

Amino Acid Sequence↗
PubMed2003

A rapid purification method for human RNA polymerase II by two-step affinity chromatography.

The molecular dissection of transcription mechanisms is greatly facilitated by constructing and manipulating defined transcription systems in vitro. This approach requires highly purified transcription factors. A major enzyme participating in the transcription reaction is RNA polymerase II (RNAPII), which is composed of at least 12 subunits (RPB1-12). Due to its complex structure, it is difficult to prepare highly pure RNAPII by the conventional purification procedure. We transfected HeLa cells with a plasmid expressing RPB3 with a double FLAG-histidine tag on its amino-terminus. A high yielding clone was isolated and its extracts were subjected to immunoaffinity purification and then Co(2+) affinity chromatography. This resulted in a preparation of RNAPII complexes that consisted of all the core subunits, including the double-tagged RPB3 protein. Transcription reactions with oligo (dC)-tailed templates and transcription assays involving general transcription factors revealed that the double-tagged RNAPII complexes are active and functional in basal and activated transcription. Our method is superior to the conventionally used purification procedure in that the final preparation is markedly more pure (92% versus 40%), and the procedures are much less time-consuming. Thus, this two-step affinity purification method is an uncomplicated and effective method by which active and functional RNAPII can be prepared.

Cell Line↗
PubMed2003

Human transcription elongation factor NELF: identification of novel subunits and reconstitution of the functionally active complex.

The multisubunit transcription elongation factor NELF (for negative elongation factor) acts together with DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF)/human Spt4-Spt5 to cause transcriptional pausing of RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, A to E. NELF-A has sequence similarity to hepatitis delta antigen (HDAg), the viral protein that binds to and activates RNAPII, whereas NELF-E is an RNA-binding protein whose RNA-binding activity is critical for NELF function. To understand the interactions of DSIF, NELF, and RNAPII at a molecular level, we identified the B, C, and D proteins of human NELF. NELF-B is identical to COBRA1, recently reported to associate with the product of breast cancer susceptibility gene BRCA1. NELF-C and NELF-D are highly related or identical to the protein called TH1, of unknown function. NELF-B and NELF-C or NELF-D are integral subunits that bring NELF-A and NELF-E together, and coexpression of these four proteins in insect cells resulted in the reconstitution of functionally active NELF. Detailed analyses using mutated recombinant complexes indicated that the small region of NELF-A with similarity to HDAg is critical for RNAPII binding and for transcriptional pausing. This study defines several important protein-protein interactions and opens the way for understanding the mechanism of DSIF- and NELF-induced transcriptional pausing.

Amino Acid Sequence↗
PubMed2003

A novel enzyme of Bacillus sp. 217C-11 that produces inulin from sucrose.

We found a bacterium that converts sucrose to a useful material, using about 6,000 samples of bacteria isolated from soil. This bacterium, Bacillus sp. 217C-11, was identified according to Bergey's manual, and produced a highly efficient enzyme that converted sucrose into inulin. So, the enzyme was purified to homogeneity through five chromatographic steps, to identify its enzymatic properties. The molecular mass of the enzyme was estimated to be 45,000, and this enzyme was a monomer protein (by SDS-PAGE). The optimum pH and temperature of this enzyme were 7-8 and 45-50 degrees C, respectively. The enzyme reacted only with sucrose, but did not with other disaccharides, fructooligosaccharides and inulin. This paper will show that our enzyme is a novel one, which is different from the other well-known enzymes concerned in inulin production.

Amino Acid Sequence↗
PubMed2003

[A device for tracheostomized children in home care].

Tracheomized children with deglutition disturbance require frequent suction--a procedure that may present problems for parents and other home-caregivers. We introduced a suction pump to drain the thoracic cavity continuously. This reduced the number of times of suction was required and raised no problems.

Child↗
PubMed2003

[Discharge and introduction to home care for 70-year-old terminally-ill patient with metastatic gastrointestinal cancer].

There are many problems associated with the discharge and management of terminally-ill patients with cancer, A 70-year-old patient with gastro-intestinal cancer obstructing his intestinal tract, who is receiving total parenteral nutrition through a central intravenous catheter is being prepared for his discharge from hospital. The effective management of the discharge of this patient and introduction to home care, consisting of visits by physicians and community nurses is described.

Aged↗
PubMed2003

[Discharge and introduction to home care for 70 year old terminally ill patient with metastatic gastrointestinal cancer].

There are many problems associated with the discharge and management of terminally ill patients with cancer. A 70-year-old patient with gastrointestinal cancer obstructing his intestinal tract, who is receiving total parenteral nutrition through a central intravenous catheter is being prepared for his discharge from hospital. The effective management of the discharge of this patient and introduction to home care, consisting of visits by physicians and community nurses is described.

Aged↗