Influenza virus M1 protein has been shown to inhibit the transcription catalyzed by viral ribonuc... more Influenza virus M1 protein has been shown to inhibit the transcription catalyzed by viral ribonucleoprotein complexes isolated from virions. Here, this inhibition mechanism was studied with the recombinant M1 protein purified from Escherichia coli expressing it from cDNA. RNA mobility shift assays indicated that both soluble and aggregate forms of the recombinant M1, which were separated by the glycerol density gradient, were bound to RNA. Once an M1-RNA complex was formed, free M1 was bound to the M1-RNA complex cooperatively rather than to free RNA. In addition, the recombinant M1 was capable of binding to preformed RNA-nucleocapsid protein complexes. The mechanism for inhibition of the viral RNA polymerase activity was analyzed by the in vitro RNA synthesis systems that depend on an exogenously added RNA template. These systems were more sensitive for evaluating the inhibition by M1 than the RNA synthesis system depending on an endogenous RNA template. The RNA synthesis inhibitio...
Using HeLa whole cell extracts, we have demonstrated that transcription in vitro of the cloned hu... more Using HeLa whole cell extracts, we have demonstrated that transcription in vitro of the cloned human and bovine corticotropin/beta-lipotropin precursor genes is initiated accurately and efficiently. DNA sequences required for promoter function have been assessed by using a series of 5'-deletion mutants of a fusion gene that contains the 5'-flanking sequence and capping site of the human corticotropin/beta-lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The results obtained have shown that the region between 22 base pairs and 35 base pairs upstream from the capping site is essential for the correct and efficient transcriptional initiation in vitro. Thus, the 'TATA box' present in this region seems to be the main promoter element for transcription of the human corticotropin/beta-lipotropin precursor gene in the HeLa cell-free system. We have also developed a transcription system in vitro from the corticotropin-producing mouse pituitary tumor cell line AtT-20 in culture. Deletion mapping of the fusion gene promoter has indicated that the 'TATA box' region is required for the accurate and efficient transcriptional initiation in this system as well. Characteristic of this system is that the deletion of the sequence lying between 53 base pairs and 59 base pairs upstream from the capping site increases the transcriptional efficiency. Because this effect is observed in the AtT-20 cell-free system, but hardly in the HeLa cell-free system, it seems reasonable to assume that the interaction of this upstream sequence with some factor(s) in the AtT-20 cell extract is responsible for the modulation of transcription of the human corticotropin/beta-lipotropin precursor gene.
The human MxA protein is one of the interferon‐inducible proteins that inhibits multiplication of... more The human MxA protein is one of the interferon‐inducible proteins that inhibits multiplication of influenza virus and other viruses. To clarify the control mechanism of its expression, we prepared a series of mutant MxA promoters and identified a 30 nucleotides long cis‐acting interferon‐responsive element by transient transfection assay. Its nucleotide sequence is somewhat similar to that of ISRE (interferon‐stimulated response element), suggesting that the regulation of MxA mRNA synthesis is under the control of some ISRE binding factor such as ISGF‐3 (interferon‐stimulated gene factor‐3).
A series of deletion mutants spanning the adenovirus early region IV (EIV) promoter were tested f... more A series of deletion mutants spanning the adenovirus early region IV (EIV) promoter were tested for transcription activity in vitro. At least three elements were found to be important for maximal transcription in HeLa whole-cell extracts. Deletion of the TATA box drastically reduced the transcription activity from the EIV promoter. Sequences between nucleotides -58 and -44 are also important for efficient transcription since deletion of this region reduced activity by 50%. More importantly, sequences residing upstream from -140 critically influence the level of EIV transcription. Deletion of sequences between nucleotides -325 (the right terminus of adenovirus genome) and -140 reduced the level of transcription more than 10-fold. It is possible that a specific cellular factor stimulates EIV transcription by recognition of these upstream sequences. The dependence of transcription from the EIV promoter on a distal upstream element may explain some aspects of the regulation of this prom...
Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et... more Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980) were tested for their efficiency and fidelity in directing protein synthesis in reticulocyte lysates. Simian virus 40 deoxyribonucleic acid (DNA), cleaved by various restriction endonucleases, was used as the template. Successful translation of the small tumor antigen t, as well as the capsid proteins VP1, VP2, and VP3, was detected by immunoprecipitation analysis. Although no synthesis of large T antigen was detected, use of this technology allows detection of large T synthesis resulting from the correct splicing of as little as 0.2% of the in vitro RNA transcripts, making it ideal for use as an in vitro splicing assay. Transcripts synthesized in vitro were used as messages at least as efficiently as were viral messenger RNA's (mRNA's) synthesized in vivo; and in the case of small t, there was more efficient translation of ...
E4TF1 was originally identified as one of the transcription factors responsible for adenovirus E4... more E4TF1 was originally identified as one of the transcription factors responsible for adenovirus E4 gene transcription. It is composed of two subunits, a DNA binding protein with a molecular mass of 60 kDa and a 53-kDa transcription-activating protein. Heterodimerization of these two subunits is essential for the protein to function as a transcription factor. In this study, we identified a new E4TF1 subunit, designated E4TF1-47, which has no DNA binding activity but can associate with E4TF1-60. We then cloned the cDNAs for each of the E4TF1 subunits. E4TF1 was purified, and the partial amino acid sequence of each subunit was determined. The predicted amino acid sequence of each cDNA clone revealed that E4TF1-60 had an ETS domain, which is a DNA binding domain common to ets-related transcription factors. E4TF1-53 had four tandemly repeated notch-ankyrin motifs. The putative cDNA of E4TF1-47 coded almost the same amino acid sequences as E4TF1-53. Three hundred and thirty-two amino acids...
Immortalized cloned human chondrocytes isolated from a normal (Ch-4, 8, N) and an osteoarthritis ... more Immortalized cloned human chondrocytes isolated from a normal (Ch-4, 8, N) and an osteoarthritis patient (Ch-8-OA) were established by introduction of recombinant SV40adenovirus vector containing SV40 early gene. These cells exhibited continuous proliferative capacity in monolayer culture and showed chondrocytic characteristics in that they were positive for alkaline phosphatase and collagen type II. When cells were treated with IL-la, the growth was inhibited. IL-la induced the production of IL-6, GM-CSF and TNFa from immortalized chondrocytes. Significantly high amounts of cytokines including IL-6, GM-CSF and TNFa were produced from Ch-8-OA cells, even in the absence of IL-la stimulation. Interestingly, TNFa, exogenously added into the culture, inhibited the growth of Ch-8-OA cells. Further studies are required to clarify the different mechanisms on chondro cytes originating from osteoarthritis cartilage underlying the biological reaction to various cytokines and the production of these cytokines as compared with chondrocytes from normal cartilages. However, the novel chondrocyte cell lines established in the present study may provide researchers with a useful model for studying the pathogenesis of osteoarthritis.
Apoptosis is a principal mechanism in metazoans by which superfluous or potentially harmful cells... more Apoptosis is a principal mechanism in metazoans by which superfluous or potentially harmful cells are eliminated. Deregulation of this process leads to a variety of diseases such as cancer and autoimmune diseases. Stimuli that can induce apoptosis are relatively diverse, and include the death factors (Fas ligand, tumor necrosis factor and TRAIL), DNA damage, and oxidative stress. Regardless of the origin of the apoptotic stimulus, commitment to apoptosis leads to activation of caspases, a family of cysteine proteases. Cleavage of a select group of cellular substrates by caspases is responsible for the morphological and biochemical changes that characterize apoptotic cell death. The degradation of nuclear DNA into nucleosomal units is one of the features of apoptotic cell death, and is mediated by a caspase-activated DNase (CAD). Cells deficient in CAD undergo cell death without the DNA fragmentation, but CAD-null mice did not show any adverse phenotypes. A close examination of the apoptotic cells in these mice indicated that apoptotic cells are always in macrophages. It seems that at an early stage of apoptosis, the dying cells present an 'eat me signal' on their surface. This signal is recognized by macrophages for engulfment, and DNase II in the lysosomes of macrophages degrades DNA of apoptotic cells. Mice deficient in both CAD and DNase II genes were established, and the development of various organs was found to be severely impaired in these mutant mice. The mice accumulated a large amount of undigested DNA in macrophages in various tissues during development. This accumulation of DNA in macrophages activated the innate immunity to induce the expression of the interferon β gene. The interferon thus produced seems to be responsible for the impaired tissue development. These results indicate that the degradation of DNA during apoptotic cell death is an essential step of apoptosis to maintain mammalian homeostasis.
Alkylphenols perturb the endocrine system and are considered to have weak estrogenic activities. ... more Alkylphenols perturb the endocrine system and are considered to have weak estrogenic activities. Although it is known that nonylphenol can bind weakly to the estrogen receptor, it is unclear whether all reported effects of nonylphenol are attributable to its estrogen receptor-binding activity. In order to examine whether alkylphenols have similar effects to the natural hormone, estradiol, we used a mouse model to examine the effects of nonylphenol on gene expression and compared it with estradiol. DNA microarray analysis revealed that, in the uterus, most of the genes activated by this alkylphenol at a high dose (50 mg/kg) were also activated by estradiol. At lower doses, nonylphenol (0.5 mg/kg and 5 mg/kg) had little effect on the genes that were activated by estradiol. Thus, we concluded that the effects of nonylphenol at a high dose (50 mg/kg) were very similar to estradiol in uterine tissue. Moreover, since evaluation of estrogenic activity by gene expression levels was comparab...
A basic-leucine zipper transcription factor, MafA, was recently identified as one of the most imp... more A basic-leucine zipper transcription factor, MafA, was recently identified as one of the most important transactivators of insulin gene expression. This protein controls the glucose-regulated and pancreatic beta-cell-specific expression of the insulin gene through a cis-regulatory element called RIPE3b/MARE (Maf-recognition element). Here, we show that MafA expression is restricted to beta-cells of pancreatic islets in vivo and in insulinoma cell lines. We also demonstrate that c-Maf, another member of the Maf family of transcription factors, is expressed in islet alpha-cells and in a glucagonoma cell line (alphaTC1), but not in gamma- and delta-cells. An insulinoma cell line, betaTC6, also expressed c-Maf, albeit at a low level. Chromatin immunoprecipitation assays demonstrated that Maf proteins associate with insulin and glucagon promoters in beta- and alpha-cell lines, respectively. c-Maf protein stimulated glucagon promoter activity in a transient luciferase assay, and activatio...
Temperature-sensitive mutants of human adenovirus types 12 and 5, defective in viral DNA synthesi... more Temperature-sensitive mutants of human adenovirus types 12 and 5, defective in viral DNA synthesis, were able to support growth of adeno-associated virus type I at the non-permissive temperature.
Gold(I)-containing compounds have long been used in the treatment of rheumatoid arthritis (RA), b... more Gold(I)-containing compounds have long been used in the treatment of rheumatoid arthritis (RA), but the molecular mechanism of their action has remained largely unknown. In this paper we have demonstrated that gold(I) drugs selectively activate the DNA binding of a heterodimer consisting of the basic-leucine zipper transcription factors Nrf2 and small Maf. Once bound to its recognition DNA sequence termed antioxidant-responsive element or Maf-recognition element, Nrf2/small Maf induces a set of antioxidative stress genes, including heme oxygenase-1 and ␥-glutamylcysteine synthetase, whose products have been demonstrated to contribute to the scavenging of reactive oxygen species and to exhibit anti-inflammatory effects. Our findings suggest that stimulation of antioxidative stress response through activation of Nrf2/small Maf may be a pharmacologically important part of the actions of gold(I) drugs for the treatment of rheumatoid arthritis. Alternatively, activation of Nrf2/small Maf may be a protective response of cells against toxic effects of the drugs.
Background: Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for ... more Background: Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA binding activators, such as FTZ-F1 and GCN4. MBF1 bridges the DNA-binding regions of these activators and the TATA-box binding protein (TBP), suggesting that MBF1 functions by recruiting TBP to promoters where the activators are bound. In addition, MBF1 stimulates DNA binding activities of the activators to their recognition sites. To date, little is known about structures of coactivators that bind to TBP. Results: The two-dimensional (2D) 1 H-15 N correlation spectrum of 15 N labeled MBF1 indicated that MBF1 consists of both flexible and well structured parts. Limited digestion of MBF1 by ␣-chymotrypsin yielded a ϳ9 kDa fragment. N-terminal sequence analysis and NMR measurements revealed that this fragment originates from the C-terminal 80 residues of MBF1 and form a well structured C-terminal domain of MBF1, MBF1 CTD. As previous deletion analyses have shown that MBF1 CTD is capable of binding to TBP, it is suggested that MBF1 CTD is the TBP binding domain of MBF1. Sequential assignments have been obtained by means of threedimensional (3D) and four dimensional (4D) heteronuclear correlation spectroscopies, and then the secondary structure of MBF1 CTD was determined. As a result, MBF1 CTD was shown to contain four amphipathic helices and a conserved C-terminal region. Asp106 which is assumed to be responsible for the binding to TBP is located at the hydrophilic side of the third helix. Conclusions: Structural analyses revealed that MBF1 consists of two structurally different domains. A N-terminal region is indispensable for the binding to activators, and does not form a well defined structure. In contrast, the C-terminal 80 residues, which is capable of binding to TBP by itself, form a well-structured domain, MBF1 CTD. MBF1 CTD is made up of four amphipathic helices and a conserved C-terminal tail. A putative TBP binding residue is located on the hydrophilic surface of the third helix.
The PAX6 gene is involved in ocular morphogenesis, and PAX6 mutations have been detected in vario... more The PAX6 gene is involved in ocular morphogenesis, and PAX6 mutations have been detected in various types of ocular anomalies, including aniridia, Peters anomaly, corneal dystrophy, congenital cataract, and foveal hypoplasia. The gene encodes a transcriptional regulator that recognizes target genes through its paired-type DNA-binding domain. The paired domain is composed of two distinct DNA-binding subdomains, the N-terminal subdomain (NTS) and the C-terminal subdomain (CTS), which bind respective consensus DNA sequences. The human PAX6 gene produces two alternative splice isoforms that have the distinct structure of the paired domain. The insertion, into the NTS, of 14 additional amino acids encoded by exon 5a abolishes the DNAbinding activity of the NTS and unmasks the DNAbinding ability of the CTS. Thus, exon 5a appears to function as a molecular switch that specifies target genes. We ascertained a novel missense mutation in four pedigrees with Peters anomaly, congenital cataract, Axenfeldt anomaly, and/or foveal hypoplasia, which, to our knowledge, is the first mutation identified in the splicevariant region. A TrA transition at the 20th nucleotide position of exon 5a results in a ValrAsp (GTCrGAC) substitution at the 7th codon of the alternative splice region. Functional analyses demonstrated that the V54D mutation slightly increased NTS binding and decreased CTS transactivation activity to almost half.
INTERMAG Asia 2005. Digests of the IEEE International Magnetics Conference, 2005.
Medical magnetic beads encapsulated in polymer coating with bioactive molecules immobilized on th... more Medical magnetic beads encapsulated in polymer coating with bioactive molecules immobilized on the surface will be used as carriers for the most advanced medical techniques such as bio-screening and proteome analyses. Magnetic separation of the beads expedites the screening and analyses. The performances will be further improved by decreasing the sizes of the beads, thus increasing their specific surface area.
Influenza virus M1 protein has been shown to inhibit the transcription catalyzed by viral ribonuc... more Influenza virus M1 protein has been shown to inhibit the transcription catalyzed by viral ribonucleoprotein complexes isolated from virions. Here, this inhibition mechanism was studied with the recombinant M1 protein purified from Escherichia coli expressing it from cDNA. RNA mobility shift assays indicated that both soluble and aggregate forms of the recombinant M1, which were separated by the glycerol density gradient, were bound to RNA. Once an M1-RNA complex was formed, free M1 was bound to the M1-RNA complex cooperatively rather than to free RNA. In addition, the recombinant M1 was capable of binding to preformed RNA-nucleocapsid protein complexes. The mechanism for inhibition of the viral RNA polymerase activity was analyzed by the in vitro RNA synthesis systems that depend on an exogenously added RNA template. These systems were more sensitive for evaluating the inhibition by M1 than the RNA synthesis system depending on an endogenous RNA template. The RNA synthesis inhibitio...
Using HeLa whole cell extracts, we have demonstrated that transcription in vitro of the cloned hu... more Using HeLa whole cell extracts, we have demonstrated that transcription in vitro of the cloned human and bovine corticotropin/beta-lipotropin precursor genes is initiated accurately and efficiently. DNA sequences required for promoter function have been assessed by using a series of 5'-deletion mutants of a fusion gene that contains the 5'-flanking sequence and capping site of the human corticotropin/beta-lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The results obtained have shown that the region between 22 base pairs and 35 base pairs upstream from the capping site is essential for the correct and efficient transcriptional initiation in vitro. Thus, the 'TATA box' present in this region seems to be the main promoter element for transcription of the human corticotropin/beta-lipotropin precursor gene in the HeLa cell-free system. We have also developed a transcription system in vitro from the corticotropin-producing mouse pituitary tumor cell line AtT-20 in culture. Deletion mapping of the fusion gene promoter has indicated that the 'TATA box' region is required for the accurate and efficient transcriptional initiation in this system as well. Characteristic of this system is that the deletion of the sequence lying between 53 base pairs and 59 base pairs upstream from the capping site increases the transcriptional efficiency. Because this effect is observed in the AtT-20 cell-free system, but hardly in the HeLa cell-free system, it seems reasonable to assume that the interaction of this upstream sequence with some factor(s) in the AtT-20 cell extract is responsible for the modulation of transcription of the human corticotropin/beta-lipotropin precursor gene.
The human MxA protein is one of the interferon‐inducible proteins that inhibits multiplication of... more The human MxA protein is one of the interferon‐inducible proteins that inhibits multiplication of influenza virus and other viruses. To clarify the control mechanism of its expression, we prepared a series of mutant MxA promoters and identified a 30 nucleotides long cis‐acting interferon‐responsive element by transient transfection assay. Its nucleotide sequence is somewhat similar to that of ISRE (interferon‐stimulated response element), suggesting that the regulation of MxA mRNA synthesis is under the control of some ISRE binding factor such as ISGF‐3 (interferon‐stimulated gene factor‐3).
A series of deletion mutants spanning the adenovirus early region IV (EIV) promoter were tested f... more A series of deletion mutants spanning the adenovirus early region IV (EIV) promoter were tested for transcription activity in vitro. At least three elements were found to be important for maximal transcription in HeLa whole-cell extracts. Deletion of the TATA box drastically reduced the transcription activity from the EIV promoter. Sequences between nucleotides -58 and -44 are also important for efficient transcription since deletion of this region reduced activity by 50%. More importantly, sequences residing upstream from -140 critically influence the level of EIV transcription. Deletion of sequences between nucleotides -325 (the right terminus of adenovirus genome) and -140 reduced the level of transcription more than 10-fold. It is possible that a specific cellular factor stimulates EIV transcription by recognition of these upstream sequences. The dependence of transcription from the EIV promoter on a distal upstream element may explain some aspects of the regulation of this prom...
Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et... more Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980) were tested for their efficiency and fidelity in directing protein synthesis in reticulocyte lysates. Simian virus 40 deoxyribonucleic acid (DNA), cleaved by various restriction endonucleases, was used as the template. Successful translation of the small tumor antigen t, as well as the capsid proteins VP1, VP2, and VP3, was detected by immunoprecipitation analysis. Although no synthesis of large T antigen was detected, use of this technology allows detection of large T synthesis resulting from the correct splicing of as little as 0.2% of the in vitro RNA transcripts, making it ideal for use as an in vitro splicing assay. Transcripts synthesized in vitro were used as messages at least as efficiently as were viral messenger RNA's (mRNA's) synthesized in vivo; and in the case of small t, there was more efficient translation of ...
E4TF1 was originally identified as one of the transcription factors responsible for adenovirus E4... more E4TF1 was originally identified as one of the transcription factors responsible for adenovirus E4 gene transcription. It is composed of two subunits, a DNA binding protein with a molecular mass of 60 kDa and a 53-kDa transcription-activating protein. Heterodimerization of these two subunits is essential for the protein to function as a transcription factor. In this study, we identified a new E4TF1 subunit, designated E4TF1-47, which has no DNA binding activity but can associate with E4TF1-60. We then cloned the cDNAs for each of the E4TF1 subunits. E4TF1 was purified, and the partial amino acid sequence of each subunit was determined. The predicted amino acid sequence of each cDNA clone revealed that E4TF1-60 had an ETS domain, which is a DNA binding domain common to ets-related transcription factors. E4TF1-53 had four tandemly repeated notch-ankyrin motifs. The putative cDNA of E4TF1-47 coded almost the same amino acid sequences as E4TF1-53. Three hundred and thirty-two amino acids...
Immortalized cloned human chondrocytes isolated from a normal (Ch-4, 8, N) and an osteoarthritis ... more Immortalized cloned human chondrocytes isolated from a normal (Ch-4, 8, N) and an osteoarthritis patient (Ch-8-OA) were established by introduction of recombinant SV40adenovirus vector containing SV40 early gene. These cells exhibited continuous proliferative capacity in monolayer culture and showed chondrocytic characteristics in that they were positive for alkaline phosphatase and collagen type II. When cells were treated with IL-la, the growth was inhibited. IL-la induced the production of IL-6, GM-CSF and TNFa from immortalized chondrocytes. Significantly high amounts of cytokines including IL-6, GM-CSF and TNFa were produced from Ch-8-OA cells, even in the absence of IL-la stimulation. Interestingly, TNFa, exogenously added into the culture, inhibited the growth of Ch-8-OA cells. Further studies are required to clarify the different mechanisms on chondro cytes originating from osteoarthritis cartilage underlying the biological reaction to various cytokines and the production of these cytokines as compared with chondrocytes from normal cartilages. However, the novel chondrocyte cell lines established in the present study may provide researchers with a useful model for studying the pathogenesis of osteoarthritis.
Apoptosis is a principal mechanism in metazoans by which superfluous or potentially harmful cells... more Apoptosis is a principal mechanism in metazoans by which superfluous or potentially harmful cells are eliminated. Deregulation of this process leads to a variety of diseases such as cancer and autoimmune diseases. Stimuli that can induce apoptosis are relatively diverse, and include the death factors (Fas ligand, tumor necrosis factor and TRAIL), DNA damage, and oxidative stress. Regardless of the origin of the apoptotic stimulus, commitment to apoptosis leads to activation of caspases, a family of cysteine proteases. Cleavage of a select group of cellular substrates by caspases is responsible for the morphological and biochemical changes that characterize apoptotic cell death. The degradation of nuclear DNA into nucleosomal units is one of the features of apoptotic cell death, and is mediated by a caspase-activated DNase (CAD). Cells deficient in CAD undergo cell death without the DNA fragmentation, but CAD-null mice did not show any adverse phenotypes. A close examination of the apoptotic cells in these mice indicated that apoptotic cells are always in macrophages. It seems that at an early stage of apoptosis, the dying cells present an 'eat me signal' on their surface. This signal is recognized by macrophages for engulfment, and DNase II in the lysosomes of macrophages degrades DNA of apoptotic cells. Mice deficient in both CAD and DNase II genes were established, and the development of various organs was found to be severely impaired in these mutant mice. The mice accumulated a large amount of undigested DNA in macrophages in various tissues during development. This accumulation of DNA in macrophages activated the innate immunity to induce the expression of the interferon β gene. The interferon thus produced seems to be responsible for the impaired tissue development. These results indicate that the degradation of DNA during apoptotic cell death is an essential step of apoptosis to maintain mammalian homeostasis.
Alkylphenols perturb the endocrine system and are considered to have weak estrogenic activities. ... more Alkylphenols perturb the endocrine system and are considered to have weak estrogenic activities. Although it is known that nonylphenol can bind weakly to the estrogen receptor, it is unclear whether all reported effects of nonylphenol are attributable to its estrogen receptor-binding activity. In order to examine whether alkylphenols have similar effects to the natural hormone, estradiol, we used a mouse model to examine the effects of nonylphenol on gene expression and compared it with estradiol. DNA microarray analysis revealed that, in the uterus, most of the genes activated by this alkylphenol at a high dose (50 mg/kg) were also activated by estradiol. At lower doses, nonylphenol (0.5 mg/kg and 5 mg/kg) had little effect on the genes that were activated by estradiol. Thus, we concluded that the effects of nonylphenol at a high dose (50 mg/kg) were very similar to estradiol in uterine tissue. Moreover, since evaluation of estrogenic activity by gene expression levels was comparab...
A basic-leucine zipper transcription factor, MafA, was recently identified as one of the most imp... more A basic-leucine zipper transcription factor, MafA, was recently identified as one of the most important transactivators of insulin gene expression. This protein controls the glucose-regulated and pancreatic beta-cell-specific expression of the insulin gene through a cis-regulatory element called RIPE3b/MARE (Maf-recognition element). Here, we show that MafA expression is restricted to beta-cells of pancreatic islets in vivo and in insulinoma cell lines. We also demonstrate that c-Maf, another member of the Maf family of transcription factors, is expressed in islet alpha-cells and in a glucagonoma cell line (alphaTC1), but not in gamma- and delta-cells. An insulinoma cell line, betaTC6, also expressed c-Maf, albeit at a low level. Chromatin immunoprecipitation assays demonstrated that Maf proteins associate with insulin and glucagon promoters in beta- and alpha-cell lines, respectively. c-Maf protein stimulated glucagon promoter activity in a transient luciferase assay, and activatio...
Temperature-sensitive mutants of human adenovirus types 12 and 5, defective in viral DNA synthesi... more Temperature-sensitive mutants of human adenovirus types 12 and 5, defective in viral DNA synthesis, were able to support growth of adeno-associated virus type I at the non-permissive temperature.
Gold(I)-containing compounds have long been used in the treatment of rheumatoid arthritis (RA), b... more Gold(I)-containing compounds have long been used in the treatment of rheumatoid arthritis (RA), but the molecular mechanism of their action has remained largely unknown. In this paper we have demonstrated that gold(I) drugs selectively activate the DNA binding of a heterodimer consisting of the basic-leucine zipper transcription factors Nrf2 and small Maf. Once bound to its recognition DNA sequence termed antioxidant-responsive element or Maf-recognition element, Nrf2/small Maf induces a set of antioxidative stress genes, including heme oxygenase-1 and ␥-glutamylcysteine synthetase, whose products have been demonstrated to contribute to the scavenging of reactive oxygen species and to exhibit anti-inflammatory effects. Our findings suggest that stimulation of antioxidative stress response through activation of Nrf2/small Maf may be a pharmacologically important part of the actions of gold(I) drugs for the treatment of rheumatoid arthritis. Alternatively, activation of Nrf2/small Maf may be a protective response of cells against toxic effects of the drugs.
Background: Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for ... more Background: Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA binding activators, such as FTZ-F1 and GCN4. MBF1 bridges the DNA-binding regions of these activators and the TATA-box binding protein (TBP), suggesting that MBF1 functions by recruiting TBP to promoters where the activators are bound. In addition, MBF1 stimulates DNA binding activities of the activators to their recognition sites. To date, little is known about structures of coactivators that bind to TBP. Results: The two-dimensional (2D) 1 H-15 N correlation spectrum of 15 N labeled MBF1 indicated that MBF1 consists of both flexible and well structured parts. Limited digestion of MBF1 by ␣-chymotrypsin yielded a ϳ9 kDa fragment. N-terminal sequence analysis and NMR measurements revealed that this fragment originates from the C-terminal 80 residues of MBF1 and form a well structured C-terminal domain of MBF1, MBF1 CTD. As previous deletion analyses have shown that MBF1 CTD is capable of binding to TBP, it is suggested that MBF1 CTD is the TBP binding domain of MBF1. Sequential assignments have been obtained by means of threedimensional (3D) and four dimensional (4D) heteronuclear correlation spectroscopies, and then the secondary structure of MBF1 CTD was determined. As a result, MBF1 CTD was shown to contain four amphipathic helices and a conserved C-terminal region. Asp106 which is assumed to be responsible for the binding to TBP is located at the hydrophilic side of the third helix. Conclusions: Structural analyses revealed that MBF1 consists of two structurally different domains. A N-terminal region is indispensable for the binding to activators, and does not form a well defined structure. In contrast, the C-terminal 80 residues, which is capable of binding to TBP by itself, form a well-structured domain, MBF1 CTD. MBF1 CTD is made up of four amphipathic helices and a conserved C-terminal tail. A putative TBP binding residue is located on the hydrophilic surface of the third helix.
The PAX6 gene is involved in ocular morphogenesis, and PAX6 mutations have been detected in vario... more The PAX6 gene is involved in ocular morphogenesis, and PAX6 mutations have been detected in various types of ocular anomalies, including aniridia, Peters anomaly, corneal dystrophy, congenital cataract, and foveal hypoplasia. The gene encodes a transcriptional regulator that recognizes target genes through its paired-type DNA-binding domain. The paired domain is composed of two distinct DNA-binding subdomains, the N-terminal subdomain (NTS) and the C-terminal subdomain (CTS), which bind respective consensus DNA sequences. The human PAX6 gene produces two alternative splice isoforms that have the distinct structure of the paired domain. The insertion, into the NTS, of 14 additional amino acids encoded by exon 5a abolishes the DNAbinding activity of the NTS and unmasks the DNAbinding ability of the CTS. Thus, exon 5a appears to function as a molecular switch that specifies target genes. We ascertained a novel missense mutation in four pedigrees with Peters anomaly, congenital cataract, Axenfeldt anomaly, and/or foveal hypoplasia, which, to our knowledge, is the first mutation identified in the splicevariant region. A TrA transition at the 20th nucleotide position of exon 5a results in a ValrAsp (GTCrGAC) substitution at the 7th codon of the alternative splice region. Functional analyses demonstrated that the V54D mutation slightly increased NTS binding and decreased CTS transactivation activity to almost half.
INTERMAG Asia 2005. Digests of the IEEE International Magnetics Conference, 2005.
Medical magnetic beads encapsulated in polymer coating with bioactive molecules immobilized on th... more Medical magnetic beads encapsulated in polymer coating with bioactive molecules immobilized on the surface will be used as carriers for the most advanced medical techniques such as bio-screening and proteome analyses. Magnetic separation of the beads expedites the screening and analyses. The performances will be further improved by decreasing the sizes of the beads, thus increasing their specific surface area.
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Papers by H. Handa