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Biomedical subjects

H Handa

Publications and source records attributed to H Handa.

At least 73 records · Page 4Linked to original sources
PubMed1999

Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro.

We examined the supportive function of stromal cells from patients with refractory anemia (RA) of myelodysplastic syndrome (MDS) on CD34-positive hematopoietic cell proliferation and differentiation using a long-term bone marrow culture (LTMC) system. Primary marrow stromal cells were obtained from 11 MDS RA patients and 12 healthy volunteers, and freshly prepared CD34-positive bone marrow cells from a normal subject were inoculated onto the stroma. There seems to be three broad patterns of hematopoietic cell growth in the LTMCs. In one group, hematopoietic cells were maintained at near normal levels (type A). In the second group, the number of hematopoietic cells increased within the first 5-10 days of culture, but declined to low levels at 15-20 days of culture as compared with normal control (type B). In the third group, the incidence of hematopoietic cells steadily declined from the beginning of the culture (type C). Furthermore, apoptotic change of hematopoietic cells was very frequently observed in cultures with the type C stroma, which were especially defective for supporting CD34 + cell proliferation and differentiation. The expression of CD95 on hematopoietic cells was induced by the type C stroma, however, production of fas ligand by the stromal cells was not observed. These findings suggest a lack of hematopoietic supportive function in some cases of MDS RA and also indicate that there is heterogeneity of stromal function among MDS RA patients.

Adult↗
PubMed1999

T-kininogen and a 45 kda proteinase from Porphyromonas gingivalis.

To clarify the pathogenic role of proteinases from Porphyromonas gingivalis, a 45 kDa proteinase was isolated from P. gingivalis culture medium by a combination of gel filtration (Bio-Gel A-0.5 m) and ion-exchange chromatographies (DEAE-Sephacel and SP-Sepharose FF). The enzyme was found to have a molecular mass of 45 kDa by SDS-PAGE and to require mercaptoethanol for its activation. The 45 kDa proteinase cleaved T-kininogen into small fragments, but failed to release kinin. In contrast, T-kininogen inhibited the Arg-amidolytic activity of the 45 kDa proteinase with a Ki of 2 nM. On the other hand, the 45 kDa proteinase did not stimulate the production of PGE2, IL-1beta, and TNF-alpha from the macrophages.

Bacterial Proteins↗
PubMed1999

Resonance assignments, secondary structure and 15N relaxation data of the human transcriptional coactivator hMBF1 (57-148).

Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator that is thought to bridge between the TATA box-binding protein (TBP) and DNA binding regulatory factors, and is conserved from yeast to human. Human MBF1 (hMBF1) can bind to TBP and to the nuclear receptor Ad4BP, and is suggested to mediate Ad4BP-dependent transcriptional activation. Here we report the resonance assignments and secondary structure of hMBF1 (57-148) that contains both TBP binding and activator binding residues. 15N relaxation data were also obtained. As a result, hMBF1 (57-148) was shown to consist of flexible N-terminal residues and a C-terminal domain. The C-terminal domain contains four helices and a conserved C-terminal region.

Amino Acid Sequence↗
PubMed1999

Identification of the core domain and the secondary structure of the transcriptional coactivator MBF1.

BACKGROUND: Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA binding activators, such as FTZ-F1 and GCN4. MBF1 bridges the DNA-binding regions of these activators and the TATA-box binding protein (TBP), suggesting that MBF1 functions by recruiting TBP to promoters where the activators are bound. In addition, MBF1 stimulates DNA binding activities of the activators to their recognition sites. To date, little is known about structures of coactivators that bind to TBP. RESULTS: The two-dimensional (2D) 1H-15N correlation spectrum of 15N labeled MBF1 indicated that MBF1 consists of both flexible and well structured parts. Limited digestion of MBF1 by alpha-chymotrypsin yielded a approximately 9 kDa fragment. N-terminal sequence analysis and NMR measurements revealed that this fragment originates from the C-terminal 80 residues of MBF1 and form a well structured C-terminal domain of MBF1, MBF1CTD. As previous deletion analyses have shown that MBF1CTD is capable of binding to TBP, it is suggested that MBF1CTD is the TBP binding domain of MBF1. Sequential assignments have been obtained by means of three-dimensional (3D) and four dimensional (4D) heteronuclear correlation spectroscopies, and then the secondary structure of MBF1CTD was determined. As a result, MBF1CTD was shown to contain four amphipathic helices and a conserved C-terminal region. Asp106 which is assumed to be responsible for the binding to TBP is located at the hydrophilic side of the third helix. CONCLUSIONS: Structural analyses revealed that MBF1 consists of two structurally different domains. A N-terminal region is indispensable for the binding to activators, and does not form a well defined structure. In contrast, the C-terminal 80 residues, which is capable of binding to TBP by itself, form a well-structured domain, MBF1CTD. MBF1CTD is made up of four amphipathic helices and a conserved C-terminal tail. A putative TBP binding residue is located on the hydrophilic surface of the third helix.

Amino Acid Sequence↗
PubMed1999

Missense mutation in the alternative splice region of the PAX6 gene in eye anomalies.

The PAX6 gene is involved in ocular morphogenesis, and PAX6 mutations have been detected in various types of ocular anomalies, including aniridia, Peters anomaly, corneal dystrophy, congenital cataract, and foveal hypoplasia. The gene encodes a transcriptional regulator that recognizes target genes through its paired-type DNA-binding domain. The paired domain is composed of two distinct DNA-binding subdomains, the N-terminal subdomain (NTS) and the C-terminal subdomain (CTS), which bind respective consensus DNA sequences. The human PAX6 gene produces two alternative splice isoforms that have the distinct structure of the paired domain. The insertion, into the NTS, of 14 additional amino acids encoded by exon 5a abolishes the DNA-binding activity of the NTS and unmasks the DNA-binding ability of the CTS. Thus, exon 5a appears to function as a molecular switch that specifies target genes. We ascertained a novel missense mutation in four pedigrees with Peters anomaly, congenital cataract, Axenfeldt anomaly, and/or foveal hypoplasia, which, to our knowledge, is the first mutation identified in the splice-variant region. A T-->A transition at the 20th nucleotide position of exon 5a results in a Val-->Asp (GTC-->GAC) substitution at the 7th codon of the alternative splice region. Functional analyses demonstrated that the V54D mutation slightly increased NTS binding and decreased CTS transactivation activity to almost half.

Abnormalities, Multiple↗
PubMed1999

A wheat cold-regulated cDNA encoding an early light-inducible protein (ELIP): its structure, expression and chromosomal location.

Sequencing and computer analysis revealed that a wcr (wheat cold-regulated) cDNA, wcr12, contained a 525 bp open reading frame encoding a polypeptide of 174 amino acids. The deduced WCR12 protein had a high sequence similarity with Early light-inducible proteins (ELIPs) which are known as nuclear-encoded chloroplast proteins induced by light stress. The accumulation of wcr12 transcripts was induced by low temperature below 8 degrees C and was also increased under a high light flux of 1,000 microEm-2 s-1, which are typical expression patterns of the genes for ELIPs in other plants. The dramatic increase in the level of wcr12 transcripts was detected at 4 degrees C even under a low light condition (50 microEm-2 s-1) and the level of transcripts became 20-fold higher than the control at 20 degrees C under a moderate light condition (250 microEm-2 s-1). Surprisingly, the accumulation of wcr12 mRNAs was also induced by cold treatment alone at 4 degrees C in the darkness. The level of wcr12 transcripts accumulated in the darkness at 4 degrees C reached the same level as that under a high light flux at 20 degrees C. These results indicate that light is not essential for the accumulation of wcr12 transcripts at a low temperature. To determine the chromosomal location of wcr12 gene, we performed Southern analysis using the nullitetrasomic lines. The data showed that the wcr12 gene or related homologues are located on the chromosomes of homoeologous group-5 in wheat.

Amino Acid Sequence↗
PubMed1999

Nuclear transport of the major capsid protein is essential for adeno-associated virus capsid formation.

Adeno-associated virus capsids are composed of three proteins, VP1, VP2, and VP3. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but the functional roles of each protein are still not well understood. By analyzing a series of deletion mutants of VP2, we identified a region necessary for nuclear transfer of VP2 and found that the efficiency of nuclear localization of the capsid proteins and the efficiency of virus-like particle (VLP) formation correlated well. To confirm the importance of the nuclear localization of the capsid proteins, we fused the nuclear localization signal of simian virus 40 large T antigen to VP3 protein. We show that this fusion protein could form VLP, indicating that the VP2-specific region located on the N-terminal side of the protein is not structurally required. This finding suggests that VP3 has sufficient information for VLP formation and that VP2 is necessary only for nuclear transfer of the capsid proteins.

Amino Acid Sequence↗
PubMed1999

Tat-SF1 protein associates with RAP30 and human SPT5 proteins.

The potent transactivator Tat recognizes the transactivation response RNA element (TAR) of human immunodeficiency virus type 1 and stimulates the processivity of elongation of RNA polymerase (Pol) II complexes. The cellular proteins Tat-SF1 and human SPT5 (hSPT5) are required for Tat activation as shown by immunodepletion with specific sera and complementation with recombinant proteins. In nuclear extracts, small fractions of both hSPT5 and Pol II are associated with Tat-SF1 protein. Surprisingly, the RAP30 protein of the heterodimeric transcription TFIIF factor is associated with Tat-SF1, while the RAP74 subunit of TFIIF is not coimmunoprecipitated with Tat-SF1. Overexpression of Tat-SF1 and hSPT5 specifically stimulates the transcriptional activity of Tat in vivo. These results suggest that Tat-SF1 and hSPT5 are indispensable cellular factors supporting Tat-specific transcription activation and that they may interact with RAP30 in controlling elongation.

Chromosomal Proteins, Non-Histone↗
PubMed1999

PAX6 expression in the developing human eye.

AIMS: To investigate the changes in PAX6 expression in the developing human eye. METHODS: Six developing human eyes from 6 to 22 weeks' gestation were evaluated. Frozen sections were immunohistochemically stained with monoclonal antibody to chick Pax6 (amino acids 1-223). To verify antibody specificity, western blot analysis was carried out using cell lysates from P19 cells transfected with the human PAX6 gene. RESULTS: Western blot analysis demonstrated that the antibody reacted to human PAX6 protein. Positive immunostainings for PAX6 were seen in the surface ectoderm, lens vesicle, inner and outer layers of the optic cup, and optic stalk at 6 weeks, and in the corneal epithelia and conjunctiva, lens, and non-pigmented ciliary epithelia from 8 to 22 weeks. In the retina, positive cells were seen in the entire retina from 8 to 10 weeks, and were restricted to the ganglion cell layer and the inner and outer portions of the inner nuclear layer after 21 weeks. CONCLUSIONS: PAX6 is expressed on the surface and neuroectoderms at an early stage, then in the differentiating cells in the cornea, lens, ciliary body, and retina through development. PAX6 may play a role in determining cell fate in the morphogenesis of various human ocular tissue.

Antibody Specificity↗
PubMed1999

Suitable image parameters and analytical method for quantitatively measuring cerebral blood flow volume with phase-contrast magnetic resonance imaging.

The aim of this study was to determine suitable image parameters and an analytical method for phase-contrast magnetic resonance imaging (PC-MRI) as a means of measuring cerebral blood flow volume. This was done by constructing an experimental model and applying the results to a clinical application. The experimental model was constructed from the aorta of a bull and circulating isotonic saline. The image parameters of PC-MRI (repetition time, flip angle, matrix, velocity rate encoding, and the use of square pixels) were studied with percent flow volume (the ratio of actual flow volume to measured flow volume). The most suitable image parameters for accurate blood flow measurement were as follows: repetition time, 50 msec; flip angle, 20 degrees; and a 512 x 256 matrix without square pixels. Furthermore, velocity rate encoding should be set ranging from the maximum flow velocity in the vessel to five times this value. The correction in measuring blood flow was done with the intensity of the region of interest established in the background. With these parameters for PC-MRI, percent flow volume was greater than 90%. Using the image parameters for PC-MRI and the analytical method described above, we evaluated cerebral blood flow volume in 12 patients with occlusive disease of the major cervical arteries. The results were compared with conventional xenon computed tomography. The values found with both methods showed good correlation. Thus, we concluded that PC-MRI was a noninvasive method for evaluating cerebral blood flow in patients with occlusive disease of the major cervical arteries.

Adult↗
PubMed1999

The relative extent and propensity of CD34+ vs. CD34- cells to undergo apoptosis in myelodysplastic marrows.

The paradox of peripheral cytopenias despite cellular bone marrow (BM) observed in myelodysplastic syndromes (MDS) has been associated with excessive intramedullary apoptosis of hematopoietic cells. Since MDS is regarded as a stem cell disorder, the present studies were undertaken to examine the relative susceptibility and propensity of early progenitor CD34+ cells to undergo apoptosis as compared to more maturing/matured CD34- cells. Five serial studies were performed on 4 independent groups of 36 newly diagnosed MDS patients. First, in 2 separate groups of 16 and 8 patients each, measurement of the extent of apoptosis in CD34+ and CD34- fractions of the BM aspirate mononuclear cells was carried out using independent biparametric flow cytometry methods, CD34 labeling/terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (n = 16), and CD34 labeling/reduced uptake of nucleic acid staining dye LDS751 (n = 8). The difference in the median degrees of apoptosis in CD34+ vs. CD34- cells was not statistically significant by either technique (P = 0.583 and P = 0.674 for TUNEL and LDS751, respectively). In the next group of 4 MDS patients, a double-labeling was performed on plastic embedded marrow biopsy sections, to detect CD34 antigen with specific monoclonal antibody and apoptosis by in situ end labeling (ISEL) of fragmented DNA. Despite high overall apoptosis (56.2% +/- 18.4%), only an occasional CD34+ cell was found to be simultaneously labeled with ISEL. Finally, in the last group of 8 MDS patients, CD34+ cells were separated from CD34- cells on affinity column and cultured in serum containing medium for 4 hours. At 0- and 4-hour time points, ISEL was carried out to label apoptotic cells. In addition, a fluorometric assay was employed to estimate the activity of a proapoptotic enzyme, Caspase 3. Both the net increase in % ISEL labeled cells (apoptotic index or AI) and Caspase-3 activity were significantly lower in CD34+ cells as compared to CD34- cells (AI, 0.87% +/- 0.5% vs. 3.97% +/- 1.4%, n = 6, P = 0.028 and Caspase-3 Units/mg protein, 46.9 +/- 25.0 vs. 71.7 +/- 23.03, n = 5, P = 0.042, respectively). We conclude that when estimated in a total population of mononuclear cells, CD34+ cells and CD34- cells show comparable degrees of apoptosis. However, once separated the CD34+ fraction demonstrates lower propensity to undergo apoptosis, thereby suggesting the CD34- fraction as being a possible source for proapoptotic signaling.

Aged↗
PubMed1999

Effects of physical therapy on cytokines and two color analysis-lymphocyte subsets in patients with cerebrovascular diseases.

The effects of physical therapy on immunological parameters were evaluated in 12 patients (8 males and 4 females, 69.2 +/- 9.0 years) with cerebrovascular diseases in a stable situation two to three months after the onset of stroke who entered in our hospital between 1994 and 1997. After a two-month physical therapy program, the proportions of helper-inducer T (Thi) cells and suppressor-inducer T (Tsi) cells were increased significantly and that of cytotoxic T (Tc) cells was decreased, although those of HLA-DR+, suppressor T (Ts) and activated T (Tac) cells were not changed. The antibody dependent cellular cytotoxicity (ADCC) was significantly increased, although natural killer (NK) cell activity was not changed. The serum levels of interleukin-2 receptor was significantly increased but those of interleukin-2, interleukin-6 and interleukin-12 were not changed. The serum levels of interleukin-10, interleukin-12 and tumor necrosis factor-alpha were not detectable, while interleukin-1beta was decreased in 2 patients and interleukin-10 was increased in 2 patients. These findings suggest that daily physical exercise may activate the immune system possibly through the cytokine network in patients with cerebrovascular diseases (CVD).

Aged↗
PubMed1998

Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro.

Recently, a positive and a negative elongation factor, implicated in 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibition of transcription elongation, has been identified. P-TEFb is a positive transcription elongation factor and the DRB-sensitive kinase that phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). PITALRE, a member of the Cdc2 family of protein kinases, is the catalytic subunit of P-TEFb. DSIF is a human homolog of the yeast Spt4-Spt5 complex and renders elongation of transcription sensitive to DRB. DRB sensitivity-inducing factor (DSIF) binds to RNA Pol II and may directly regulate elongation. Here we show a functional interaction between P-TEFb and DSIF. The reduction of P-TEFb activity induced by either DRB, antibody against PITALRE, or immunodepletion resulted in a negative effect of DSIF on transcription. DSIF acts at an early phase of elongation, and the prior action of P-TEFb makes transcription resistant to DSIF. The state of phosphorylation of CTD determines the DSIF-RNA Pol II interaction, and may provide a direct link between P-TEFb and DSIF. Taken together, this study reveals a molecular basis for DRB action and suggests that P-TEFb stimulates elongation by alleviating the negative action of DSIF.

Cyclin-Dependent Kinase 9↗
PubMed1998

Cloning and characterization of the human interleukin-3 (IL-3)/IL-5/ granulocyte-macrophage colony-stimulating factor receptor betac gene: regulation by Ets family members.

High-affinity receptors for interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are composed of two distinct subunits, a ligand-specific chain and a common beta chain (betac). Whereas the mouse has two homologous beta subunits (betac and betaIL-3), in humans, only a single beta chain is identified. We describe here the isolation and characterization of the gene encoding the human IL-3/IL-5/GM-CSF receptor beta subunit. The gene spans about 25 kb and is divided into 14 exons, a structure very similar to that of the murine betac/betaIL-3 genes. Surprisingly, we also found the remnants of a second betac chain gene directly downstream of betac. We identified a functional promoter that is active in the myeloid cell lines U937 and HL-60, but not in HeLa cells. The proximal promoter region, located from -103 to +33 bp, contains two GGAA consensus binding sites for members of the Ets family. Single mutation of those sites reduces promoter activity by 70% to 90%. The 5' element specifically binds PU.1, whereas the 3' element binds a yet-unidentified protein. These findings, together with the observation that cotransfection of PU.1 and other Ets family members enhances betac promoter activity in fibroblasts, reinforce the notion that GGAA elements play an important role in myeloid-specific gene regulation.

Animals↗
PubMed1998

Functional interactions of transcription factor human GA-binding protein subunits.

The transcription factor human GA-binding protein (hGABP) is composed of two subunits, the Ets-related hGABPalpha, which binds to a specific DNA sequence, and either one of two hGABPalpha-associated subunits, hGABPbeta or hGABPgamma. The DNA-binding protein hGABPalpha cannot affect transcription by itself, but can modify hGABP-dependent transcription in vitro and in vivo in the presence of its associated subunits. In this study, co-transfection assays showed that the ratio of hGABPbeta to hGABPgamma affected transcription from a promoter containing hGABP binding sites. Biochemical analysis showed that they bind to hGABPalpha competitively, indicating that the ratio of hGABPbeta to hGABPgamma is important for hGABP complex formation. Kinetic analysis of the protein-protein interaction using the surface plasmon resonance system showed that hGABPalpha binds to hGABPbeta or hGABPgamma with similar equilibrium constants. Kinetic analysis of the DNA-hGABP interaction showed that the binding of hGABPgamma to hGABPalpha stabilized the interaction of hGABPalpha with its DNA binding site. In addition, the kinetic analysis revealed that this was due to a slower dissociation of the protein complex from the DNA. These results suggest that hGABPalpha-associated subunits influence the DNA binding stability of hGABPalpha and regulate hGABP-mediated transcription by competing with each other.

Base Sequence↗
PubMed1998

Involvement of MAP kinase-independent protein kinase C signaling pathway in the EGF-induced p21(WAF1/Cip1) expression and growth inhibition of A431 cells.

Previous studies have revealed that the growth inhibition of A431 cells overexpressing epidermal growth factor (EGF) receptors by a high concentration of EGF is mainly due to the expression of cycline dependent kinase (CDK) inhibitor p21(WAF1/Cip1). However, the signal transduction mechanism from the activated EGF receptor to the induction of p21(WAF1/Cip1) gene is still poorly understood. We investigated which signaling pathway plays an important role in p21(WAF1/Cip1) expression and growth inhibition by using specific inhibitors of the signaling molecules. A broad PKC inhibitor, PKC delta inhibitor, but not the conventional PKC inhibitor suppressed the EGF-induced p21(WAF1/Cip1) expression and the growth inhibition of A431 cells. These inhibitors did not alter either the activation of EGF receptor or the stimulation of MAP kinase at detectable levels. Furthermore, we found that the induction of p21(WAF1/Cip1) at the early phase (within 12 hr after stimulation) by a high concentration of EGF was independent of the MAP kinase activation by using dominant negative Ras. These results suggest that PKC, especially PKC delta plays a crucial role in the EGF-induced p21(WAF1/Cip1) expression, resulting in the growth inhibition of A431 cells.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

Inhibition of macrophage migration inhibitory factor secretion from macrophages by vitamin E.

Macrophage migration inhibitory factor (MIF) was identified in rat peritoneal macrophages by Western blot analysis and its secretion into culture medium by enzyme-linked immunosorbent assay. We investigated the effect of vitamin E on MIF production in macrophages in response to phorbol 12-myristate-13-acetate (PMA), calcium ionophore A23187, and lipopolysaccharide (LPS). Intraperitoneal injections of vitamin E (5 mg per rat) for 6 successive days resulted in a significant increase of alpha-tocopherol content in peritoneal macrophages (478.3+/-90.7 ng/106 cells) compared with the control (1.5+/-0.5 ng/10(6) cells). For the control macrophages, MIF content of the medium (2.5x10(6) cells/18 ml) without stimulation was 2.27+/-0.20 ng/ml after 14 h culture, whereas stimulation with calcium ionophore A23187 (400 nM) and LPS (5.0 microg/ml) induced the elevation of MIF content to 3. 66+/-0.41 and 4.12+/-0.58 ng/ml, respectively. On the other hand, vitamin E-enriched macrophages without stimulation showed less MIF content (0.77+/-0.23 ng/ml) than the control. Similarly, the increase of MIF of vitamin E-treated macrophages was significantly suppressed after stimulation with calcium ionophore A23187 or LPS, compared with the control macrophages. From analysis of intracellular MIF content by Western blot, we found no alteration of intracellular MIF content of vitamin E-macrophages, in contrast to the decreased content of control stimulated-macrophages. Taken together, these results indicate that vitamin E may contribute to the regulation of immune responses through regulation of MIF secretion.

Animals↗