Biomedical subjects

T Wada

Publications and source records attributed to T Wada.

At least 217 records · Page 12Linked to original sources

PubMed2000

Chemical synthesis of hydroxymethylphosphonate alpha-DNA.

In this paper, we report the synthesis of hydroxymethylphosphonate alpha-DNAs and related compounds by use of the H-phosphonate method. These modified alpha-DNAs were designed to improve the inherent poor solubility of well-known methylphosphonate alpha-DNAs by introduction of a more hydrophilic hydroxymethylphosphonate function. The hybridization ability of hydroxymethylphosphonate alpha-DNAs was studied. We also report a novel strategy for the synthesis of alpha-thymidine by use of C1'-epimerization. The details of the neighboring effect of various 5'- and 3'-hydroxyl protective groups such as carbamoyl groups on the beta-->alpha conversion will be described.

DNA↗

PubMed1999

Structure and stability of the consecutive stereoregulated chiral phosphorothioate DNA duplex.

The duplex structures of the stereoregulated phosphorothioate DNAs, [R(p),R(p)]- and [S(p),S(p)]-[d(GC(ps)T(ps)ACG)] (ps, phosphorothioate; PS-DNA), with their complementary RNA have been investigated by combined use of (1)H NMR and restrained molecular dynamics calculation. Compared to those obtained for the unmodified duplex structures (PO-DNA.RNA), the NOE cross-peak intensities are virtually identical for the PS-DNA.RNA hybrid duplexes. The structural analysis on the basis of the NOE restraints reveals that all of the three DNA.RNA duplexes take a A-form conformation and that there is no significant difference in the base stacking for the DNA.RNA hybrid duplexes. On the other hand, the NOE cross-peak intensities of the protons around the central T(ps)A step of the PS-DNA.DNA duplexes are apparently different from those of PO-DNA. DNA. The chemical shifts of H8/6 and H1' at the T(ps)A step are also largely different among PS-DNA.DNAs and PO-DNA.DNA, suggesting that the DNA.DNA structure is readily changed by the introduction of the phosphorothioate groups to the central T(p)A step. The structure calculations indicate that all of these DNA.DNA duplexes are B-form although there exist some small differences in helical parameters between the [R(p),R(p)]- and [S(p),S(p)]PS-DNA.DNA duplexes. The melting temperatures (T(m)) were determined for all of the duplexes by plotting the chemical shift change of isolated peaks as a function of temperature. For the PS-DNA.RNA hybrid duplexes, the [S(p),S(p)] isomer is less stable than the [R(p),R(p)] isomer while this trend is reversed for the PS-DNA.DNA duplexes. Consequently, although the PS-DNA.RNA duplexes take the similar A-form structure, the duplex stability is different between PS-DNA.RNA duplexes. The stability of the DNA.RNA duplexes may not be governed by the A-form structure itself but by some other factors such as the hydration around the phosphorothioate backbone, although the T(m) difference of the DNA.DNA duplexes could be explained by the structural factor.

DNA↗

PubMed1999

The role of human MBF1 as a transcriptional coactivator.

Multiprotein bridging factor 1 (MBF1) is a coactivator which mediates transcriptional activation by interconnecting the general transcription factor TATA element-binding protein and gene-specific activators such as the Drosophila nuclear receptor FTZ-F1 or the yeast basic leucine zipper protein GCN4. The human homolog of MBF1 (hMBF1) has been identified but its function, especially in transcription, remains unclear. Here we report the cDNA cloning and functional analysis of hMBF1. Two isoforms, which we term hMBF1alpha and hMBF1beta, have been identified. hMBF1alpha mRNA was detected in a number of tissues, whereas hMBF1beta exhibited tissue-specific expression. Both isoforms bound to TBP and Ad4BP/SF-1, a mammalian counterpart of FTZ-F1, and mediated Ad4BP/SF-1-dependent transcriptional activation. While hMBF1 was detected in the cytoplasm by immunostaining, coexpression of the nuclear protein Ad4BP/SF-1 with hMBF1 induced accumulation of hMBF1 in the nucleus, suggesting that hMBF1 is localized in the nucleus through its binding to Ad4BP/SF-1. hMBF1 also bound to ATF1, a member of the basic leucine zipper protein family, and mediated its activity as a transcriptional activator. These data establish that the coactivator MBF1 is functionally conserved in eukaryotes.

Amino Acid Sequence↗

PubMed1999

Ischemic "cross" tolerance in hypoxic ischemia of immature rat brain.

The phenomenon of ischemic tolerance has been closely associated with the expression of heat shock proteins but recently, stress tolerance not related to hsp72 has been reported. In the present study, we focused on ischemic tolerance induced by hypoxia and hyperthermia in neonatal rat brain and analyzed the expression of hsp72. In a neonatal rat model of hypoxic ischemia (H-I), preconditioning by whole-body hyperthermia or hypoxia was induced 24 h prior to the ischemia. Brain damage was histologically evaluated and the expressions of hsp72 were analyzed. Hyperthermic preconditioning at 41 degrees C for 15 min, as well as hypoxic preconditioning with 8% hypoxia for 3 h, had almost complete neuroprotective effects. However, we failed to detect the expression of hsp72 in any of preconditioning. Only the H-I insult itself induced hsp72 in the dorsal striatum and slightly in the thalamus and the hippocampus. Hyperthermic preconditioning has neuroprotective effects which are comparable to hypoxic preconditioning in immature brain. The expression of hsp72 is not likely necessary for the ischemic tolerance in immature brain.

Animals↗

PubMed1999

Topological analysis of an RND family transporter, MexD of Pseudomonas aeruginosa.

The membrane topology of a resistance-nodulation-division (RND) family transporter, MexD of Pseudomonas aeruginosa, was determined. Although it had been predicted previously that most RND proteins contain 12 transmembrane helices, three independent computer programs used in the present study predicted that MexD possessed 11, 14 or 17 transmembrane segments. To investigate the topology of MexD more thoroughly, 25 MexD-PhoA (alkaline phosphatase) and 18 MexD-Bla (beta-lactamase) fusion plasmids were constructed and analyzed. The resulting topological model had just 12 transmembrane helices and two periplasmic loops of about 300 residues between helices 1 and 2 and helices 7 and 8. It is therefore proposed that the N- and C-termini are located in the cytoplasm and the predicted orientation is consistent with the 'positive-inside rule'. This topological model can be applied to other RND proteins.

Alkaline Phosphatase↗

PubMed1999

The DNA-binding and transcriptional activities of MAZ, a myc-associated zinc finger protein, are regulated by casein kinase II.

Myc-associated zinc finger protein (MAZ) is a transcription factor that contains proline-rich, alanine repeats and six C(2)H(2)-type zinc finger motifs, as well as five putative sites of phosphorylation by casein kinase II (CKII). Site-specific mutagenesis of MAZ revealed that the serine residue at position 480 was the major site of phosphorylation by CKII both in vitro and in vivo. Phosphorylation of MAZ by CKII at this serine residue was required for maximum binding of MAZ to the pyrimidine-rich DNA of the nuclease-hypersensitive element (NHE) in the 5'-end promoter region of the c-myc gene. Mutation of serine at position 480 to alanine eliminated the DNA-binding activity of MAZ to this element. Moreover, the mutated MAZ was unable to enhance the expression of luciferase encoded by a c-myc promoter/luciferase reporter gene in HeLa cells in the presence of CKII. These results suggest that phosphorylation of the serine residue at position 480 of MAZ by CKII can control the function of MAZ by altering its DNA-binding activity.

Amino Acid Substitution↗

PubMed1999

His(15) of subunit a of the Escherichia coli ATP synthase is important for the structure or assembly of the membrane sector F(o).

Approximately 37 amino acids at the amino-terminus of subunit a of the Escherichia coli ATP synthase are found localized to the periplasm. Results indicate that a single amino acid substitution, H15D, disrupts assembly of subunit a and causes a loss of ATP synthase function. In this study, a conserved region of nine amino acids, 11-19, was initially mutagenized randomly, generating no mutants that could grow on succinate-minimal medium. Subsequent mutagenesis, confined to residues His(14), His(15), and Asn(17), indicated that constructs containing H15D were the most deleterious. Four single mutants were constructed and analyzed: H15A, H14D, H15A, and H15D. Only H15D was significantly impaired, with respect to ATP-driven proton translocation, passive proton permeability through F(o), and sensitivity of membrane-bound ATPase to DCCD. Immunoblot analysis indicated very low levels of subunit a from H15D. Cysteine mutations were constructed at positions 14, 15, 17, and 18. Residues 14, 15, and 17 were shown to be accessible in the periplasmic space, while residue 18 was not, indicating that this region was stably folded. While both His(14) and His(15) are conserved among a group of bacteria, results presented here indicate that they are not equivalent, and that a specific role for His(15) in the assembly or structure of the ATP synthase is supported.

Amino Acid Sequence↗

PubMed1999

Cloning, expression, and chromosomal mapping of a human ganglioside sialidase.

Here we report the cDNA sequence of a human ganglioside sialidase. The cDNA was isolated from a human brain cDNA library by screening with a 240 bp probe generated by polymerase chain reaction using primers based on the sequences of rat cytosolic and bovine membrane sialidases which we previously cloned. The 3.0 kb cDNA encodes an open reading frame of 436 amino acids containing a putative transmenbrane domain and an Arg-Ile-Pro and three Asp-box sequences characteristic of sialidases and showing overall 83% and 39% identities to the bovine and rat enzymes, respectively. Northern blot analysis revealed high expression in skeletal muscle and testis, but low level in kidney, placenta, lung, and digestive organs. Transient expression of the cDNA in COS-1 cells resulted in a 130-fold increase in sialidase activity compared to the control level, and the activity was found to be almost specific for gangliosides. Fluorescent in situ hybridization allowed the human sialidase gene localized to chromosome 11 at q 13.5.

Amino Acid Sequence↗

PubMed1999

Cloning and expression of Der f 6, a serine protease allergen from the house dust mite, Dermatophagoides farinae.

House dust mite allergen is thought to be a major cause of asthma. Characterization of these allergen molecules is therefore an important step for the development of effective diagnostic and therapeutic agents against mite-associated allergic disorders. Here we report molecular cloning and expression of the group 6 (chymotrypsin-like) allergen from the house dust mite, Dermatophagoides farinae. Sequencing analysis indicates that cloned cDNA, designated Der f 6, encodes a 279 amino acid polypeptide which conserves a primary structure characteristic for chymotrypsin-like serine proteases found in mammals. Recombinant Der f 6 expressed in Escherichia coli bound IgE in a pool made of 20 sera, and induced histamine release from patients' peripheral blood cells.

Allergens↗

PubMed1999

Molecular cloning of rat SH2-containing inositol phosphatase 2 (SHIP2) and its role in the regulation of insulin signaling.

SH2-containing inositol 5'-phosphatase (SHIP) plays a negative regulatory role in hematopoietic cells. We have now cloned the rat SHIP isozyme (SHIP2) cDNA from skeletal muscle, which is one of the most important target tissue of insulin action. Rat SHIP2 cDNA encodes a 1183-amino-acid protein that is 45% identical with rat SHIP. Rat SHIP2 contains an amino-terminal SH2 domain, a central 5'-phosphoinositol phosphatase activity domain, and a phosphotyrosine binding (PTB) consensus sequence and a proline-rich region at the carboxyl tail. Specific antibodies to SHIP2 were raised and the function of SHIP2 was studied by stably overexpressing rat SHIP2 in Rat1 fibroblasts expressing human insulin receptors (HIRc). Endogenous SHIP2 underwent insulin-mediated tyrosine phosphorylation and phosphorylation was markedly increased when SHIP2 was overexpressed. Although overexpression of SHIP2 did not affect insulin-induced tyrosine phosphorylation of the insulin receptor beta-subunit and Shc, subsequent association of Shc with Grb2 was inhibited, possibly by competition between the SH2 domains of SHIP2 and Grb2 for the Shc phosphotyrosine. As a result, insulin-stimulated MAP kinase activation was reduced in SHIP2-overexpressing cells. Insulin-induced tyrosine phosphorylation of IRS-1, IRS-1 association with the p85 subunit of PI3-kinase, and PI3-kinase activation were not affected by overexpression of SHIP2. Interestingly, although both PtdIns-(3,4,5)P3 and PtdIns(3,4)P2 have been implicated in the regulation of Akt activity in vitro, overexpression of SHIP2 inhibited insulin-induced Akt activation, presumably by its 5'-inositol phosphatase activity. Furthermore, insulin-induced thymidine incorporation was decreased by overexpression of SHIP2. These results indicate that SHIP2 plays a negative regulatory role in insulin-induced mitogenesis, and regulation of the Shc. Grb2 complex and of the downstream products of PI3-kinase provides possible mechanisms of SHIP2 action in insulin signaling.

Adaptor Proteins, Signal Transducing↗

PubMed1999

A novel labeling approach supports the five-transmembrane model of subunit a of the Escherichia coli ATP synthase.

Cysteine mutagenesis and surface labeling has been used to define more precisely the transmembrane spans of subunit a of the Escherichia coli ATP synthase. Regions of subunit a that are exposed to the periplasmic space have been identified by a new procedure, in which cells are incubated with polymyxin B nonapeptide (PMBN), an antibiotic derivative that partially permeabilizes the outer membrane of E. coli, along with a sulfhydryl reagent, 3-(N-maleimidylpropionyl) biocytin (MPB). This procedure permits reaction of sulfhydryl groups in the periplasmic space with MPB, but residues in the cytoplasm are not labeled. Using this procedure, residues 8, 27, 37, 127, 131, 230, 231, and 232 were labeled and so are thought to be exposed in the periplasm. Using inside-out membrane vesicles, residues near the end of transmembrane spans 1, 64, 67, 68, 69, and 70 and residues near the end of transmembrane spans 5, 260, 263, and 265 were labeled. Residues 62 and 257 were not labeled. None of these residues were labeled in PMBN-permeabilized cells. These results provide a more detailed view of the transmembrane spans of subunit a and also provide a simple and reliable technique for detection of periplasmic regions of inner membrane proteins in E. coli.

Amino Acid Sequence↗

PubMed1999

NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation.

DRB is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). Since DRB generally affects class II genes, factors involved in this process must play fundamental roles in pol II elongation. Recently, two elongation factors essential for DRB action were identified, namely DSIF and P-TEFb. Here we describe the identification and purification from HeLa nuclear extract of a third protein factor required for DRB-sensitive transcription. This factor, termed negative elongation factor (NELF), cooperates with DSIF and strongly represses pol II elongation. This repression is reversed by P-TEFb-dependent phosphorylation of the pol II C-terminal domain. NELF is composed of five polypeptides, the smallest of which is identical to RD, a putative RNA-binding protein of unknown function. This study reveals a molecular mechanism for DRB action and a regulatory network of positive and negative elongation factors.

Amino Acid Sequence↗

PubMed1999

Structure and function of the human transcription elongation factor DSIF.

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). We have previously identified and purified a novel transcription elongation factor, termed DSIF (for DRB sensitivity-inducing factor), that makes transcription sensitive to DRB. DSIF is composed of 160- and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4. DSIF may either repress or stimulate transcription in vitro, depending on conditions, but its physiological function remains elusive. Here we characterize the structure and function of DSIF p160. p160 is shown to be a ubiquitous nuclear protein that forms a stable complex with p14 and interacts directly with the pol II largest subunit. Mutation analysis of p160 is used to identify structural features essential for its in vitro activity and to map the domains required for its interaction with p14 and pol II. Finally, a p160 mutant that represses DSIF activity in a dominant-negative manner is identified and used to demonstrate that DSIF represses transcription from various promoters in vivo.

Amino Acid Sequence↗

PubMed1999

Correlation of antiangiogenic and antitumor efficacy of N-biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA), an orally-active, selective matrix metalloproteinase inhibitor.

The antiangiogenic activity and antitumor efficacy of a newly developed matrix metalloproteinase (MMP) inhibitor were examined. N-biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA) potently inhibits MMP-2, -9, and -14, but not MMP-1, -3, or -7. In contrast, (-)BPHA, an enantiomer of BPHA, was inactive against all MMPs tested. Daily oral administration of 200 mg/kg BPHA, but not (-)BPHA in mice resulted in potent inhibition of tumor-induced angiogenesis, primary tumor growth, and liver metastasis. The growth inhibition activity of BPHA was 48% and 45% in a B16-BL6 melanoma and F2 hemangio-endothelioma model, respectively. BPHA also showed 42% inhibition of the liver metastasis of C-1H human colon carcinoma cells. These results indicate that selective MMP inhibition is correlated with antiangiogenic and antitumor efficacy and that the selective MMP inhibitor BPHA has therapeutic potential.

Animals↗

PubMed1999

Molecular cloning and characterization of a plasma membrane-associated sialidase specific for gangliosides.

Gangliosides are plasma membrane components thought to play important roles in cell surface interactions, cell differentiation, and transmembrane signaling. A mammalian sialidase located in plasma membranes is unique in specifically hydrolyzing gangliosides, suggesting crucial roles in regulation of cell surface functions. Here we describe the cloning and expression of a cDNA for the ganglioside sialidase, isolated from a bovine brain cDNA library based on the amino acid sequence of the purified enzyme from bovine brain. This cDNA encodes a 428-amino acid protein containing a putative transmembrane domain and the three Asp boxes characteristic of sialidases and sharing 19-38% sequence identity with other sialidases. Northern blot and polymerase chain reaction analyses revealed a general distribution of the gene in mammalian species, including man, and the mouse. In COS-7 cells transiently expressing the sialidase, the activity was found to be 40-fold that of the control level with ganglioside substrates in the presence of Triton X-100, and the hydrolysis was almost specific to gangliosides other than GM1 and GM2, both alpha2-->3 and alpha2-->8 sialyl linkages being susceptible. The major subcellular localization of the expressed sialidase was assessed to be plasma membrane by Percoll density gradient centrifugation of cell homogenates and by immunofluorescence staining of the transfected COS-7 cells. Analysis of the membrane topology by protease protection assay suggested that this sialidase has a type I membrane orientation with its amino terminus facing to the extracytoplasmic side and lacking a signal sequence.

Amino Acid Sequence↗

PubMed1999

Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin.

Calcification of vascular tissue is a common complication in aging, atherosclerosis, diabetes, renal failure, aortic stenosis, and prosthetic valve replacement. Osteopontin is a noncollagenous adhesive protein routinely found at sites of dystrophic calcification and synthesized at high levels by macrophages in calcified aortic valves and atherosclerotic plaques. In the present study, we have characterized the calcification of bovine aortic smooth muscle cell (BASMC) cultures in vitro and have studied the effects of exogenous osteopontin on mineral deposition. Induction of calcification in BASMC cultures was alkaline phosphatase-dependent and was characterized by a multilayer cell morphology. Mineral deposition occurred in the basal matrix of multilayered areas as indicated by von Kossa staining, and transmission electron microscopy and electron diffraction identified the mineral as apatite. Ultrastructural analysis of the cultures showed the presence of extracellular matrix vesicles, calcifying collagen fibrils, and nodular-type calcifications similar to those found in calcified heart valves and atherosclerotic plaques. Purified osteopontin (0.05 to 5 microgram/mL) dose dependently inhibited calcification of BASMC cultures, whereas vitronectin and fibronectin had no effect. In contrast to the inhibitory mechanism of levamisole on mineral deposition, osteopontin did not inhibit alkaline phosphatase activity or reduce phosphorus levels in the culture medium. Addition of calcium to the cultures overcame the inhibitory effect of osteopontin on BASMC culture calcification and resulted in decreased levels of calcium in the culture medium and increased levels in the cell layer. Moreover, using high-resolution, colloidal-gold immunocytochemistry, osteopontin was found intimately associated with growing apatite crystals. These data indicate that the effect of osteopontin, although calcium-dependent, was not mediated by simple calcium chelation but most likely by direct interaction of osteopontin with crystal surfaces. These studies suggest that BASMCs can be used to model vascular calcification in vitro and that soluble osteopontin released near sites of vascular calcification may represent an adaptive mechanism aimed at preventing vascular calcification.

Adaptation, Physiological↗

PubMed1999

Effects of mental stress on cardiac and motor rhythms.

To identify whether spontaneous cardiac rhythm and voluntary motor rhythm are modified in parallel or influenced separately when imposing mental stress, we recorded simultaneously the two rhythms during finger tapping as a simple model of rhythmical motion in 10 healthy human subjects (6 males, 4 females each). Each subject performed finger tapping with an arbitrary tapping rhythm. Mental stress was given intermittently three times for 10-15 s at intervals of 40 s during tapping for 150 s. Heart rate (HR) and tapping rate (TR) and their variations (standard deviation; SD) during finger tapping with and without mental stress were compared. HR and TR increased significantly in response to mental stress during tapping. After mental stress was ended, HR returned rapidly to the initial level, but TR remained at a higher level. Moreover, SD of TR, but not SD of HR, during tapping was increased by mental stress. The present results indicate that the cardiac and motor rhythms are influenced simultaneously by mental stress. However, a difference was seen about the sustained effect of mental stress on the two rhythms.

Adult↗

PubMed1999

Enhanced phosphoinositide hydrolysis via overexpression of phospholipase C beta1 or delta1 inhibits stimulus-induced insulin release in insulinoma MIN6 cells.

To study the effects of enhanced phosphoinositide hydrolysis on insulin secretion, phosphoinositide-specific phospholipase Cbeta1 (PLCbeta1) or PLCdelta1 was overexpressed in insulinoma MIN6 cells via adenoviral vectors. Inositol phosphate production stimulated by NaF (with AlCl3) in PLCbeta1-overexpressing cells and that stimulated by KCl or glucose in both PLCbeta1- and PLCdelta1-overexpressing cells were greater than that in control cells. In addition, reduced phosphatidylinositol-4,5-bisphosphate levels were observed in these cells stimulated by NaF or KCl. The greater phosphoinositide hydrolysis was accompanied by 25-45% inhibition of insulin secretion. These data suggest that excessive phosphoinositide hydrolysis inhibits secretagogue-induced insulin release in MIN6 cells.

Adenoviridae↗