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S Ishii

Publications and source records attributed to S Ishii.

At least 19 recordsLinked to original sources
PubMed1992

Transcriptional regulation by a point mutant of adenovirus-2 E1a product lacking DNA binding activity.

The adenovirus E1a protein (E1A) regulates transcription through interaction with transcription factors bound to DNA, like cAMP response element BP1/ATF2, or through dissociating E2F transcription factor complex. However, it was also reported that E1A can bind to DNA (Chatterjee, P. K., Bruner, M., Flint, S. J., and Harter, M. L. (1988) EMBO J. 7, 835-841), and it is not clear whether DNA binding of E1A is involved in a part of the process of transcriptional regulation by E1A. In this paper, the small region of E1A that is responsible for DNA binding was identified and a point mutant lacking DNA binding activity was constructed. Analysis of deletion mutants of E1A proteins expressed in bacteria showed that a basic region between amino acids 201 and 216 of E1A is essential for DNA binding. Point mutation of arginines at amino acid numbers 205 and 206 to aspartic acids completely abolished the DNA binding activity of E1A. Using this mutant, the requirement of the E1A DNA binding for E1A-dependent transcriptional regulation was examined. trans-Activation of the adenovirus E4 promoter and trans-repression of the human c-erbB-2 promoter by this point mutant were examined by cotransfection experiments. Mutations of the E1A DNA-binding domain affected neither the E1A-induced trans-activation nor trans-repression at all. These results give complete proof that the DNA binding activity of E1A is not required for transcriptional regulation by E1A.

Adenovirus E1A Proteins↗
PubMed1992

Solution structure of a DNA-binding unit of Myb: a helix-turn-helix-related motif with conserved tryptophans forming a hydrophobic core.

The DNA-binding domain of the c-myb protooncogene product consists of three imperfect tandem repeats of 51 or 52 amino acids, each of which contains three conserved tryptophans, spaced 18 or 19 amino acids apart. The structure of the third repeat, which is essential for sequence-specific DNA binding, has been determined by NMR with distance geometry calculation. It includes three well-defined helices (residues 149-162, 166-172, and 178-187) maintained by a hydrophobic core that includes the three conserved tryptophans, together with two histidines. Helices 2 and 3 form a structure related to but distinct from a canonical helix-turn-helix motif. In particular, the turn between these helices is one amino acid longer than the corresponding turn in bacterial repressors and homeodomains and contains a proline residue. In addition, the architecture of the three helices is different from those of homeodomains and DNA-binding domains of bacterial repressors. Based on the present structure, the binding mode of Myb repeat 3 with a specific DNA is also discussed.

Amino Acid Sequence↗
PubMed1992

Hemoglobin-binding site on human haptoglobin. Identification of lysyl residues participating in the binding.

The locus of human haptoglobin (Hp) molecules that interacts with human hemoglobin (Hb) was examined by the differential labeling technique. First, amino groups of Hp were extensively labeled with ethyl acetimidate (EAI) either in the free state (experiment A) or in the equimolar complex with Hb (experiment B). Each of the labeled Hp samples was reduced and S-carboxymethylated, and the remaining amino groups were then allowed to react with trinitrobenzenesulfonic acid (TNBS) under denatured conditions. Only 0.6 mol/mol of the trinitrophenyl (TNP) group was introduced into the heavy chain of Hp in experiment A, whereas 2.5 mol/mol of TNP groups were into the same chain in experiment B. The extent of TNP modification for the light chain was very low in either of the experiments. The amino acid residues carrying TNP groups in the heavy chain were identified by the peptide mapping procedure. They were Ile1, Lys136, and Lys218 in experiment B, and only Ile1 in experiment A. These findings suggest that epsilon-amino groups of Lys136 and Lys218 in the heavy chain are both situated within the Hb-binding locus of Hp, and that the alpha-amino group of Ile1 is buried inside the molecule either in the presence or absence of Hb.

Amino Acid Sequence↗
PubMed1992

Overlap of the p53-responsive element and cAMP-responsive element in the enhancer of human T-cell leukemia virus type I.

The wild-type p53 protein suppresses transformation, but certain missense mutants of p53 can transform cells. Although the wild-type p53 protein contains a transcriptional activation domain, no p53-responsive element has been identified. Here, we identified the p53-responsive element within the Tax-responsive element [21-base-pair (bp) enhancer] of human T-cell leukemia virus type I. Mutation analysis of the 21-bp enhancer indicated that the 16-bp sequence containing the cAMP-responsive element and its surrounding sequence was responsible for p53-induced transactivation. This 16-bp sequence was demonstrated to bind specifically to wild-type human p53 protein in vitro. Using a series of deletion mutants of p53, we showed that almost the entire region of p53 is needed for the transactivating capacity. Furthermore, the transforming mutants of p53 were unable to act as transcriptional activators. The p53-responsive element identified here should be useful to analyze the mechanism by which p53 regulates expression of a set of genes with a negative effect on cellular growth.

Animals↗
PubMed1992

A pSC101-par sequence-mediated study on the intracellular state of supercoiling of the pBR322 genome in Escherichia coli DNA topoisomerase I deletion mutant.

In Escherichia coli DNA topoisomerase I deletion mutant DM800, transcription of the tetracycline-resistance gene (tet) in the pBR322 genome is thought to create and maintain two domains of positive supercoils ahead, and negative supercoils behind, the transcription complex. To assess the actual intracellular state of twin-supercoiled domains, par sequence (365 bp) of plasmid pSC101, which shows a high affinity for DNA gyrase, was inserted into the EcoRI site upstream, or the AvaI site downstream, of the tet gene on the pBR322 genome. Analysis of the oxolinic acid-induced sites of cleavage by gyrase in DM800 revealed that the pBR322 derivatives are highly preferentially cleaved at the par sequence of the EcoRI site as well as the AvaI site and efficiently linearized when compared with pBR322. Assessment of the state of negative supercoiling of the pBR322 derivatives isolated suggested that the DNA (containing the AvaI site) ahead of the tet transcripts, is not so positively supercoiled and preferential interaction of gyrase with the EcoRI-par sequence does not result in removing negative superhelical turns so effectively as DNA topoisomerase I does on pBR322 DNA in the isogenic wild-type cells.

Base Sequence↗
PubMed1992

Dynorphin-degrading cysteine protease is highly specific for paired arginine residues.

The cleavage of dynorphin and three analogs containing paired basic residues by several proteases was investigated. The cysteine protease of neuroblastoma cells cleaved only the bond between Arg-Arg residues. Submandibular arginyl-endopeptidase, however, cleaved bonds between both Arg-Arg and Arg-Lys residues, and pancreatic trypsin at the carboxyl sides of both arginine and lysine residues. This shows that the cysteine protease is highly specific for paired arginine residues.

Amino Acid Sequence↗
PubMed1992

Transactivation and transformation by Myb are negatively regulated by a leucine-zipper structure.

The negative regulatory domain of the c-myb protooncogene product (c-Myb) normally represses transcriptional activation by c-Myb. We show here that a leucine-zipper structure is a component of the negative regulatory domain, because its disruption markedly increases both the transactivating and transforming capacities of c-Myb. We also demonstrate that this leucine-zipper structure can interact with cellular proteins. Our results suggest that an inhibitor that suppresses transactivation binds to c-Myb through the leucine zipper and that c-Myb can be oncogenically activated by missense mutation.

3T3 Cells↗
PubMed1992

Interaction of the Myb protein with specific DNA binding sites.

c-myb is expressed predominantly in proliferating immature hemopoietic cells and, like v-myb, functions as a transcriptional activator that displays sequence-specific DNA binding. Oncogenic activation of the c-myb protein (Myb) is associated with carboxyl-terminal and/or amino-terminal truncations, the former of which also potentiates Myb's transcriptional activation capacity. We show here that a carboxyl-truncated Myb protein binds with a 7-fold higher affinity to an oligonucleotide bearing a Myb recognition sequence than does the full-length form. In addition, data are presented which show that Myb binds with different apparent affinities to variants of the recognition site and that Myb binds independently to adjacent sites regardless of the orientation of these sites.

Animals↗
PubMed1992

Mutagenesis studies on the amino acid residues involved in the iron-binding and the activity of human 5-lipoxygenase.

Human 5-lipoxygenase contains a non-heme iron essential for its activity. In order to determine which amino acid residues are involved in the iron-binding and the lipoxygenase activity, nine amino acid residues in highly homologous regions among the lipoxygenases were individually replaced by means of site-directed mutagenesis. Mutant 5-lipoxygenases in which His-367 or His-550 was replaced by either Asn or Ala, His-372 by either Asn or Ser, or Glu-376 by Gln were completely devoid of the activity. Though mutants containing an alanine residue instead of His-390 or His-399 lacked the activity, the corresponding asparagine substituted mutants exhibited. The other mutants retained the enzyme activity. These results strongly suggest that His-367, His-372, His-550 and Glu-376 are crucial for 5-lipoxygenase activity and coordinate to the essential iron.

Amino Acid Sequence↗
PubMed1992

c-Jun represses the human insulin promoter activity that depends on multiple cAMP response elements.

Glucose is known to increase the cAMP concentration in pancreatic beta cells. To determine the mechanism by which cAMP augments insulin gene expression, we first identified the cAMP response elements (CREs) of the human insulin gene. In DNase I footprint analysis, the bacterially synthesized CRE-binding protein, CRE-BP1, protected four sites: two sites in the region upstream from the insulin core promoter, one site in the first exon, and one site in the first intron. To examine the roles of those four sites, we constructed a series of DNA plasmids in which the wild-type and mutant insulin promoters were linked to the chloramphenicol acetyl-transferase gene. Studies of the transcriptional activity of these plasmids after transfection into hamster insulinoma (HIT) cells showed that these four sites contributed additively to the cAMP inducibility of the insulin promoter. Surprisingly, the c-jun protooncogene product (c-Jun) repressed the cAMP-induced activity of the insulin promoter in a cotransfection assay with the c-Jun expression plasmid. Northern blot analysis demonstrated that the level of c-jun mRNA was dramatically increased by glucose deprivation in HIT cells. These results suggest that glucose may regulate expression of the human insulin gene through multiple CREs and c-Jun.

Animals↗
PubMed1992

Point mutations in the upstream region of the alpha-galactosidase A gene exon 6 in an atypical variant of Fabry disease.

Single point mutations in the upstream region of exon 6 of the alpha-galactosidase A gene were found in two Japanese cases of the cardiac form of Fabry disease; 301Arg----Gln (902G----A) in a case that has already been published and 279Gln----Glu (835C----G) in a new case. They both expressed markedly low, but significant, amounts of residual activity in COS-1 cells. In contrast, two unrelated cases with classic Fabry disease were found to have different point mutations, which showed a complete loss of enzyme activity in a transient expression assay; 328Gly----Arg (982G----A) in the downstream region of exon 6 in one case and two combined mutations, 66Glu----Gln (196G----C)/112Arg----Cys (334C----T), in exon 2 in the other. We conclude, on the basis of the results recorded in this study and those in previous reports, that the pathogenesis of atypical Fabry disease is closely associated with point mutations in the upstream region of exon 6 of the alpha-galactosidase A gene.

Adolescent↗
PubMed1992

Usefulness of a novel monoclonal antibody against human osteocalcin in immunohistochemical diagnosis.

A novel monoclonal antibody against human osteocalcin, recently established in our laboratory, was shown by immunoblotting and immunohistochemistry to react specifically with human osteoblasts. In the present study, the antibody was applied to the immunohistochemical diagnosis of human bone tumours, especially osteoblastic tumours. The antibody reacted with all 27 osteosarcomas. No positive reaction was found either in chondrosarcoma, giant cell tumours of bone, soft tissue tumours or epithelial tumours. A positive reaction was found preferentially in the cytoplasm of most of the osteosarcoma cells, but not in the extracellular matrix. Since the antibody reacted with formalin-fixed and paraffin-embedded tissues, it will be a useful tool for routine immunohistochemical diagnosis of osteoblastic lesions.

Animals↗
PubMed1992

Amplexus induces surge of luteinizing hormone in male toads, Bufo japonicus.

At mating time in the wild, male toads (Bufo japonicus) exhibit a surge in circulating luteinizing hormone (LH) that is thought to result in spermiation. Experiments were performed to identify proximate control of this gonadotropin surge. Male toads migrating to a breeding pond were captured and put in plastic containers either with equal numbers of females or without females. All the males placed with the females clasped them in amplexus that lasted for about 12 hr. During this period, plasma LH and follicle-stimulating hormone levels increased progressively to about 25 and 2 times the initial levels, respectively, and then returned to the initial levels. No significant increases in plasma gonadotropin levels were observed in the males kept without females. These results suggest that the amplexus directly induced the LH surge, but do not preclude control by pheromonal or visual stimuli. To isolate effects of these factors, solitary male toads were kept with dummies of the female. The dummy was a block of "konnyaku," Japanese food whose component is mannan extracted from the root of a plant. Konnyaku is highly elastic, white in color, and virtually odorless. Thus, these dummies felt, but did not look or smell, realistic. All the males clasped their dummy and formed amplexus for 12 hr or more; an LH surge indistinguishable from that with the females was observed. It is concluded that the amplexus itself is the stimulus that induces the LH surge, and subsequent spermiation, in male toads. A strongly male-biased sex ratio, combined with a need to time spermiation precisely with actual mating, may have resulted in the evolution of this neuroendocrine reflex in male Bufo.

Animals↗
PubMed1992

Changes in luteinizing hormone receptors in the granulosa and theca layers of the ovarian follicle during follicular maturation in the Japanese quail.

Attempts were made to reveal changes in number and affinity of LH receptors in the avian ovary during the course of the final maturation of follicles. First, we characterized the hormone binding properties of LH receptors in ovarian follicles of the Japanese quail by incubating a crude plasma membrane preparation of follicle layer with radioiodinated chicken LH prepared by a recently devised method. Specific LH receptors with common binding properties were detected in both granulosa and theca layers of large preovulatory follicles. The binding affinity of the receptors in the quail ovary to chicken LH (Ka: 2.9-17.2 x 10(9) M-1) was similar to that of gonadal LH receptors of other vertebrates. Optimal temperature and pH for the binding of chicken LH to the receptors of quail were also similar to those reported for LH receptors of mammals. Secondly, we compared the bindings of chicken LH to the receptors in the follicular layers of the largest three follicles. Specific binding to the crude plasma membrane preparation of the theca layer differed slightly between the third and second largest follicles, but decreased abruptly to about one-third in the largest follicle that was expected to ovulate within 24 hr. This change was explained mainly by a decrease in the number of receptors. The specific binding of chicken LH to receptors in the granulosa layer did not differ significantly among the largest three follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Annual changes in the binding of follicle-stimulating hormone to gonads and plasma gonadotropin concentrations in Indian weaver birds inhabiting the subtropical zone.

In birds, annual changes in gonadal weight are much more pronounced in the male than in the female. To analyze the mechanism inducing such a sex difference in the gonadal responsiveness to natural environmental conditions, we measured annual changes in the binding of follicle-stimulating hormone (FSH) to the gonads and the plasma gonadotropin concentration in adult Indian weaver birds inhabiting the subtropical zone. The binding of FSH was highly specific for mammalian FSHs and located primarily in the gonad. The testicular weight and FSH binding showed marked changes during the annual breeding cycle. The testicular weight was maximal in the breeding phase (June, 1987) and minimal in the nonbreeding phase (December, 1987). FSH binding per unit testicular weight was greatest in the nonbreeding phase, while the total FSH binding per two testes was maximal in the breeding phase and minimal in the regressive phase (October, 1987). In contrast, the changes in the ovarian weight and FSH binding to the ovary were less pronounced than those in the testis. Although FSH binding per unit ovarian weight showed a peak in the nonbreeding phase, there was no significant change in the total FSH binding per ovary during the year, indicating the presence of a clear sex difference in the total FSH binding. Scatchard plot analyses of the binding suggested that the dissociation constant (Kd) ranged from 0.35 to 1.53 nM regardless of sex and season and that the changes in FSH binding were due to changes in the number of binding sites. Plasma FSH and luteinizing hormone (LH) levels markedly changed during the year in both sexes. Both FSH and LH levels were maximal in the breeding phase and minimal in the nonbreeding phase regardless of sex, although the rate of change in either hormone tended to be greater in the male than in the female.

Animals↗
PubMed1992

Induction of ovarian growth and ovulation by administration of a chicken gonadotrophin preparation to Japanese quail kept under a short-day regimen.

1. Chronic administration of a glycoprotein fraction from chicken pituitary using an ALZET osmotic pump at 12.5 micrograms/h for 2 weeks induced growth of ovarian follicles to a mature size in the ovary and deposition of yolk in sexually immature 6-week-old Japanese quail females which were kept under 8L:16D short-day conditions. However, no ovulation was induced in these birds. 2. Injection of 500 micrograms of the glycoprotein into other immature short-day females was performed following the above chronic administration, 12 or 14 and 15 d after implantation of the osmotic pump. About 24 h after injection, 3 of 14 females laid one or two eggs. Ovulation was confirmed in 4 females by autopsy. A total of 5 of 14 females ovulated 6 eggs, and significant development of the oviduct and the cloacal opening were observed in all of the treated females. 3. Thus, complete ovarian function could be induced in sexually immature female Japanese quail by administration of avian gonadotrophin using a combination of an osmotic pump and an injection.

Animals↗