Biomedical subjects

H Handa

Publications and source records attributed to H Handa.

At least 109 records · Page 6Linked to original sources

PubMed1997

Characterization of the human thrombopoietin gene promoter. A possible role of an Ets transcription factor, E4TF1/GABP.

Thrombopoietin (TPO), the ligand for c-Mpl, is a cytokine that regulates megakaryocyte growth and development. We have cloned the 5'-flanking region of the human TPO gene and analyzed its promoter activity. The human TPO gene promoter lacks a TATA box and directs transcription initiation at multiple sites over a 50-nucleotide region. Transient expression in a human liver cell line (PLC) of promoter fragment-luciferase reporter gene constructs containing a series of 5'-truncated sequences or site-directed mutations identified a sequence 5'-ACTTCCG-3' from -69 to -63 as a positive cis-acting element for high level expression of TPO gene. This sequence contains a core motif (C/A)GGA(A/T) for Ets family proteins in the noncoding strand. Gel mobility shift assays performed with nuclear protein from PLC cells identified a DNA binding protein(s) specific for the element. Anti-E4TF1-60(GABPalpha) or anti-E4TF1-53/47(GABPbeta) antibodies supershifted the complex in gel shift assay. Furthermore, co-expression of E4TF1-60 and E4TF1-53/47 squelched TPO gene expression in PLC and HepG2 cells. It is concluded that Ets family transcription factor E4TF1(GABPalpha/beta), an ubiquitously expressed protein, is required for high level expression of the TPO gene in liver.

Base Sequence↗

PubMed1997

Inhibition of HIV-1 protease by oxim derivatives.

In cell-free proteolytic processing using recombinant HIV-1 protease and Gag precursor polypeptide, certain simple oxim derivatives containing halogenomethylketone and phenyl moieties displayed HIV-1 protease inhibitory activity. Their Ki values ranged from 2.1 microM to 6.3 microM and they did not inhibit significantly other aspartic acid proteases. Both the halogenomethylketone moiety and the oxim structure were essential for the observed inhibition. Molecular modeling analysis suggested that these compounds are recognized by the HIV-1 protease as the P1 and P1' part of the substrate. In addition, one potent derivative showed inhibition of viral maturation in HIV-1IIIB chronically infected Molt-4 cells. These results indicate that it is possible to develop new and specific nonpeptidyl HIV protease inhibitors of low molecular weight.

Binding, Competitive↗

PubMed1997

Development of HIV-1 protease expression methods using the T7 phage promoter system.

New and simple human immunodeficiency virus type 1 (HIV-1) protease expression methods in Escherichia coli were developed using the T7 phage promoter system. In order to suppress leaky HIV-1 protease expression under the control of the T7 polymerase, two new methods were tested. One involved the introduction of supplementary T7 promoter regions into host cells [E. coli BL-21 (DE3)] containing the HIV-1 protease gene under the control of the T7 promoter. It was expected that the supplementary T7 promoter regions would compete with the HIV-1 protease expression vector for the T7 polymerase binding. The other involved the infection of late-log-phase cultures of E. coli JM109 harboring the same HIV-1 protease expression vector with the M13 phage expressing T7 polymerase. Both methods were effective, and transformants with the mature HIV-1 protease expression vector showed ten times higher HIV-1 protease activity than activities obtained with the autoprocessing vector. The expression systems described here are convenient and are also easily applicable for the expression of other proteins toxic for E. coli.

Bacteriophage T7↗

PubMed1997

The rapeseed mitochondrial gene for subunit 2 of the NADH dehydrogenase complex: a trans-spliced structure is conserved in one of the smallest plant mitochondrial genomes.

The single-copy gene encoding NADH dehydrogenase subunit 2 (nad2) has been identified in the mitochondrial genome of rapeseed (Brassica napus L.). The rapeseed nad2 gene has the same gene organization in Oenothera and wheat: it consists of five exons located in two loci encoding the two first exons and the last three exons respectively. All exons are separated by group-II introns. A trans-splicing event is required to join exons B and C. Putative splicing intermediates were identified by transcriptional analysis of the nad2 gene. The complexity of organization of the nad2 gene is completely conserved even in one of the smallest mitochondrial genomes of higher plants.

Amino Acid Sequence↗

PubMed1997

Bcl-2 and c-myc expression, cell cycle kinetics and apoptosis during the progression of chronic myelogenous leukemia from diagnosis to blastic phase.

Chronic myelogenous leukemia (CML) has a progressive course but little is known about the biologic characteristics of disease progression. This study was designed to assess the changes in cell proliferative characteristics, apoptosis, the expression of the bcl-2 and c-myc genes between the time of initial diagnosis and entrance into the blastic phase of the disease. We observed that the rate of cell proliferation decreased and the cell death rate did not significantly change as the disease accelerated. The level of bcl-2 expression was significantly higher in accelerated/blastic phase cells than in the chronic phase cells in the population as a whole, however, the bcl-2 expression level did not change in blast cell subpopulation. c-myc Expression was significantly higher in the blast cell subpopulation of accelerated/blastic phase than in that of earlier phases of the disease. In conclusion, the characteristics of CML cells, namely proliferation rate, c-myc and bcl-2 change during the course of the disease. It is possible that the change in c-myc expression plays a causative role in evolution of the blastic phase from the chronic phase.

Adult↗

PubMed1997

The promoter of an androgen dependent gene in the hamster flank organ.

Hamster flank organs are useful for studying androgen-dependent growth of hair follicles and sebaceous glands. A cDNA clone (FAR-17a) was isolated from the hamster flank organ, whose expression was highly sensitive to androgen. The mRNA level of this gene was reduced after castration but reappeared after testosterone treatment. To elucidate the mechanism of expression of this gene regulated by androgen we isolated a genomic clone, from a hamster genomic library, that includes the promoter and upsteam region. The promoter region was used to drive a luciferase reporter gene in Cos 7 cells. This construct was activated five to six times higher over a control plasmid lacking the promoter region. We tested the effects of testosterone by transfection of the reporter plasmid into androgen dependent SC-3 cells. The results showed up to fivefold stimulation after the addition of androgen. The sequence of this promoter region was analyzed and the transcription factor binding sites were predicted. Since no obvious androgen responsive elements were included in the promoter region, we suggest that the stimulation of the reporter construct has to be mediated indirectly by androgen-dependent transcription factor(s).

Amino Acid Sequence↗

PubMed1997

Transcriptional activation through interaction of MBF2 with TFIIA.

BACKGROUND: Transcriptional activation of the Drosopohila melanogaster fushi tarzu gene by FTZ-F1 or its silkworm counterpart BmFTZ-F1 requires two cofactors MBF1 and MBF2 which do not directly bind to DNA. MBF1 is a bridging molecule that connects FTZ-F1 (or BmFTZ- F1), MBF2 and TATA binding protein TBP. MBF2 is a positive cofactor that activates transcription. RESULTS: To elucidate the mechanism of transcriptional activation by MBF2, we isolated a cDNA coding for the factor. Northern blot analyses showed temporally restricted expression of MBF2 mRNA similar to that of BmFTZ-F1 mRNA. The cDNA sequence predicts a polypeptide of 10 kDa whereas natural MBF2 is a glycoprotein of 22 kDa. The deduced amino acid sequence of the factor showed no homology with proteins in the databases. Farwestern analyses and glutathione S-transferase interaction assays demonstrated that MBF2 makes a direct contact with the beta-subunit of TFIIA. In a HeLa cell nuclear extract, bacterially expressed recombinant MBF2 activated transcription from various promoters as natural MBF2 did. This activation requires the MBF2-TFIIA interaction. When recombinant MBF2 was added to the HeLa cell nuclear extract in the presence of MBF1 and FTZ622 bearing the DNA-binding region of FTZ-F1, it selectively activated transcription of the fushi tarazu gene. This selective activation also requires the MBF2-TFIIA interaction. CONCLUSION: MBF2 activates transcription through its interaction with TFIIA. Selective transcriptional activation occurs when MBF2 is recruited to a promoter carrying the FTZ-F1 binding site by FTZ-F1 and MBF1.

Amino Acid Sequence↗

PubMed1997

Autoregulation of Pax6 transcriptional activation by two distinct DNA-binding subdomains of the paired domain.

BACKGROUND: Pax6 is a transcription factor that plays a central role in eye development. Pax6 contains a DNA-binding domain called paired domain, which consists of a highly conserved N-terminal subdomain and a variable C-terminal subdomain. Recent findings have suggested that both subdomains possess distinct DNA-binding activities. RESULTS: To understand the mechanism of DNA-binding and transcriptional activation by Pax6 via these subdomains, we employed Pax6 paired domain mutants previously isolated from patients with ocular disorders. Analysis of these mutants by gel shift assay revealed that the N-terminal and C-terminal subdomains can independently bind to their respective cognate sites, P6CON and 5aCON. Results from a luciferase assay, however, showed that the two functional subdomains negatively regulate their transactivation potentials each other. Wild-type Pax6 and its hyperactive variants show different patterns of DNA contact. CONCLUSION: These results support a new model for the regulation of Pax6 transactivation: When one DNA-binding subdomain binds to its cognate site, the other subdomain also interacts with the flanking sequences nonspecifically, and this interaction constrains its structure to give a reduced level of transactivation.

Animals↗

PubMed1997

Possible involvement of bone marrow stromal cells in agranulocytosis caused by vesnarinone treatment.

Vesnarinone, an oral therapeutic agent for cardiac failure, causes agranulocytosis as a side effect. To elucidate the mechanism of occurrence of the agranulocytosis, we examined the effect of vesnarinone on granulopoiesis using an in vitro human long-term bone marrow culture system. Addition of vesnarinone to the culture decreased the total number of hematopoietic cells, mainly composed of mature granulocytes and macrophages, but increased the number of granulocyte-macrophage progenitor cells (CFU-GM) and CD33-CD34+ cells as compared with an untreated control. Differentiation of CFU-GM was induced by removing the agent from the culture medium, indicating that the effect of vesnarinone was reversible. The agent did not directly affect CFU-GM in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Furthermore, treatment of stromal cells with vesnarinone repressed the production of G, GM, M-CSF, suggesting that the agent may cause a hematopoietic disorder, agranulocytosis, through the impairment of stromal cell function.

Agranulocytosis↗

PubMed1997

In vitro and ex vivo anti-human immunodeficiency virus (HIV) activities of a new water-soluble HIV protease inhibitor, R-87366, containing (2S,3S)-3-amino-2-hydroxy-4-phenylbutanoic acid.

In a series of compounds containing (2S,3S)-3-amino-2-hydroxy-4-phenylbutanoic acid (AHPBA), a transitionstate mimetic, R-87366:(2S,3S)-3-[N-(quinoxaline-2-carbonyl)-L-asparaginyl]amino- 2-hydroxy-4-phenylbutanoyl-L-proline tert-butylamide, was found to be a potent human immunodeficiency virus protease inhibitor (Ki value was 11 nM) and anti-HIV agent (IC90 value was 0.5 microM for HIV-1IIIB acutely infected cells) with moderate water-solubility (4.2 mg/ml at 25 degrees C). The compound was also active in chronically infected Molt-4/HIV-1IIIB cells, and inhibited the proteolytic processing of p55 into p17, suggesting that its anti-HIV activity was derived from HIV protease inhibition. The compound showed more potent activity (IC90 value was 0.03-0.25 microM) against clinical isolates of HIV in 5 out of 6 patients examined with varying clinical status in an ex vivo assay. One isolate, however, from the sixth patient, was less sensitive to R-87366 (IC90 value was 0.5 microM). In experiments with this strain, R-87366 showed comparatively low efficacy in acutely infected peripheral blood mononuclear cell (PBMC). This result suggests that the diversity of sensitivity shown in the ex vivo assay could be caused by the viral property itself. As a result of the determination of nucleic acid sequences in the clinical isolates, some amino acids were found to be substituted in the protease region, in contrast to the HIV-1 clade B consensus sequence, and some of them have been reported to contribute to the susceptibility of HIV protease inhibitors.

Amino Acid Sequence↗

PubMed1997

Effects of vesnarinone on the bone marrow stromal cell-dependent proliferation and differentiation of HL60 cells in vitro.

It has been reported that vesnarinone, a new inotropic agent used in the treatment of cardiac failure, causes agranulocytosis as a side effect. To study the mechanisms by which this complication occurs, vesnarinone was introduced into a coculture system of HL60 and bone marrow (BM) stromal cells, in which HL60 cells were able to differentiate into mature granulocytes with no inducible exogenous factors added to the culture. When HL60 cells were cocultured with the human BM-derived stromal cell line LP101, HL60 cells were induced to differentiate into mature granulocytes, and expression of the mature granulocyte-macrophage surface antigen, CD11b was increased. Conditioned medium (CM) obtained from LP101 cells also showed the capacity to induce the maturation of HL60 cells, in a dose- and time-dependent manner. The differentiation of HL60 cells induced by CM was also determined by morphological analysis, expression of myeloperoxidase, and a nitroblue tetrazolium (NBT) reduction test. When HL60 cells were cocultured with LP101 in the presence of vesnarinone, the CD11b expression was greatly suppressed. CM obtained from vesnarinone-treated LP101 (ves-CM) lost the capacity to induce the differentiation of HL60 cells, at a concentration of 1 microg/mL of vesnarinone. Vesnarinone itself did not affect the proliferation of HL60 cells. Furthermore, the addition of vesnarinone or ves-CM to HL60 cultures incubated with CM did not alter the induction of CD11b expression, suggesting that vesnarinone has no effect on HL60 cells, but that it inhibits stromal cells from producing soluble factor(s) required for the differentiation of HL60 cells to mature granulocytes. All these findings indicate that vesnarinone causes the hematopoietic disorder agranulocytosis, via impairment of stromal function.

Bone Marrow↗

PubMed1997

[Motor functional MRI by spiral scan--compared with GRE].

Recently, functional MRI (fMRI) has been performed in two methods using 1.5 tesla machine. One was performed using gradient recalled echo (GRE), the other was performed using echo planner imaging (EPI). There is a third method for fMRI called spiral scan. This method is applicable without strong gradient magnetic field such as required by EPI. In this study, motor fMRI was performed in ten normal volunteers by both spiral scan and GRE. Less than 1% risk (t-test), spiral scan showed the reactive area more clearly than GRE. And GRE showed higher signal intensity changing ratio than spiral scan, but there was no significant difference between spiral scan and GRE. The acquisition time of spiral scan was 2 seconds per image, and that of GRE was 10.5 seconds. Spiral scan has good temporal resolution and contrast-noise ratio. These are very advantageous for fMRI.

Adult↗

PubMed1996

Construction and characterization of a recombinant adenovirus vector carrying the human preproinsulin gene under the control of the metallothionein gene promoter.

A new adenovirus vector carrying human-preproinsulin (h-PPI) genomic DNA, which was placed under the control of the mouse metallothionein gene promoter, was constructed. In the recombinant virus-infected cells, h-PPI gene expression increased as a function of ZnSO4 concentration. Reversed-phase high-performance liquid chromatography analysis revealed that the recombinant adenovirus-infected cells secreted immature insulin containing proinsulin and incorrectly processed insulin. Tyrosyl phosphorylation of human insulin receptor substrate 1 occurred when HepG2 cells were treated with the cultured medium, indicating that the h-PPI gene product was functionally active in vitro. We also examined the biological activity of the product using diabetic severe combined immunodeficient mice and confirmed that the h-PPI gene product reduced the blood glucose concentration in vivo. This study suggests that the adenovirus vector can be used to express a foreign gene under the control of an external promoter in various human cells.

Adenoviridae↗

PubMed1996

Functional domains of transcription factor hGABP beta1/E4TF1-53 required for nuclear localization and transcription activation.

Transcription factor E4TF1 is the human homolog of GABP and has been renamed hGABP (human GABP). hGABP is composed of two types of subunits; hGABP beta1/E4TF1-53 and the ets-related protein hGABP alpha/E4TF1-60. Both bind together to form an (alpha)2(beta1)2 heterotetrameric complex on DNA and activate transcription at specific promoters in vitro. Tetramer formation depends on two regions of hGABP beta1; the N-terminal region containing the Notch/ankyrin-type repeats is necessary for binding to hGABP alpha and the C-terminal region is necessary for homodimerization. In this report, we constructed various deletion mutants of hGABP beta1 in order to delimit the functional regions required for nuclear localization and transcription activity. We found that hGABP beta1 localization in the nucleus is dependent on a region located between amino acids 243 and 330 and that the presence of hGABP beta1 influences the efficiency of hGABP alpha transport into the nucleus. Next, we demonstrated that the hGABP complex composed of alpha and beta1 subunits activates transcription from the adenovirus early 4 promoter in vivo. This transcription activation needs the C-terminal region of hGABP beta1 and is consistent with results obtained with the in vitro assay. Furthermore, site-directed mutagenesis analysis of the C-terminal region reveals that the alpha-helix structure and the leucine residues are important for formation of a heterotetrameric complex with hGABP alpha in vitro and for transcription activation in vivo. These results suggest that hGABP beta1 stimulates transcription as part of a heterotetrameric complex with hGABP alpha in vivo.

Animals↗

PubMed1996

Effect of vitamin E on expression of cyclooxygenase-2 in lipopolysaccharide-stimulated rat macrophages.

To clarify the role of vitamin E (alpha-tocopherol) for the induction of cyclooxygenase-2 (COX-2) in rat macrophages stimulated by lipopolysaccharide (LPS), vitamin E-enriched macrophages were prepared by intraperitoneal injection of vitamin E for 6 days at a rate of 5 mg per day. The production of PGE2 was increased in dose- and time-dependent manners by addition of LPS in both control and vitamin E-enriched peritoneal macrophages. The maximum effect of LPS was observed in 12 h at concentration of 5 micrograms/ml. By analyzing COX-2 mRNA level by Northern blot and COX-2 enzyme mass and phosphotyrosine by Western blot, it was revealed that the increase of PGE2 production reflected the induction of COX-2 expression through activation of tyrosine kinase. Vitamin E failed to inhibit PGE2 production in LPS-stimulated macrophages; however, genistein, a tyrosine kinase inhibitor, completely inhibited the production at 100 microM. These results suggest that vitamin E does not inhibit COX-2 expression via LPS-mediated tyrosine kinase signal transduction pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

The rapeseed mitochondrial gene encoding a homologue of the bacterial protein Ccl1 is divided into two independently transcribed reading frames.

In the rapeseed mitochondrial genome we identified sequences that have a high similarity to those of a bacterial gene involved in the biogenesis of cytochromes c designated ccl1. The structure of this gene is quite unusual. In rapeseed mitochondria, the ccl1-homologous (orf577) sequence is divided into two parts, which are at least 45 kb apart. These two parts are transcribed separately and their transcripts are edited similarly to the homologous transcripts of wheat and Oenothera. However it was impossible to identify a mature transcript covering the whole coding region, a result that excludes a trans-splicing event. No other copy of this gene was found in either the nuclear genome or the mitochondrial genome. The protein product of orf577 is present in rapeseed mitochondria. These results raise the possibility that this divided gene might be functional and active in rapeseed mitochondria through a novel mechanism of gene expression.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of virus-like particles and pentamers produced by the expression of SV40 capsid proteins in insect cells.

Three capsid proteins of SV40 (VP1, VP2, and VP3) were expressed in insect cells using recombinant baculoviruses. When the VP1 capsid protein was expressed alone or co-expressed with VP2 and VP3, virus-like particles (VLP) were produced. In the latter case, the minor capsid proteins, VP2 and VP3, were incorporated into the VLP. VLPs with and without VP2 and VP3, and the wild type SV40 virions were indistinguishable under electron microscope. The sedimentation coefficient, S20,w' obtained for the VLP consisting of VP1 alone (VP1-VLP) was 170 S, and that for the VLP consisting of all of the capsid proteins (VP1/2/3-VLP) was 174 S. Treatment of the VP1-VLP with a calcium ion chelating agent and a reducing agent caused dissociation of the VP1-VLP. The dissociated and purified VP1 proteins were identified as pentamers of VP1 based on the molecular weight determination by sedimentation equilibrium. The pentamers were shown to possess the ability to re-assemble into VLP which had the S20,w of 141S. The results are discussed in relation to the morphogenesis of SV40.

Animals↗

PubMed1996

Activation of the retinoblastoma gene expression by Bcl-3: implication for muscle cell differentiation.

The retinoblastoma (Rb) protein is a master regulator of cell cycle. Accumulating evidence suggests that elevation of Rb expression is a key event in differentiation of various cell types. However the mechanism of regulation of Rb expression is poorly understood. Here we report that the candidate oncoprotein Bcl-3, previously characterized as a member of the IkappaB family, activates transcription of the Rb gene, whose promoter has no typical kappaB sites. A target element for Bcl-3 that matches the consensus for the E4TF1/GABP transcription factor was identified. Bcl-3 was shown to promote tetramer formation of E4TF1. During muscle cell differentiation, increased bcl-3 expression was observed before the induction of the Rb mRNA. Transient expression of Bcl-3 in myoblasts was shown to induce expression of the endogenous Rb. Furthermore, expression of the antisense bcl-3 RNA in myoblasts suppressed induction of Rb and myogenic differentiation. These results suggest that Bcl-3 is an upstream regulator of Rb expression during differentiation of muscle cells.

B-Cell Lymphoma 3 Protein↗