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Biomedical subjects

Hiroshi Handa

Publications and source records attributed to Hiroshi Handa.

At least 73 records · Page 4Linked to original sources
PubMed2004

[Thalidomide treatment of patients with refractory myeloma in the institutes participating in the Japan Myeloma Study Group].

Thalidomide was used in 73 patients with refractory myeloma in 15 of 45 institutes participating in the Japan Myeloma Study Group. The mean age and male/female ratio were 63.8 years and 0.92 (35/38), respectively. Thirty-four patients (47%) were treated with only thalidomide, 27 patients (37%) were treated with thalidomide and steroids, and 12 (16%) were treated with thalidomide and chemotherapy. The mean initial, maximum, and maintenances dose of thalidomide were 111.0, 204.8, and 163.0 mg/day, respectively. Almost all of the patients were maintained on low-dose thalidomide between 100-200 mg/day. Complete, near complete and partial response was obtained in 31 patients (42.5%). The progression-free and overall survivals after thalidomide therapy were 9.8 and 21.3 months, respectively. The most common adverse effects were gastrointestinal disturbance, peripheral neuropathy, psychological signs, and skin eruption. In contrast to reports from Europe and America, no deep vein thrombosis was observed in this study. On the other hand, leukopenia was relatively frequently observed, and might be recognized as a serious adverse effect in myeloma patients. In conclusion, low-dose thalidomide is a useful and safe tool for the treatment of refractory myeloma.

Female↗
PubMed2004

Identification of differentiation-inducing activity produced by human bone marrow stromal cell line LP101.

We have previously reported that human promyelocytic leukemia HL-60 cells can be induced to differentiate into mature granulocytes when HL-60 co-cultivated with human bone marrow stromal LP101 cells. In the present study, we investigated which factors produced by LP101 cells induce HL-60 cells to differentiate into mature granulocytes. The expression of the cell surface antigen CD11b on HL-60 cells was increased after a 72-h culture with the conditioned medium (CM) obtained from LP101 cells. LP101 cells were observed to produce various cytokines, including TNF-alpha, GM-CSF and IL-6. The neutralizing antibodies against these cytokines partially suppressed the CM-induced differentiation of HL-60 cells. Recombinant TNF-alpha induced the differentiation of HL-60 cells, and GM-CSF and IL-6 additionally enhanced the effect of TNF-alpha. When the CM was divided into a low molecular weight (LMW) fraction and a high molecular weight (HMW) fraction by ultrafiltration, the LMW fraction synergistically enhanced the differentiation inducible activity of TNF-alpha. These results demonstrate that LP101 cells induce the differentiation of HL-60 cells by producing various cytokines including TNF-alpha, IL-6, and GM-CSF, and that unknown low molecular weight factors also participate.

Antibodies↗
PubMed2004

[Urinary 8-hydroxy-2'-deoxyguanosin (8-OHdG) in patients with chronic liver diseases].

Urinary 8-hydroxy-2'-deoxyguanosin(8-OHdG) has been reported as sensitive biomarker of oxidative DNA damage and also of oxidative stress. We measured the urinary 8-OHdG in patients with chronic liver diseases by competitive ELISA, and analyzed the relationship with clinical characteristics. Fifty patients (male/female: 22/28) with chronic liver disease were enrolled this study. The mean concentration of urinary 8-OHdG in healthy control and patients with liver cirrhosis, chronic hepatitis C, chronic hepatitis B, and autoimmune hepatitis were 10.40+/-3.14, 10.14+/-4.19, 11.79+/-5.58, 14.99+/-4.46, and 13.64+/-3.84 microg/gCr, respectively. There were no significant differences among the five group. The mean concentration of urinary 8-OHdG in inveterate drinker was significantly higher than that in non-drinker (16.67+/-4.29 vs. 11.19+/-4.80 microg/gCr, p<0.05). The smoking enhanced the elevation of urinary 8-OHdG in drinkers. In clinical characteristics, serum y-GTP, a marker of alcoholic liver disease, had significant positive correlation with urinary 8-OHdG on the drinker with chronic hepatitis. In addition, there was a positive correlation between serum ferritin levels and urinary 8-OHdG levels. Iron in the liver suggested oxidative damage of hepatocytes through the fenton reaction in patients with chronic liver disease. In conclusion, drinking and smoking induced liver damage by oxidative stress, and urinary 8-OHdG may be reliable marker of oxidative stress in patients with chronic liver disease.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2003

A novel hydrogen peroxide-induced phosphorylation and ubiquitination pathway leading to RNA polymerase II proteolysis.

DNA damage-induced ubiquitination of the largest subunit of RNA polymerase II, Rpb1, has been implicated in transcription-coupled repair for years. The studies so far, however, have been limited to the use of bulky helix-distorting DNA damages caused by UV light and cisplatin, which are corrected by the nucleotide excision repair pathway. Non-bulky, non-helix-distorting damages are caused at high frequency by reactive oxygen species in cells and corrected by the base excision repair pathway. Contrary to a classic view, we recently found that the second type of DNA lesions also causes RNA polymerase II stalling in vitro. In this paper, we show that hydrogen peroxide (H(2)O(2)) causes significant ubiquitination and proteasomal degradation of Rpb1 by mechanisms that are distinct from those employed after UV irradiation. UV irradiation and H(2)O(2) treatment cause characteristic changes in protein kinases phosphorylating the carboxyl-terminal domain at Ser-2 and -5. The H(2)O(2)-induced ubiquitination is likely dependent on unusual Ser-5 phosphorylation by ERK1/2. Moreover, the H(2)O(2)-induced ubiquitination occurs on transcriptionally engaged polymerases without the help of Cockayne syndrome A and B proteins and von Hippel-Lindau tumor suppressor proteins, which are all required for the UV-induced ubiquitination. These results suggest that stalled polymerases are recognized and ubiquitinated differentially depending on the types of DNA lesions. Our findings may have general implications in the basic mechanism of transcription-coupled nucleotide excision repair and base excision repair.

Cell Death↗
PubMed2003

Differential requirement of EGFR signaling for the expression of defective proventriculus gene in the Drosophila endoderm and ectoderm.

A homeobox gene, defective proventriculus (dve), is expressed in various tissues including the ventral ectoderm and midgut. Here, we show the expression pattern of dve in the ventral ectoderm, in which dve expression is induced by Spitz, a ligand for Drosophila epidermal growth factor receptor (EGFR). In spitz mutants, dve expression is only lost in the ventral ectoderm and overexpression of Spitz induces ectopic dve activation in the ventral ectoderm. Dve expression in the middle midgut depends on Decapentaplegic (Dpp) signaling, while expression of a dominant-negative form of Drosophila EGFR (DER(DN)) also causes a marked decrease in dve expression in the middle midgut. Furthermore, heterozygous mutation of thick veins (tkv), a Dpp receptor, strongly enhances the effect of DER(DN). These results indicate that EGFR signaling is crucial for dve expression in the ventral ectoderm and is required in the middle midgut where it cooperates with Dpp signaling.

Animals↗
PubMed2003

Specific interaction with transcription factor IIA and localization of the mammalian TATA-binding protein-like protein (TLP/TRF2/TLF).

TBP-like protein (TLP) is structurally similar to the TATA-binding protein (TBP) and is thought to have a transcriptional regulation function. Although TLP has been found to form a complex with transcription factor IIA (TFIIA), the in vivo functions of TFIIA for TLP are not clear. In this study, we analyzed the interaction between TLP and TFIIA. We determined the biophysical properties for the interaction of TLP with TFIIA. Dissociation constants of TFIIA versus TLP and TFIIA versus TBP were 1.5 and 10 nm, respectively. Moreover, the dissociation rate constant of TLP and TFIIA (1.2 x 10(-4)/m.s was significantly lower than that of TBP (2.1 x 10(-3)/m.s). These results indicate that TLP has a higher affinity to TFIIA than does TBP and that the TLP-TFIIA complex is much more stable than is the TBP-TFIIA complex. We found that TLP forms a dimer and a trimer and that these multimerizations are inhibited by TFIIA. Moreover, TLP mutimers were more stable than a TBP dimer. We determined the amounts of TLPs in the nucleus and cytoplasm of NIH3T3 cells and found that the molecular number of TLP in the nucleus was only 4% of that in the cytoplasm. Immunostaining of cells also revealed cytoplasmic localization of TLP. We established cells that stably express mutant TLP lacking TFIIA binding ability and identified the amino acids of TLP required for TFIIA binding (Ala-32, Leu-33, Asn-37, Arg-52, Lys-53, Lys-78, and Arg-86). Interestingly, the level of TFIIA binding defective mutant TLPs in the nucleus was much higher than that of the wild-type TLP and TFIIA-interactable mutant TLPs. Immunostaining analyses showed consistent results. These results suggest that the TFIIA binding ability of TLP is required for characteristic cytoplasmic localization of TLP. TFIIA may regulate the intracellular molecular state and the function of TLP through its property of binding to TLP.

Animals↗
PubMed2003

Cell cycle-dependent regulation of the Skp2 promoter by GA-binding protein.

Skp2 is the F-box protein component of an SCF-type ubiquitin ligase that interacts specifically with p27(Kip1) and thereby promotes its ubiquitylation and degradation. The abundance of Skp2 mRNA oscillates in a cell cycle-dependent manner, being maximal in S and G(2) phases. The regulation of Skp2 transcription was investigated by cloning the promoter region of the mouse gene and determination of its activity in a luciferase reporter assay. Deletion analysis identified a minimal approximately 0.3-kb promoter region with marked transcriptional activity and a 105-bp essential sequence within this region. Electrophoretic mobility shift assays indicated the presence in nuclear extracts of proteins that bind to this sequence. Site-directed mutagenesis revealed that the core binding motif, CACTTCCG, which is similar to that of GA-binding protein (GABP), is essential for Skp2 transcription. "Supershift" analysis indicated that the protein-probe complexes detected by electrophoretic mobility shift assays contain GABP. Endogenous GABP bound to Skp2 promoter element in a cell cycle-dependent manner. Furthermore, overexpression of GABPbeta increased Skp2 promoter activity, and suppression of GABPalpha or GABPbeta by a small interfering RNA resulted in the reduction of Skp2 promoter activity. These data suggest that the cell cycle-dependent binding of GABP to the Skp2 promoter plays an important role in the regulation of Skp2 expression and cell cycle progression from G(1) to S phase.

3T3 Cells↗
PubMed2003

Drosophila FACT contributes to Hox gene expression through physical and functional interactions with GAGA factor.

Chromatin structure plays a critical role in the regulation of transcription. Drosophila GAGA factor directs chromatin remodeling to its binding sites. We show here that Drosophila FACT (facilitates chromatin transcription), a heterodimer of dSPT16 and dSSRP1, is associated with GAGA factor through its dSSRP1 subunit, binds to a nucleosome, and facilitates GAGA factor-directed chromatin remodeling. Moreover, genetic interactions between Trithorax-like encoding GAGA factor and spt16 implicate the GAGA factor-FACT complex in expression of Hox genes Ultrabithorax, Sex combs reduced, and Abdominal-B. Chromatin immunoprecipitation experiments indicated the presence of the GAGA factor-FACT complex in the regulatory regions of Ultrabithorax and Abdominal-B. These data illustrate a crucial role of FACT in the modulation of chromatin structure for the regulation of gene expression.

Amino Acid Sequence↗
PubMed2003

NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila.

NELF and DSIF collaborate to inhibit elongation by RNA polymerase IIa in extracts from human cells. A multifaceted approach was taken to investigate the potential role of these factors in promoter proximal pausing on the hsp70 gene in Drosophila. Immunodepletion of DSIF from a Drosophila nuclear extract reduced the level of polymerase that paused in the promoter proximal region of hsp70. Depletion of one NELF subunit in salivary glands using RNA interference also reduced the level of paused polymerase. In vivo protein-DNA cross-linking showed that NELF and DSIF associate with the promoter region before heat shock. Immunofluorescence analysis of polytene chromosomes corroborated the cross-linking result and showed that NELF, DSIF, and RNA polymerase IIa colocalize at the hsp70 genes, small heat shock genes, and many other chromosomal locations. Finally, following heat shock induction, DSIF and polymerase but not NELF were strongly recruited to chromosomal puffs harboring the hsp70 genes. We propose that NELF and DSIF cause polymerase to pause in the promoter proximal region of hsp70. The transcriptional activator, HSF, might cause NELF to dissociate from the elongation complex. DSIF continues to associate with the elongation complex and could serve a positive role in elongation.

Animals↗
PubMed2003

Mutations of the PAX6 gene detected in patients with a variety of optic-nerve malformations.

The PAX6 gene is involved in ocular morphogenesis and is expressed in the developing central nervous system and numerous ocular tissues during development. PAX6 mutations have been detected in various ocular anomalies, including aniridia, Peters anomaly, corneal dystrophy, congenital cataracts, and foveal hypoplasia. However, it has not been identified in patients with optic-nerve malformations. Here, we identified novel mutations in eight pedigrees with optic-nerve malformations, including coloboma, morning glory disc anomaly, optic-nerve hypoplasia/aplasia, and persistent hyperplastic primary vitreous. A functional assay demonstrated that each mutation decreased the transcriptional activation potential of PAX6 through the paired DNA-binding domain. PAX6 and PAX2 are each thought to downregulate the expression of the other. Four of the detected mutations affected PAX6-mediated transcriptional repression of the PAX2 promoter in a reporter assay. Because PAX2 gene mutations were detected in papillorenal syndrome, alternation of PAX2 function by PAX6 mutations may affect phenotypic manifestations of optic-nerve malformations.

Adult↗
PubMed2003

NTP-driven translocation by human RNA polymerase II.

We report a "running start, two-bond" protocol to analyze elongation by human RNA polymerase II (RNAP II). In this procedure, the running start allowed us to measure rapid rates of elongation and provided detailed insight into the RNAP II mechanism. Formation of two bonds was tracked to ensure that at least one translocation event was analyzed. By using this method, RNAP II is stalled briefly at a defined template position before restoring the next NTP. Significantly, slow reaction steps are identified both before and after phosphodiester bond synthesis, and both of these steps can be highly dependent on the next templated NTP. The initial and final NTP-driven events, however, are not identical, because the slow step after chemistry, which includes translocation and pyrophosphate release, is regulated differently by elongation factors hepatitis delta antigen and transcription factor IIF. Because recovery from a stall and the processive transition from one bond to the next can be highly NTP-dependent, we conclude that translocation can be driven by the incoming substrate NTP, a model fully consistent with the RNAP II elongation complex structure.

Adenosine Triphosphate↗
PubMed2003

Inhibition by (+/-)-indenestrol A of interferon gamma-stimulated nitric oxide formation in murine macrophage RAW 264.7 cells.

We examined the effects of (+/-)-indenestrol A (IA), an antioxidative and superoxide-producing metabolite of diethylstilbestrol (DES), on the activation of murine macrophages (RAW 264.7 cells) in vitro, particularly with regard to interferon (IFN)-gamma-induced nitric oxide (NO) production. (+/-)-IA inhibited NO production more strongly than DES as assessed by a nitrite assay. The inhibitory effect of (+/-)-IA on IFN-gamma-induced intracellular NO production was confirmed by direct staining of intracellular NO with diaminofluorescein-2 diacetyl. Inhibition of NO production was confirmed by Western blot analysis of IFN-gamma-induced NO synthase. Under IFN-gamma-stimulated conditions, the IFN-gamma activation site (GAS), which was the most important transcription factor, was significantly inhibited by (+/-)-IA. (+/-)-IA also promoted the activation of NF-kappaB. (+/-)-IA at 1 and 3 microM delayed the onset of apoptosis. Our results suggest that (+/-)-IA inhibited the activation of macrophages, resulting in the suppression of NO-mediated apoptosis. These results suggest a novel mechanism for the carcinogenic promoting activity of DES via its metabolite, (+/-)-IA.

Animals↗
PubMed2003

High Th1/Th2 ratio in patients with chronic idiopathic thrombocytopenic purpura.

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by increased platelet clearance because of antiplatelet antibodies. It was recently reported that the balance of T helper 1 (Th1) and T helper 2 (Th2) subsets has been implicated in the regulation of many immune responses. In this study, the intracellular interleukin-4 and interferon-gamma production in CD4+ T-lymphocytes activated by phorbol 12-myristate 13-acetate and ionomycin was assessed via flow cytometry in order to determine the clinical significance of the Th1/Th2 ratio in 42 patients with ITP. The study cohort included 28 untreated patients, seven postprednisolone therapy patients and seven postsplenectomy patients. The mean level of the Th1/Th2 ratio in the untreated group was 36.9 (95% CI 25.8-47.9), and significantly higher than in the control group (mean 12.8, 95% CI 9.5-16.1). The mean levels of the Th1/Th2 ratio in the postprednisolone therapy and postsplenectomy groups were 20.5 (95% CI 8.4-32.6) and 16.4 (95% CI 3.1-29.7), respectively, but were no significant differences as compared with control subjects. When untreated patients were divided into two subgroups by Th1/Th2 ratio, the mean level of platelet associated IgG in the high Th1/Th2 subgroup (higher than upper limit of control group) tended to be higher than in the normal Th1/Th2 subgroup. In conclusion, the high Th1/Th2 ratio was closely related to the etiology and disease status of chronic ITP.

Adult↗
PubMed2003

TCR Vbeta repertoire analysis in CD56+ CD16(dim/-) T-cell large granular lymphocyte leukaemia: association with CD4 single and CD4/CD8 double positive phenotypes.

We report 10 patients with T-cell large granular lymphocyte (LGL) leukaemia: four patients had CD16+ CD56- LGL lymphocytes (typical for LGL leukaemia), and six patients had CD56+ CD16(dim/-) LGL lymphocytes (atypical). Among the CD56+ CD(dim/-) patients, LGL lymphocytes were CD4+ CD8- in one patient, CD4/CD8 double positive (DP) in three, and CD4- CD8+ in two. The CD4+ CD8dim DP cells expressed a CD8alphaalpha homodimer. T-cell receptor (TCR) Vbeta complementarity-determining region 3 (CDR3) size distribution analysis and direct sequencing identified at least 1 in-frame clonal TCR Vbeta transcript in each patient; three patients had two or three different clonal sequences. To determine whether these transcripts were translated into cell surface TCR, we performed flow cytometric analysis using Vbeta monoclonal antibodies (mAbs). A single Vbeta protein was identified in patients, even those with multiple in-frame transcripts. Previous and present results suggest that CD56+ CD16(dim/-) LGL leukaemia is more common than previously thought, and is associated with unusual phenotypes. When assessed using only molecular techniques, the monoclonal status of this disease may be misinterpreted as oligoclonal; thus, flow cytometric analysis using Vbeta mAb is quite useful. Because mAbs do not cover the entire Vbeta repertoire, assessing clonality using a combination of molecular methods and mAbs is preferable.

Adult↗
PubMed2003

Suppression of NF-kappaB-dependent gene expression by a hexamethylene bisacetamide-inducible protein HEXIM1 in human vascular smooth muscle cells.

BACKGROUND: Neointima formation is a characteristic feature of atherosclerosis and post-angioplasty restenosis, in which various soluble factors and mechanical injury stimulate signalling pathways in vascular smooth muscle cells (VSMC), promoting their migration and proliferation, and the eventual formation of the neointima. The transcription factor NF-kappaB has been shown to play a pivotal role in this process. Hexamethylene bisacetamide, an inhibitor of VSMC proliferation, induces the mRNA expression of HEXIM1 (hexamethylene bisacetamide-inducible protein 1). However, the protein expression and function of HEXIM1 remain unknown. RESULTS: In the present study, we demonstrated that HEXIM1 localizes in the cytoplasm and nucleus, and its nuclear expression is restricted to discrete speckled areas. Treatment of VSMC with hexamethylene bisacetamide up-regulated HEXIM1 expression, not only in mRNA but also protein levels. Moreover, HEXIM1 is shown to suppress the transcriptional activity of NF-kappaB via its C-terminal leucine-rich domain. A glutathione-S-transferase pull down assay indicated that HEXIM1 interacts with the p65 subunit of NF-kappaB. In VSMC, treatment with hexamethylene bisacetamide resulted in a down-modulation of the transcription of NF-kappaB target genes. CONCLUSION: We may therefore conclude that HEXIM1 plays an inhibitory role in NF-kappaB-dependent gene expression in VSMC and is the candidate of a novel therapeutic target for inhibition of VSMC proliferation.

Acetamides↗
PubMed2003

Mechanism of H-8 inhibition of cyclin-dependent kinase 9: study using inhibitor-immobilized matrices.

BACKGROUND: Positive transcription elongation factor b (P-TEFb), which phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII), is comprised of the catalytic subunit cyclin-dependent kinase 9 (CDK9) and the regulatory subunit cyclin T. The kinase activity and transcriptional activation potential of P-TEFb is sensitive to various compounds, including H-8, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), and flavopiridol. RESULTS: We investigated the molecular mechanism of the H-8 inhibition of CDK9 using matrices to which H-9, an amino derivative of H-8, was immobilized. CDK9 bound specifically to H-9, and this interaction was competitively inhibited by ATP and DRB, but not by flavopiridol. Mutational analyses demonstrated that the central region of CDK9, which encompasses the T-loop region, was important for its binding to H-9. CONCLUSIONS: H-9-immobilized latex beads are useful for trapping CDK9 and a subset of kinases from crude cell extracts. The flavopiridol-binding region of CDK9 is most likely different from its H-9-binding region. These biochemical data support previously reported observations which were based on crystallographic data.

Adenosine Triphosphate↗