Biomedical subjects

Tomoko Yamada

Publications and source records attributed to Tomoko Yamada.

29 records · Page 2Linked to original sources

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

Giant acquired digital fibrokeratoma.

We describe a 33-year-old patient with a giant skin tumor on her right big toe. The tumor was characterized by unusual clinical manifestations including huge size and papillomatous surface. Chronic long-standing irritation as a result of aerobic exercise may have been involved in the development of giant fibrokeratoma with papillomatous surface.

Adult↗

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 case of nonscarring subepidermal blistering disease associated with autoantibodies reactive with both type VII collagen and laminin 5.

A 35-year-old Japanese woman had recurrent, pruritic, vesicular lesions on the face, neck and upper back as well as erosive lesions of the oral cavity and genitalia. The skin and mucosal lesions healed without scarring upon the systemic administration of corticosteroid and azathioprine. Direct immunofluorescence revealed linear deposits of IgG, IgA and C3 at the cutaneous basement membrane zone. Indirect immunofluorescence on 1 M NaCl-split human skin sections demonstrated that the patient's IgG antibodies reacted with the dermal side of the split, while IgA antibodies weakly reacted with the epidermal side. By immunoblot analyses, the patient's serum reacted with the NC1 domain of type VII collagen as well as both the alpha3- and beta3-subunits of laminin 5. We regarded our case as a nonscarring subepidermal blistering disease with autoantibodies to both type VII collagen and two different subunits of laminin 5. Such a case has not been previously reported.

Adult↗

PubMed2003

Pathophysiologic characteristics of the activity-stress paradigm in animal models: inhibitory effect of glucose on these responses.

This review provides a discussion of the pathophysiologic significance of animal models of the activity-stress paradigm and the role of plasma glucose level in the appearance of physical stress responses of those models. Many research reports have demonstrated that animal models exposed to activity-stress are useful as a "symptomatic model" of anorexia nervosa and obsessive-compulsive disorder as well as peptic ulcer. Our findings show that a decrease in plasma glucose concentration is an important factor in determining the activity-stress-induced physical responses. Further investigation of the pathophysiology of activity-stressed animal models may contribute to the development of new therapeutics for diseases such as anorexia nervosa and obsessive-compulsive disorder.

Animals↗

PubMed2003

Embryonic lethal effect of expressing a dominant negative mutant human thyroid hormone receptor alpha1 in mice.

Resistance to thyroid hormone (RTH) is caused mainly by mutations of the thyroid hormone receptor (TR) beta gene. Although, in vitro, TRalpha1 and TRbeta1 mutants exhibit similar dominant negative effects against wild-type TR, no TRalpha mutants have ever been identified in RTH patients. It has been postulated that mutations in TRalpha gene may be lethal, compensated completely by intact TRbeta or associated with phenotypic manifestations different from RTH. To investigate the consequences of mutant TRalpha1 expression in vivo, we tried to generate two different lines of transgenic mice which express a strong or a weak dominant negative mutant TR alpha1, respectively. First, we expressed betaF451X identified in a patient with severe RTH and alphaF397X, which has an identical C-terminal truncation and a similarly strong dominant negative potency to betaF451X, under the control of human polypeptide chain elongation factor 1alpha promoter. Six betaF451X-transgenic mice were born from 223 transferred embryos, giving a transgenic frequency of 2.7%. By contrast, expression of alphaF397X resulted in quite a low transgenic frequency with 0.39% of the transferred embryos bearing the transgene. Only three transgenic mice were born with no apparently overt abnormalities, of which one male produced F1 offspring. The transgenic progeny expressed alphaF397X in the testis but we did not succeed in generating transgenic mice expressing alphaF397X in multiple organs. To avoid toxic effects mediated by a strong dominant negative activity of mutant TRalpha1, we exchanged alphaF397X for alphaK389E, which has an identical missense mutation and a relatively weak transdominant potency as betaK443E identified in a patient with mild RTH. When expressed by cytomegalovirus immediate early enhancer-chicken beta-actin promoter, we did not succeed in creating alphaK389E-transgenic mice despite three independent transgene-injections. These findings define crucial in vivo functions of mutant TRalpha1s during mouse fetal development and suggest the possibility that the expression of a dominant negative mutant TRalpha1 in extensive tissues from the early embryonal stages might be lethal.

Animals↗

PubMed2002

REST4-mediated modulation of REST/NRSF-silencing function during BDNF gene promoter activation.

Neural-restrictive silencer element (NRSE)/repressor element-1 (RE1) regulates neuron-specific gene expression by binding the transcriptional factor REST/NRSF which functions as a silencer in nonneuronal cells. In neuronal cells, a truncated, neuronal-specific REST/NRSF isoform, REST4, has been found but little is known about its function. To address this, we investigated the effect of REST/NRSF and REST4 on the activity-dependent activation of BDNF gene promoter I (BDNFp-I) using cultured rat cortical neurons. REST/NRSF markedly repressed the transcriptional activation of BDNFp-I, whereas the effect of REST4 was weak, depending upon the NRSE/RE1 sequence. In addition, REST4 enhanced the basal transcriptional activity of BDNFp-I. Coexpression of REST4 with REST/NRSF competitively inhibited the silencing effect of REST/NRSF on the activation of BDNFp-I. Although REST4 itself has a weak repressive effect on activation of the BDNF gene via NRSE/RE1, it can compete the silencing effect of REST/NRSF, suggesting a primary role for REST4 in preventing the neuron-specific gene from being inactivated by REST/NRSF and allowing gene activation in response to a variety of neuronal stimuli.

Animals↗

PubMed2002

Prognostic value of thymidylate synthase expression in patients with p-stage I adenocarcinoma of the lung.

We investigated thymidylate synthase (TS) expression in tumor tissues and examined the relationship between TS expression and post-operative survival in patients with p-stage I adenocarcinoma of the lung. A total of 104 patients, who underwent complete resection for p-stage I adenocarcinoma of the lung, were retrospectively reviewed. TS expression in tumor tissues was evaluated by immunohistochemical staining using rhTS polyclonal antibody. The intensity of immunohistochemical staining was classified into four categories using a visual grading system from 0 to 3. The percentage of each grade of TS staining was 9.6% for Grade 0, 18.3% for Grade 1, 35.6% for Grade 2 and 36.5% for Grade 3. Five-year survival rates of patients with Grade 0 to Grade 3 were 90.0, 83.9, 70.3 and 73.7%, respectively with no significant difference among all groups (P=0.236). When divided into two groups, according to the intensity of the grade, 5-year survival rates of TS low expression group (Grade 0 and Grade 1) and TS high expression group (Grade 2 and Grade 3) were 86.1 and 72.0%, respectively, with a significant difference (P=0.048). In conclusion, high level of TS expression was associated with poor prognosis. Immunohistochemical evaluation of TS expression may be useful to predict survival after complete resection in p-stage I adenocarcinoma of the lung.

Adenocarcinoma↗

PubMed2002

Apoptotic tumor-cell death in response to cell proliferation is influenced by p53 status in resected non-small cell lung cancer.

To evaluate the influence of p53 status on postoperative survival and incidence of apoptosis (apoptotic index, AI) in non-small cell lung cancer (NSCLC), a total of 185 pathologic stage I patients were retrospectively analyzed. It was demonstrated by univariate and multivariate analyses that aberrant expression of p53 was a significant factor to predict a poor prognosis, which was caused by a significantly higher proliferative index (PI) in tumor with aberrant expression of p53 (52.7%) than that in tumor without aberrant expression of p53 (37.9%, P < 0.001). In addition, for tumor without aberrant expression of p53, mean AIs of the lowest-, the lower-, the higher-, and the highest-PI groups were 12.6, 12.9, 31.3, and 35.1, respectively, showing that incidence of apoptosis was markedly increased in response to cell proliferation (P = 0.002). In contrast, for tumor with aberrant expression of p53, no increase in incidence of apoptosis in response to cell proliferation was documented. These results clearly demonstrated that active cell proliferation and reduced apoptotic cell death in response to cell proliferation resulted in the poor postoperative prognosis in tumor with aberrant expression of p53.

Adenocarcinoma↗

PubMedDate not supplied

Inhibition of LPS-stimulated NO production in mouse macrophage-like cells by Barbados cherry, a fruit of Malpighia emarginata DC.

The extract of Barbados cherry (acerola fruit), a fruit of Malpighia emarginata DC., has been reported to display diverse biological activities such as prevention of age-related diseases. We investigated here the possible effect of Barbados cherry extract on nitric oxide (NO) production by activated macrophages. Barbados cherry was roughly separated into 4 or 5 fractions by two different methods, using various organic solvents such as hexane, acetone, methanol (70% and 100%) and water, and assayed for its ability to inhibit NO production by lipopolysaccharide (LPS)-stimulated mouse macrophage-like Raw 264.7 cells. Among these fractions, AcOEt extracts (AE0) in Method I and acetone extract (A0) in Method II showed the highest inhibitory activity of NO production (SI > 20 and SI = 31, respectively). When these fractions were subjected to silica gel column chromatography, higher inhibitory activity for NO production was concentrated in AcOEt (AE6) (SI = 64) and benzene-AcOEt (1:4) (A10) fractions (SI > 59). Western blot analysis demonstrated that AE6 and A10 fractions reduced the intracellular concentration of inducible NO synthase (iNOS) by approximately one-third. ESR spectroscopy showed that these fractions scavenged various radical species such as superoxide anion (O2-) and NO radicals. These data suggest that the inhibitory effect on NO production by Barbados cherry extracts is partly due to the inhibition of iNOS expression, and scavenging of O2- and NO radicals.

Animals↗