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Biomedical subjects

H Handa

Publications and source records attributed to H Handa.

At least 37 records · Page 2Linked to original sources
PubMed1998

Nuclear targeted suppression of NF-kappa B activity by the novel quinone derivative E3330.

The activation of NF-kappa B consists of at least three steps: degradation of I kappa B alpha, translocation of NF-kappa B into the nucleus, ai post-translational modification of NF-kappa B (e.g., phosphorylation of p65). In the present study, we found that a novel quinone derivative E3330 selectively inhibited NF-kappa B-mediated gene expression without affecting any of these steps. E3330, when included in the culture medium, suppressed NF-kappa B DNA-binding activity in PMA-induced Jurkat cell nuclear extracts, suggesting that the inhibition by E3330 of NF-kappa B-mediated gene expression was due to its ability to suppress NF-kappa B DNA-binding activity. Fractionation of the nuclear extracts by column chromatography revealed that a nuclear factor enhanced NF-kappa B DNA-binding activity and that this enhancing activity was interrupted after treatment with E3330. Moreover, a major polypeptide with a molecular mass of 40 kDa was found to be in the highly purified fraction containing the NF-kappa B-enhancing activity and predominantly bind E3330. Taken together, these results suggest that the NF-kappa B activity, after dissociation from I kappa B, is enhanced by a nuclear factor that is active irrespective of PMA treatment, and the nuclear factor-mediated enhancement is selectively inhibited by E3330. Thus, we conclude that E3330 may belong to a novel class of anti-NF-kappa B drugs.

Benzoquinones↗
PubMed1998

Rapeseed mitochondrial ccb206, a gene involved in cytochrome c biogenesis, is co-transcribed with the nad3 and rps12 genes: organization, transcription, and RNA editing of the nad3/rps12/ccb206 locus.

We describe here the structure and expression of the nad3/rps12/ccb206 locus in the mitochondrial genomes of rapeseed (Brassica napus L.) and related species. In the rapeseed mitochondrial genome there are three tandemly located open reading frames for subunit 3 of NADH dehydrogenase (nad3), the S12 subunit protein of the mitochondrial ribosome (rps12), and a homologue of one subunit of the ABC-heme transporter (ccb206). The gene organization of the nad3/rps12/ccb206 locus found in rapeseed mitochondria is not conserved in Arabidopsis thaliana mitochondria, where the ccb206 and nad3/rps12 loci are separated from each other. We show here that the rapeseed gene-organization pattern is also present in other Brassica species, but not in radish, a species very closely related to Brassica. Northern hybridization and RT-PCR analyses indicate that these three mitochondrial genes are possibly co-transcribed. Ten, seven, and 39 C-to-U RNA editing events occur in the rapeseed nad3, rps12, and ccb206 transcripts, respectively. Finally we show that codon position 21 of nad3 is edited in a highly species-specific manner among related Brassica species, including rapeseed and radish.

ATP-Binding Cassette Transporters↗
PubMed1998

Genes for the ribosomal S4 protein encoded in higher plant mitochondria are transcribed, edited, and translated.

Here we describe the structure and expression of the mitochondrial gene that encodes the S4 subunit of the mitochondrial ribosome (rps4) in two higher plants, rapeseed and rice. The ORFs of rapeseed and rice rps4 genes are 362 and 352 codons long, respectively, and their corresponding transcripts are edited (C-to-U conversions) in 19 and 15 positions, respectively. The amino acid sequences deduced from the sequences of the two rps4 genes are 78.1% identical to each other and show about 50% identity to the liverwort (Marchantia polymorpha) mitochondrial RPS4 polypeptide. Both higher plant mitochondrial genes have an inserted sequence (of 137 and 141 codons for rapeseed and rice, respectively) relative to the liverwort rps4 gene. In rice rps4, the 3' portion of the inserted sequence can potentially form two stem-loop structures that resemble two domains characteristic of group II introns, i.e., domains V and VI. However, RT-PCR analysis showed that these sequences were not spliced out during maturation of the rps4 transcripts. Antibodies specific for the plant RPS4 proteins were prepared. Protein blot analysis revealed that RPS4 polypeptides are present in both rapeseed and rice mitochondria, whose sizes are in agreement with the molecular weights calculated from the non-spliced sequences. The rps4 genes and RPS4 polypeptides have been also identified in the mitochondria of wheat, maize, soybean, and turnip by Southern and Western analyses, respectively.

Amino Acid Sequence↗
PubMed1998

Interplay between positive and negative elongation factors: drawing a new view of DRB.

DRB is a classic inhibitor of transcription by RNA polymerase II (pol II). Although it has been demonstrated that DRB inhibits the elongation step of transcription, its mode of action has been elusive. DRB also markedly inhibits human immunodeficiency virus (HIV) transcription, by targeting the elongation which is enhanced by the HIV-encoded transactivator Tat. Two factors essential for DRB action have recently been identified. These factors, positive transcription elongation factor b (P-TEFb) and DRB sensitivity-inducing factor (DSIF), positively and negatively regulate pol II elongation, and are likely to be relevant to the function of Tat. In this review, we summarize the recent findings on these factors, and discuss a possible model for the molecular mechanism of DRB action.

Dichlororibofuranosylbenzimidazole↗
PubMed1998

Functional interference of Sp1 and NF-kappaB through the same DNA binding site.

Gene activation by NF-kappaB/Rel transcription factors is modulated by synergistic or antagonistic interactions with other promoter-bound transcription factors. For example, Sp1 sites are often found in NF-kappaB-regulated genes, and Sp1 can activate certain promoters in synergism with NF-kappaB through nonoverlapping binding sites. Here we report that Sp1 acts directly through a subset of NF-kappaB binding sites. The DNA binding affinity of Sp1 to these NF-kappaB sites, as determined by their relative dissociation constants and their relative efficiencies as competitor DNAs or as binding site probes, is in the order of that for a consensus GC box Sp1 site. In contrast, NF-kappaB does not bind to a GC box Sp1 site. Sp1 can activate transcription through immunoglobulin kappa-chain enhancer or P-selectin promoter NF-kappaB sites. p50 homodimers replace Sp1 from the P-selectin promoter by binding site competition and thereby either inhibit basal Sp1-driven expression or, in concert with Bcl-3, stimulate expression. The interaction of Sp1 with NF-kappaB sites thus provides a means to keep an elevated basal expression of NF-kappaB-dependent genes in the absence of activated nuclear NF-kappaB/Rel.

Animals↗
PubMed1998

Stimulatory effects of substance P on CD34 positive cell proliferation and differentiation in vitro are mediated by the modulation of stromal cell function.

Substance P (SP) is a neuropeptide widely distributed in the nervous system. Extensive study has shown SP stimulates production of various cytokines by bone marrow stromal cells, although, the role of SP in hematopoietic phenomena is still unclear. Recently, we established a human cloned stromal cell line, HAS303, which can support hematopoietic stem cell proliferation and differentiation in vitro. We used this culture system to examine the effects of SP. Expression of the mRNAs of neurokinin (NK)-1R, NK-2R and NK-3R, specific SP receptors, on HAS303 cells was demonstrated by the RT-PCR. CD34+ cells isolated from bone marrow were co-cultivated with HAS303 cells in the presence and absence of SP and the total hematopoietic cells and progenitors were counted every 5 days. Introducing SP (10(-8) M) to the co-cultures significantly increased the number of total cells and progenitors compared with control cultures. SP showed no enhancing activity on CD34+ cells cultured alone. SP also stimulated IL-3-dependent colony formation of whole bone marrow MNCs in a soft agar culture system, but showed no such activity on isolated CD34+ cells in this system. These observations suggest that SP stimulated HAS303 cells, activated HAS303 cells, and stimulated the proliferation and differentiation of CD34+ cells. Treating HAS303 cells with SP increased the intracellular Ca2+ concentration and stimulated production of G-CSF, GM-CSF, SCF and IL-6, but not IL-1alpha, IL-1beta and TNF-alpha, but did not enhance proliferation. All these findings suggest that SP mediates hematopoietic cell proliferation and differentiation in vitro by activating stromal cell function.

Antigens, CD34↗
PubMed1998

Acute idiopathic thrombocytopenic purpura presenting a high serum level of immunoglobulin E and eosinophilia in an elderly patient.

An 80-year-old Japanese woman having acute idiopathic thrombocytopenic purpura presented with an increased serum level of IgE and eosinophilia. No evidence for allergic, parasitic, collagen or malignant diseases was observed. The disease subsided with prednisolone therapy and she has been in remission for three years. Our case is one of the oldest ITP patients with increased IgE and eosinophilia.

Acute Disease↗
PubMed1998

Intussuception as a complication of chronic lymphocytic leukemia.

The case of a 58-year-old man with chronic lymphocytic leukemia (B-cell type) who later developed an intussuception of the small intestine due to a tumor is described. The histopathological findings of the removed tumor were compatible with those of diffuse small lymphocytic lymphoma (B-cell type). The residual tumor became smaller with CHOP therapy. It is considered that CLL cell infiltration into the small intestine resulted in intussuception. Since many tumors and lymphomas can form polypoid lesions causing an intussuception. This is a possible complication of CLL and it could occur even when the WBC count is well controlled.

Humans↗
PubMed1998

[Outpatient chemotherapy with weekly high-dose infusional 5-fluorouracil (weekly HD-FU) in advanced gastrointestinal cancer].

This study was designed to evaluate the efficacy, toxicity, and quality of life (QOL) of outpatient chemotherapy with weekly HD-FU in advanced gastrointestinal cancer. Treatment consisted of weekly 24-h infusion of 5-FU 2,600 mg/m2, 5-FU was administered using the Groshong catheter and the Baxter infusor LV 5 (5 ml/hr). Eight patients (pts) with advanced gastrointestinal cancer were treated 64 times (mean 8.0 times), and 21 pts were treated 168 times (mean 8.0 times) with adjuvant chemotherapy. Grade 3 or 4 toxicities were not observed. Catheter occlusions and balloon rupture were observed in 1 case each. Responses were PR 2 and NC 6. Mean times to progression were 2.0 and 3.5 months, respectively. Mean scores of QOL were: 70.5 +/- 8.2 before treatment, 78.0 +/- 13.0 after 3 weeks, 79.8 +/- 7.9 after 5 weeks, and 75.8 +/- 11.3 after treatment completion. In adjuvant cases, these scores were 80.6 +/- 11.4, 83.1 +/- 10.7, 85.1 +/- 11.5, and 91.8 +/- 6.9, respectively. In conclusion, a schedule of 5-FU is feasible for outpatient chemotherapy in advanced gastrointestinal cancer.

Adult↗
PubMed1997

Promoter structure of the MxA gene that confers resistance to influenza virus.

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).

Antiviral Agents↗
PubMed1997

Retinoblastoma binding factor 1 site in the core promoter region of the human RB gene is activated by hGABP/E4TF1.

We previously reported two oncogenic point mutations present in the RB (retinoblastoma) gene promoter region, found at consensus Sp1 and ATF sites, respectively, and in two separate hereditary RB families. However, Sp1 protein was shown not to bind to the Sp1 site; this indicated that the Sp1 consensus site mutation was blocking the action of an alternative transcription factor, which we called RBF-1 (retinoblastoma binding factor 1). Subsequent purification of RBF-1 revealed it to be hGABP/E4TF1, a transactivator from the adenovirus early-region 4 promoter. In this study, we directly examined the effects of hGABP/E4TF1 on transactivation of the RB gene promoter through the RBF-1 site. As expected, hGABP/E4TF1 enhanced the core RB promoter activity, whereas it did not stimulate a mutant RBF-1 site. We therefore conclude that the most essential transcription factor in the human RB gene is likely to be hGABP/E4TF1.

Animals↗
PubMed1997

Phosphorylation and high level expression of Fra-2 in v-src transformed cells: a pathway of activation of endogenous AP-1.

Chicken embryo fibroblasts (CEF) transformed with v-src were previously reported to revert to normal phenotype after the introduction of dominant-negative mutants of Fos or Jun, indicating that endogenous AP-1 activity is essential for the cellular transformation. The major changes in the expression levels of fos and jun family genes induced by v-src were the elevation of fra-2 and c-jun transcripts. We show here that extensive phosphorylation of the AP-1 component Fra-2 is a major qualitative change in v-src transformed CEF and that several Ser and Thr residues in a C-terminal region of Fra-2 (amino acids 266-323) are phosphorylated specifically. The induced kinase activity was detected at the position of 42 kDa by in gel kinase assay using the Fra-2 C-terminal region as a substrate, and it was identified as chicken ERK2. JNK1 and JNK2, other members of the MAP kinase family, were not significantly activated in v-src transformed CEF and Fra-2 was not a good substrate for JNKs. fra-2 promoter analysis indicated that this promoter activity is elevated in v-src transformed CEF via two AP-1 binding sites and CRE-like sequence. We propose that phosphorylation of Fra-2 by ERK2 converts it from an inefficient transcriptional activator to an active one and further that fra-2 expression is autoregulated in response to the phosphorylation status of its gene product.

Animals↗
PubMed1997

ERC-55, a binding protein for the papilloma virus E6 oncoprotein, specifically interacts with vitamin D receptor among nuclear receptors.

VDR regulates gene expression in a ligand-dependent way by binding to cognate enhancer elements of target gene promoters. The ligand-dependent activation function, AF-2, of VDR is thought to require transcriptional co-activators/co-repressors together with basal transcriptional machinery. Using a yeast two hybrid system with VDR, we have isolated a mouse Ca(2+)-binding protein (designated as VAF1) specifically interacting in vivo and in vitro with VDR among nuclear receptors like RAR, RXR, ER and GR. VAF1 is a mouse homologue to human ERC-55, which has recently been shown to interact with human papillomavirus oncogenic protein, E6[1]. Unlike those of many previously identified co-activators, the VDR-VAF1 interaction was ligand-independent. Thus, VAF1 seems a putative VDR-specific cofactor modulating its function.

Amino Acid Sequence↗
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↗