Biomedical subjects

H Handa

Publications and source records attributed to H Handa.

At least 127 records · Page 7Linked to original sources

PubMed1996

Copurification of casein kinase II with transcription factor ATF/E4TF3.

We have developed a simple method to purify sequence-specific DNA-binding proteins directly from crude cell extracts by using DNA affinity latex beads. The method enabled us to purify not only DNA-binding proteins, but also their associated proteins. Using beads bearing the ATF/E4TF3 site from the adenovirus E4 gene promoter, a protein kinase activity was copurified with the ATF/E4TF3 family. We found that the kinase interacted with ATF1 in vitro efficiently. The kinase did not bind directly to DNA. The kinase mainly phosphorylated ATF1 on serine 36, which was one of target amino acids for casein kinase (CK) II. Biological features of the kinase were the same as those of CKII and an anti-CKII serum reacted with the kinase, indicating that the kinase was CKII. Moreover, it was clearly shown that one of CKII subunits, the CKII alpha protein bound to glutathione-S-transferase (GST) fusion ATF1 but not GST in vitro. It has been reported that a specific CKII inhibitor, 5,6-dichloro-1-beta-D-ribo-furanosylbenzimidazole (DRB) inhibits transcription by RNA polymerase II [Zandomeni et al., (1986) J. Biol. Chem. 261, 3414-3419]. Taken together, these results suggest that ATF/E4TF3 may recruit the CKII activity to a transcription initiation machinery and stimulate transcription.

Activating Transcription Factors↗

PubMed1996

Selective Isolation of DNA or RNA Using Single-Stranded DNA Affinity Latex Particles.

We developed latex particles with a diameter of about 0.22 µm on which single-stranded (ss) DNA was covalently coupled to select or enrich its complementary DNA or mRNA. DNA was first covalently coupled to the latex particles in the double-stranded (ds) form with both blunt and protruding ends. More than 80% of the dsDNA was coupled through the ssDNA stretch at its protruding end. The presence of NaCl in the immobilization reaction severely inhibited DNA from coupling to the particles. The particles were then treated with alkali or heated to denature the dsDNA and sedimented by a brief centrifugation to yield ssDNA immobilized particles. They allowed the selective and efficient isolation of a desired RNA from total cellular RNA.

Journal Article↗

PubMed1996

Detection of amplified genomic sequences in human small-cell lung carcinoma cells by arbitrarily primed-PCR genomic fingerprinting.

The arbitrarily primed-PCR (AP-PCR) genomic fingerprinting method was applied to evaluate its effectiveness in detecting and characterizing amplified DNA fragments in two small-cell lung carcinoma (SCLC) cell lines, NCI-H69 and NCI-H82. Of the 2428 DNA fragments detected by AP-PCR using 62 arbitrary primers, 2 (0.08%) DNA fragments were amplified in NCI-H69 and 6 (0.25%) DNA fragments were amplified in NCI-H82. Based on these results, we estimate the total size of the amplified genomic regions in these cell lines to be 3000 megabase pairs (Mb) x 0.0008 = 2.4 Mb in NCI-H69 and 3000 Mb x 0.0025 = 7.5 Mb in NCI-H82. The 2 amplified fragments in NCI-H69 were mapped to chromosome 2, and all 6 amplified fragments in NCI-H82 were mapped to chromosome 8. This strongly suggests that restricted chromosomal regions are specifically amplified in these SCLC cell lines. Since the N-myc gene at 2p24 is amplified in NCI-H69 and the c-myc gene at 8q24 is amplified in NCI-H82, it is possible that these DNA fragments are co-amplified with N-myc or c-myc in these cell lines. However, since the 2 amplified fragments in NCI-H69 were not amplified in 42 other human cancer cell lines including 11 cell lines carrying amplified N-myc genes, it is also possible that there are amplified regions on chromosome 2 other than the N-myc locus at 2p24 in NCI-H69. In contrast, all 6 amplified fragments in NCI-H82 were amplified in several other human cancer cell lines carrying amplified c-myc genes. This result further indicates that these fragments were derived from an amplification unit that includes the c-myc gene. Our results show the ability of the AP-PCR method to analyze the fraction of the genome with amplification in human cancer cells.

Base Sequence↗

PubMed1996

Degradation of T-kininogen by cathepsin D and matrix metalloproteinases.

Cathepsin D, matrix metalloproteinase (MMP)-2, MMP-3 (stromelysin), and MMP-9 were isolated from rat granulomatous tissues. HT1080 human fibrosarcoma cells and rheumatoid synovial cell CM. At acidic conditions, cathepsin D cleaved T-kininogen into small peptides and released Met-T-kinin-Leu (kinin precursor), but failed to release kinin. MMP-3 cleaved T-kininogen into a 57 kDa fragment as measured by SDS-PAGE and Western blot analysis using anti-T-kininogen antiserum. On the other hand, no degradation of T-kininogen occurred during incubation with MMP-2 or MMP-9100/1) at pH 7.5 for 7 h.

Cathepsin D↗

PubMed1996

Structure-activity relationships of HIV-1 PR inhibitors containing AHPBA--II. Modification of pyrrolidine ring at P1' proline.

Systematic replacement in the 3- or 4-position of the pyrrolidine ring at P1' proline was carried out. Compound 26, which has a Cl atom in the 4(S)-position was the most active among inhibitors substituted with other halogen atoms or other substituents. Furthermore, the replacement of the Z group in compound 26 with five- or six-membered fused aromatic heterocycle carbonyl groups produced more potent inhibitors. 7-Methoxybenzofuran-2-carbonyl derivative (44) was the best of these and showed Ki = 4.5 nM against HIV PR and IC90S 0.58 microM and 0.06 microM in chronic and acute infections, respectively. These results suggest that the combination of the 4(S)-CI atom and fused bicyclic heterocycles may be effective in improving their cellular penetration.

Cells, Cultured↗

PubMed1996

Identification of host factors that regulate the influenza virus RNA polymerase activity.

Transcription and replication of the influenza virus RNA genome take place in the nuclei of infected cells. Ribonucleoprotein (RNP) complexes consisting of viral RNA, RNA polymerase, and nucleocapsid protein (NP) are proven to be the catalytic unit for RNA synthesis, while it has been indicated that the viral RNA polymerase activity is modulated by host-derived nuclear factors. Here we have identified such host factors present in nuclear extracts prepared from uninfected HeLa cells with biochemical complementation assays using the in vitro RNA synthesis system. The stimulatory activity was not absorbed to phosphocellulose but was tightly bound to Q-Sepharose. The eluate recovered from Q-Sepharose was able to stimulate the RNA synthesis catalyzed by both RNP complexes and purified RNA polymerase and NP. The stimulatory activity was further separated into two distinct fractions, designated RAF-1 (RNA polymerase activating factor-1) and RAF-2 fractions, through phenyl-Sepharose column chromatography. When these fractions were fractionated through a gel filtration column, RAF-1 and RAF-2 activities were recovered in fractions corresponding to the molecular mass of 350 kDa and 60 kDa, respectively. Furthermore, the RAF-2 fraction was shown to contain an inhibitory activity, tentatively designated RIF-1 (RNA polymerase inhibitory factor-1). RIF-1 sedimented as fast as bovine serum albumin in glycerol density gradient centrifugation. Roles of these host factors are discussed in the context of viral RNA transcription and replication.

Biological Factors↗

PubMed1996

NAP-I is a functional homologue of TAF-I that is required for replication and transcription of the adenovirus genome in a chromatin-like structure.

BACKGROUND: For the activation of replication and transcription from DNA in a chromatin structure, a variety of factors are thought to be needed that alter the chromatin structure. Template activating factor-I (TAF-I) has been identified as such a host factor required for replication of the adenovirus (Ad) genome complexed with viral basic core proteins (Ad core). TAF-I also stimulates transcription from the Ad core DNA. RESULTS: Using mutant TAF-I proteins, we have demonstrated that the acidic stretch present in the carboxyl terminal region is essential for the stimulation of transcription from the Ad core. A genomic footprinting experiment with restriction endonuclease has revealed that TAF-I causes a structural change in the Ad core. TAF-I has been shown to have significant amino acid similarity to nucleosome assembly protein-I (NAP-I), which is involved in the formation of the chromatin structure. We have shown that TAF-I can be substituted by NAP-I in the activation of the cell-free Ad core transcription system. Two of the tripartite acidic regions and the region homologous to TAF-I in NAP-I are required for the maximal TAF-I activity of NAP-I. Furthermore, TAF-I has been shown to have NAP-I activity, and the acidic region of TAF-I is required for this activity. CONCLUSIONS: Since TAF-I causes the structural change of the Ad core and thereby activates transcription, TAF-I is thought to be one of the proteins which is involved in chromatin remodeling. NAP-I is structurally related to TAF-I and functionally substitutes for TAF-I. Furthermore, TAF-I has NAP-I activity. These observations suggest that this type of molecule has dual functions, possibly by participating in facilitating the assembly of the chromatin structure as well as perturbing the chromatin structure to allow transcription to proceed.

Adenoviridae↗

PubMed1996

Induction of the transcription factor AP-1 in cultured human colon adenocarcinoma cells following exposure to bile acids.

We studied the effects of bile acids on inducibility of the transcription factor AP-1 in human colon carcinoma LoVo cells. Firstly, cells were treated with chenodeoxycholic acid and the nuclear extracts from those cells were processed by electrophoretic mobility shift assays to analyze nuclear AP-1 DNA-binding activity. We demonstrated that chenodeoxycholic acid induced AP-1 DNA-binding activity in a dose- and time-dependent fashion. Antibody supershift experiments clearly revealed that the majority of protein components in induced AP-1 DNA-binding activity were the products of oncogenes c-fos and c-jun. On the other hand, DNA-binding activity in the nuclear extracts for either NF kappa B, Sp1, or ATF/CREB was not affected by bile acids, suggesting that the effect of bile acids was rather specific for AP-1. Transient transfection experiments supported this notion: expression of the AP-1-luciferase reporter construct was induced by bile acids in a dose-dependent manner, and expression of either reporter construct for NF kappa B, Sp1, or ATF/CREB was not influenced by treatment of the cells with bile acids. We also demonstrated that those bile acids efficiently activated AP-1-dependent promoter in DLD-1 cells, which (as well as LoVo cells), are derived from colon adenocarcinoma, but not in COLO320DM cells which are from colon carcinoid tumor. Thus, we may indicate that bile acids exclusively induce nuclear AP-1 activity in colon adenocarcinoma cells.

Activating Transcription Factor 1↗

PubMed1996

A transcription factor with a leucine-zipper motif involved in light-dependent inhibition of expression of the puf operon in the photosynthetic bacterium Rhodobacter sphaeroides.

In the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides the synthesis of components of the photosystem is regulated in response to oxygen tension and light intensity. We have purified and cloned a trans-acting protein (SPB) that binds to the promoter region of the puf operon, which encodes the apoproteins of light-harvesting complex I and the reaction center. The SPB was composed of a single polypeptide with an apparent molecular mass of 15.0 kDa. The nucleotide sequence of the spb gene was determined. The gene encoded 104 amino acid residues, which correspond to a molecular mass of 11.5 kDa. SPB exhibited 53% homology to HvrA in Rhodobacter capsulatus. The deduced amino acid sequence indicated that SPB contained a region with homology to the leucine-zipper motif of c-JUN, a transcription factor in eukaryotes, and SPB also had a DNA-binding domain on the amino-terminal side of the leucine-zipper motif. The leucine-zipper motif of SPB might contribute to the formation of a dimer. Northern analysis indicated that spb was constitutively and monocistronically transcribed in R. sphaeroides, irrespective of growth conditions. Structural and functional differences between SPB and HvrA are discussed.

Amino Acid Sequence↗

PubMed1996

Mechanism for inhibition of influenza virus RNA polymerase activity by matrix protein.

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 inhibition was examined at four steps: cleavage of capped RNA; incorporation of the first nucleotide, GMP; limited elongation; and synthesis of full-size product. M1 inhibited RNA synthesis mainly at the early steps. The experiments with M1 mutant proteins containing amino acid deletions suggested that the M1 region between amino acid residues 91 and 111 was essential for anti-RNA synthesis activity, RNA binding, and oligomerization of M1 on RNA.

Base Sequence↗

PubMed1996

Effect of carnitine on decrease of branched chain amino acids and glutamine in serum of septic rats.

Blood concentrations of branched chain amino acids (BCAA; leucine, isoleucine, and valine) and glutamine (Gln) decrease markedly in sepsis. We investigated the effect of carnitine on serum concentrations of BCAA and Gln in fasted septic rats. Rats were made septic by cecal ligation and puncture. They developed extremely high blood concentrations of endotoxin, and serum concentrations of BCAA and Gln were markedly decreased 2 d after the operation. When L-carnitine was administered subcutaneously to the rats at 500 mg/kg body weight every 12 h for 2 d starting at the operation, no decrease in the serum concentrations of BCAA and Gln was observed. This indicates that the administration of carnitine can prevent the decrease of serum concentrations of BCAA and Gln in septic animals.

Amino Acids, Branched-Chain↗

PubMed1996

Cervical osteoplastic laminotomy using autogenous bone.

Cervical foraminotomies are performed from the fifth to the eighth cervical nerve roots on both sides. On the side with more marked radiculopathies the laminae are osteotomized to the full extent, but not so completely on the other side. The mass of laminae from C-3 to C-7 is lifted up using the incompletely severed side of the laminae as the fulcrum. The removed spinous processes of C-6 and C-7 are adjusted and fixed between the articular joints and the lifted laminae from C-4 to C-6. This procedure prevents extradural scar formation, there are no complications caused by foreign bodies, postoperative neuroimaging is possible because no metallic artifact is formed, and extensive decompression is achieved for the cervical cord and nerve roots.

Adult↗

PubMed1996

Accuracy of immunohistochemistry and flow cytometry in estimating the proportion of leukemic cells in S phase in chronic myeloid leukemia patients.

We compared the proportion of S phase cells in bone marrow and peripheral blood samples obtained from 17 patients with chronic myeloid leukemia (CML). Before sampling all patients received a one hour i.v. infusion of iododeoxyuridine (IdUrd). The proportion of S phase cells was studied by immunohistochemistry (IHC) in bone marrow biopsies, and by flow cytometry (FCM) in bone marrow aspirates and peripheral blood samples. The IdUrd labelling index (LI) in bone marrow biopsy sections (27.5 +/- 1.8%) was significantly higher than the proportion of IdUrd labelled cells in bone marrow aspirate (15.1 +/- 2.0%). The percentage of S phase cells in peripheral blood was approximately the same as that in the aspirate (12.4 +/- 1.3%) and was correlated with that of bone marrow aspirate indicating a high degree of the aspirate dilution by peripheral blood. It is likely that the differences in % S phase cells in the aspirate and the biopsy result from this dilution. Estimates of the % S phase cells in the peripheral blood study by IHC and FCM were essentially the same. Samples labelled for one hour in vitro resulted in 1.5 fold higher LI than the same samples labelled in vivo. We conclude that estimates of the 8% S phase cells in the bone marrow of patients with CML should be made by infusing patients with IdUrd or BrdUrd with immunohistochemical evaluation of a marrow biopsy. Additionally in vitro labelling is not reflective of the percent S phase cells in vivo in patients.

Antibodies, Monoclonal↗

PubMed1996

[The structure and function of general transcription factor TFIIA].

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Amino Acid Sequence↗

PubMed1995

Assignment of the E4TF1-60 gene to human chromosome 21q21.2-q21.3.

The gene encoding human transcription factor E4TF1-60 was previously mapped to chromosome 21q21. We analyzed the localization of the E4TF1-60 gene in more detail by genomic Southern hybridization and determined the sequence of the exons and the regions surrounding the intron boundaries. We report here that E4TF1-60 locates in the long arm of chromosome 21 at q21.2-q21.3 and contains a total of ten exons.

Chromosomes, Artificial, Yeast↗

PubMed1995

Construction of a cDNA library for a specific region of a chromosome using a novel cDNA selection method utilizing latex particles.

A novel method is described for the rapid concentration of particular cDNAs and their mapping to specific regions of a genome. The strategy for 'cDNA scanning' is based on the hybridization of an entire library of cDNAs to a large fragment of genomic DNA that is covalently bound to latex particles. The hybridized cDNAs are eluted, amplified by PCR and cloned into a lambda vector. Selected cDNAs that hybridized to the genomic DNA are cloned, with subsequent sequence analysis. Region-specific DNA fragments prepared from a yeast artificial chromosome (YAC) clone, EG10D9, which maps to chromosome 5 of the small cruciferous plant Arabidopsis thaliana (At), were used to prepare a model system and were covalently bound to latex particles. cDNAs that hybridized to EG10D9 were concentrated by hybridization to the immobilized DNA. The hybridized cDNAs were recovered and amplified by PCR. The resultant sub-library of cDNAs of 0.5-2 kb in length was enriched about 1000-fold. The partial sequences of the cDNAs provided information about genes that are located on the EG10D9 region of the At genome. The cDNA scanning strategy provides an efficient method for the mapping of expressed genes which could be used as expressed sequence tags (EST) within a genome.

Arabidopsis↗

PubMed1995

Potentiation of glucocorticoid-mediated gene expression by the novel benzoquinone derivative (2E)-3-[5-(2,3-dimethoxy-o-methyl-1, 4-benzoquinoyl)]-2-nonyl-2-propenoic acid (E3330).

We examined the effects of the novel benzoquinone derivative (2E)-3-[5-(2,3-dimethoxy-o-methyl-1,4-benzoquinoyl)]-2-nonyl -2-propenoic acid, E3330, on the functional activity of the glucocorticoid receptors. For that purpose we used a cloned CHOpMTGR cells, in which human glucocorticoid receptor cDNA was stably transfected and glucocorticoid receptor was expressed at high levels. After treatment of CHOpMTGR cells with E3330, neither the ligand binding activity nor immunoreactivity of the glucocorticoid receptor was affected. Moreover, E3330 did not affect the sequence-specific DNA binding activity of partially-purified glucocorticoid receptor in vitro. However, a glucocorticoid-inducible promoter was activated by E3330 in a dose-dependent fashion in the presence of the synthetic ligand dexamethasone. Interestingly, E3330 increased nuclear translocation of the glucocorticoid receptor in a ligand-independent fashion, indicating that E3330, through facilitation of the translocation of the glucocorticoid receptor, augments glucocorticoid-mediated gene transcription.

Animals↗

PubMed1995

GABP and PU.1 compete for binding, yet cooperate to increase CD18 (beta 2 leukocyte integrin) transcription.

CD18 (beta 2 leukocyte integrin) is a leukocyte-specific adhesion molecule that plays a crucial role in immune and inflammatory responses. A 79-nucleotide fragment of the CD18 promoter is sufficient to direct myeloid transcription. The CD18 promoter is bound by the B lymphocyte- and myeloid-restricted ets factor, PU.1, and disruption of the PU.1-binding sites significantly reduces promoter activity. However, PU.1 alone cannot fully account for the leukocyte-specific and myeloid-inducible transcription of CD18. We identified a ubiquitously expressed nuclear protein complex of extremely low electrophoretic mobility that also binds to this region of the CD18 promoter. This binding complex is a heterotetramer composed of GABP alpha, and ets factor, and GABP beta, a subunit with homology to Drosophila Notch. GABP alpha competes with the lineage restricted factor, PU.1, for the same critical CD18 ets sites. The CD18 promoter is activated in myeloid cells by transfection with both GABP alpha and GABP beta together, but not by either factor alone. Transfection of non-hematopoietic cells with the two GABP subunits together with PU.1 is sufficient to activate CD18 transcription in otherwise non-permissive cells. Thus, GABP and PU.1 compete for the same binding sites but cooperate to activate CD18 transcription.

Animals↗