Biomedical subjects

K Adachi

Publications and source records attributed to K Adachi.

At least 91 records · Page 5Linked to original sources

PubMed2000

A novel anti-rheumatic drug suppresses tumor necrosis factor-alpha and augments interleukin-10 in adjuvant arthritic rats.

Tumor necrosis factor-alpha (TNF-alpha) plays an important role in the pathology of rheumatoid arthritis. When N-[1-(4-¿[4-(pyrimidin-2-yl)piperazin-1-yl]methyl¿phenyl)cycloprop yl] acetamide (Y-39041) (3-30 mg/kg) was orally administered to rats with established arthritis from day 15 to day 20, hindpaw volume was significantly reduced. This inhibitory activity of Y-39041 was kept up after administration was stopped. On day 17 Y-39041 suppressed lipopolysaccharide-induced TNF-alpha and interleukin-6 production in serum at doses of 3-30 mg/kg, and augmented interleukin-10 production at doses of 10 and 30 mg/kg. The finding that Y-39041 suppresses TNF-alpha and interleukin-6 production and augments interleukin-10 production could be beneficial in the therapy of chronic inflammatory diseases.

Acetamides↗

PubMed2000

Up-regulation of glial cell line-derived neurotrophic factor (GDNF) following traumatic spinal cord injury.

We investigated the temporal and spatial expression patterns of the GDNF gene after subjecting rats to an acute contusion injury of the spinal cord using the weight drop technique. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that GDNF transcription in the spinal cord began to increase within 30 min after injury and peaked within 3 h. Immunohistochemical analysis showed GDNF immunoreactivity to be present mainly in microglia and macrophages 1 day after injury, but not in neurons or astrocytes. This immediate upregulation of GDNF gene expression may be a component of an inflammatory process and probably exerts a protective effect on neurons following spinal cord injury (SCI).

Animals↗

PubMed2000

Inhibition of BAD phosphorylation either at serine 112 via extracellular signal-regulated protein kinase cascade or at serine 136 via Akt cascade sensitizes human ovarian cancer cells to cisplatin.

We studied the roles of the phosphatidylinositol 3-kinase (PI-3K)-protein kinase B/Akt-BAD cascade in both cisplatin-resistant Caov-3 and -sensitive A2780 human ovarian cancer cell lines. Treatment of both Caov-3 and A2780 cells with cisplatin but not with the trans-diaminodichloroplatinum (transplatin) isomer stimulated the activation of Akt, and the PI-3K inhibitor wortmannin blocked the cisplatin-induced activation of Akt. Treatment of both Caov-3 and A2780 cells with cisplatin but not with the trans-diaminodichloroplatinum isomer also stimulated the phosphorylation of BAD at both the Ser-112 and Ser-136 sites. Whereas the phosphorylation of BAD at Ser-136 was blocked by treatment with wortmannin, its phosphorylation at Ser-112 was blocked by a MAP/ERK kinase inhibitor, PD98059. Exogenous expression of a dominant-negative Akt in both Caov-3 and A2780 cells decreased the cell viability after treatment with cisplatin. In contrast, no sensitization to cisplatin was observed in cells expressing wild-type Akt. We further examined the role of BAD in the viability after cisplatin treatment using BAD mutants. Exogenous expression of each of the singly substituted BADS112A or BADS136A in both Caov-3 and A2780 cells decreased the viability after treatment with cisplatin to a degree intermediate between that caused by exogenous expression of wild-type BAD and doubly substituted BAD2SA. Cisplatin did not stimulate the phosphorylation of BAD Ser-136, but did stimulate the phosphorylation of BAD Ser-112 in cells expressing a dominant-negative Akt, suggesting that BAD Ser-136 but not Ser-112 was phosphorylated by Akt. Our findings suggest that cisplatin-induced DNA damage causes the phosphorylation of both BAD Ser-112 via an extracellular signal-regulated protein kinase (ERK) cascade and BAD Ser-136 via a PI-3K-protein kinase B/Akt cascade and that inhibition of either of these cascades sensitizes ovarian cancer cells to cisplatin.

Adenocarcinoma, Papillary↗

PubMed2000

Estrogen induces the Akt-dependent activation of endothelial nitric-oxide synthase in vascular endothelial cells.

Although estrogen is known to activate endothelial nitric oxide synthase (eNOS) in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells (HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), 17beta-estradiol (E2), but not 17alpha-E2, caused acute activation of eNOS that was unaffected by actinomycin D and was specifically blocked by the pure estrogen receptor antagonist ICI-182,780. Treatment of both TRLECs and HUVECs with 17beta-E2 stimulated the activation of Akt, and the PI3K inhibitor wortmannin blocked the 17beta-E2-induced activation of Akt. 17beta-E2-induced Akt activation was also inhibited by ICI-182,780, but not by actinomycin D. Either treatment with wortmannin or exogenous expression of a dominant negative Akt in TRLECs decreased the 17beta-E2-induced eNOS activation. Moreover, 17beta-E2-induced Akt activation actually enhances the phosphorylation of eNOS. 17beta-E2-induced Akt activation was dependent on both extracellular and intracellular Ca(2+). We further examined the 17beta-E2-induced Akt activity in Chinese hamster ovary (CHO) cells transiently transfected with cDNAs for estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta). 17beta-E2 stimulated the activation of Akt in CHO cells expressing ERalpha but not in CHO cells expressing ERbeta. Our findings suggest that 17beta-E2 induced eNOS activation through an Akt-dependent mechanism, which is mediated by ERalpha via a nongenomic mechanism.

Animals↗

PubMed2000

Synthesis and immunosuppressive activity of 2-substituted 2-aminopropane-1,3-diols and 2-aminoethanols.

A series of 2-substituted 2-aminopropane-1,3-diols was synthesized and evaluated for their lymphocyte-decreasing effect and immunosuppressive effect on rat skin allograft. A phenyl ring was introduced into the alkyl chain of the lead compound 3, which is an immunosuppressive agent structurally simplified from myriocin (1, ISP-I) via compound 2. The potency of the various compounds was dependent upon the position of the phenyl ring within the alkyl side chain. The most suitable length between the quaternary carbon atom and the phenyl ring was two carbon atoms. 2-Substituted 2-aminoethanols were successively synthesized and evaluated for their T-cell-decreasing effect and immunosuppressive effect using a popliteal lymph node gain assay in rats. The absolute configuration at the quaternary carbon affected the activity, and the (pro-S)-hydroxymethyl group of compound 6 was essential for potent immunosuppressive activity. Favorable substituents for the (pro-R)-hydroxymethyl group of 6 were hydroxyalkyl (hydroxyethyl and hydroxypropyl) or lower alkyl (methyl and ethyl) groups. 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride (6, FTY720) was found to possess considerable activity and is expected to be useful as an immunosuppressive drug for organ transplantation.

Animals↗

PubMed2000

Isolation and characterization of the 5'-upstream and untranslated regions of the mouse type II iodothyronine deiodinase gene.

The type II iodothyronine deiodinase (D2) catalyzes the 5'-deiodination of thyroxine to yield the biologically active form, 3,3',5,-tri-iodothyronine, and is a member of the selenoproteins. We isolated a 17.5 kb mouse genomic clone containing the entire coding and 5'-untranslated regions of the D2 gene (mdio2). We also isolated the entire 5'-UTR of the mouse D2 cDNA, which was 753 bp in length and contained five ATG codons. An additional 258 bp ORF from the fourth ATG codon was found in the same reading frame as the coding region reported previously, and this additional ORF contained a TGA codon, which could encode selenocysteine. The proximal promoter of mdio2 contained a TATA box and several potential transcription factor-binding sequences, including CRE, C/EBP and GATA binding sites. The 1.3 kb 5'-upstream region exhibited a promoter activity by reporter assay using Mm5MT and JAR cells, which have a D2 transcript, but not HepG2 cells that have no detectable level of D2 transcript.

5' Untranslated Regions↗

PubMed2000

Stepping rotation of F1-ATPase visualized through angle-resolved single-fluorophore imaging.

Orientation dependence of single-fluorophore intensity was exploited in order to videotape conformational changes in a protein machine in real time. The fluorophore Cy3 attached to the central subunit of F(1)-ATPase revealed that the subunit rotates in the molecule in discrete 120 degrees steps and that each step is driven by the hydrolysis of one ATP molecule. These results, unlike those from the previous study under a frictional load, show that the 120 degrees stepping is a genuine property of this molecular motor. The data also show that the rate of ATP binding is insensitive to the load exerted on the rotor subunit.

Adenosine Triphosphate↗

PubMed2000

Novel phenylpiperazine derivatives as dual cytokine regulators with TNF-alpha suppressing and IL-10 augmenting activity.

Phenylpiperazine derivatives were synthesized as dual cytokine regulators with TNF-alpha suppressing and IL-10 augmenting activity. Lead optimization led to compound 5k having the potent regulatory activity and demonstrating remarkable protective effects against the lethal challenge of LPS in mice. suggesting that 5k would be a promising drug candidate for the treatment of TNF-alpha associated diseases including septic shock.

Animals↗

PubMed2000

Novel DMARDs on the basis of a new concept of dual cytokine regulation, TNF-alpha suppression and IL-10 augmentation.

A series of arylpiperazine derivatives was synthesized to obtain agents showing apparent therapeutic effects in a chronic inflammatory animal model, starting from a lead possessing potent dual cytokine regulatory activity in vivo. We found a pyrimidylpiperazine derivative 17c showing the dual regulatory activity and an excellent therapeutic effect in an adjuvant-induced arthritis model.

Animals↗

PubMed2000

A rotary molecular motor that can work at near 100% efficiency.

A single molecule of F1-ATPase is by itself a rotary motor in which a central gamma-subunit rotates against a surrounding cylinder made of alpha3beta3-subunits. Driven by the three betas that sequentially hydrolyse ATP, the motor rotates in discrete 120 degree steps, as demonstrated in video images of the movement of an actin filament bound, as a marker, to the central gamma-subunit. Over a broad range of load (hydrodynamic friction against the rotating actin filament) and speed, the F1 motor produces a constant torque of ca. 40 pN nm. The work done in a 120 degree step, or the work per ATP molecule, is thus ca. 80 pN nm. In cells, the free energy of ATP hydrolysis is ca. 90 pN nm per ATP molecule, suggesting that the F1 motor can work at near 100% efficiency. We confirmed in vitro that F1 indeed does ca. 80 pN nm of work under the condition where the free energy per ATP is 90 pN nm. The high efficiency may be related to the fully reversible nature of the F1 motor: the ATP synthase, of which F1 is a part, is considered to synthesize ATP from ADP and phosphate by reverse rotation of the F1 motor. Possible mechanisms of F1 rotation are discussed.

Animals↗

PubMed2000

Assembly of gamma- with alpha-globin chains to form human fetal hemoglobin in vitro and in vivo.

Soluble gamma-globin chains were expressed in bacteria and purified to assess the mechanism of gamma- and alpha-chain assembly to form Hb F. Formation of Hb F in vitro following incubation of equimolar mixtures of gamma and alpha chains was about 4 x 10(5)-fold slower than assembly of alpha and beta chains to form Hb A in vitro. Results of assembly for gamma(116Ile-->His) and gamma(112Thr-->Asp) chains with alpha chains were similar to that of beta chains, whereas assembly of gamma(112Thr-->Cys) and alpha chains was similar to wild type gamma chains, indicating that amino acid differences at alpha1beta1 and alpha1gamma1 interaction sites between gamma116 Ile and beta116 His are responsible for the different assembly rates in vitro in the formation of Hb F and Hb A. Homoassembly in vitro of individual gamma chains as assessed by size-exclusion chromatography shows that gamma and gamma(112Thr-->Cys) chains form stable dimers like alphabeta and alphagamma that do not dissociate readily into monomers like beta chains. In contrast, gamma(116Ile-->His) chains form monomers and dimers upon dilution. These results are consistent with the slower assembly rate in vitro of gamma and gamma(112Thr-->Cys) with alpha chains, whereas the faster rate of assembly of gamma(116Ile-->His) and gamma(112Thr-->Asp) chains with alpha chains, like beta chains, may be caused by dissociation to monomers. These results suggest that dissociation of gamma(2) dimers to monomers limits formation of Hb F in vitro. However, yields of soluble Hb F expressed in bacteria were similar to Hb A, and no unassembled alpha and gamma chains were detected. These results indicate that gamma chains assemble in vivo with alpha chains prior to forming stable gamma(2) dimers, possibly binding to alpha chains as partially folded nascent gamma-globin chains prior to release from polyribosomes.

Chromatography, Agarose↗

PubMed2000

Surface and interface beta-chain residues synergistically affect hemoglobin assembly.

Homo- and heterotetramer formations of beta112 variants (beta(112Cys-->Asp), beta(112Cys-->Ser), beta(112Cys-->Thr), and beta(112Cys-->Val)) of hemoglobin were characterized in the presence and absence of beta(16Gly-->Asp) in vitro. In all cases an alteration in overall surface charge (beta(16Gly-->Asp)) decreased the beta(4) homotetramer stability (association constants as determined by gel-permeation chromatography) albeit to differing extents. In contrast, competition experiments of hemoglobin subunits showed that heterotetramer formation was promoted by this substitution. Order of increase in tetramer formation by the additional negative surface charge in the beta112 variants was as follows: Hb betaG16D, C112D > Hb betaG16D, C112S > Hb betaG16D > Hb G16D, C112T > Hb betaG16D, C112V. Thus, the overall surface charge of the beta chain and its contribution to electrostatic interaction in these instances appear to act in synergy with alpha(1)beta(1) interface residues to affect the assembly of hemoglobin molecules.

Amino Acid Substitution↗

PubMed2000

Estrogen suppresses transcription of lipoprotein lipase gene. Existence of a unique estrogen response element on the lipoprotein lipase promoter.

Estrogen exerts a variety of effects not only on female reproductive organs but also on nonreproductive organs, including adipose tissue. Estrogen inhibits obesity triggered by ovariectomy in rodents. We studied the mechanism underlying this estrogen-dependent inhibition of obesity. Estrogen markedly decreased the amounts of fat accumulation and lipoprotein lipase (LPL) mRNA as well as triglyceride accumulation in genetically manipulated 3T3-L1 adipocytes stably expressing the estrogen receptor (ER). A pLPL(1980)-CAT construct, along with an ER expression vector, was introduced into differentiated 3T3-L1 cells, and CAT activities were determined. ER, mostly ligand-dependently, inhibited the basal LPL promoter activity by 7-fold. We searched the LPL promoter for an estrogen-responsive suppressive element by employing a set of 5'-deletion mutants of the pLPL-CAT reporter. Although there was no classical estrogen response element, it was demonstrated that an AP-1-like TGAATTC sequence located at (-1856/-1850) was responsible for the suppression of the LPL gene transcription by estrogen. An electrophoretic mobility shift assay probed with the TGAATTC sequence demonstrated formation of a specific DNA-nuclear protein complex. Interestingly, this complex was not affected by the addition of any antibodies against ER, c-Jun, c-Fos, JunB, or JunD. Because this TGAATTC element responded to phorbol ester and overexpression of CREB-binding protein abrogated the suppressive effect of estrogen on the LPL promoter, we conclude that a unique protein that is related to the AP-1 transcription factor families may be involved in the complex that binds to the TGAATTC element.

3T3 Cells↗

PubMed2000

A novel dual regulator of tumor necrosis factor-alpha and interleukin-10 protects mice from endotoxin-induced shock.

A pyrimidylpiperazine derivative, N-[1-(4-¿[4-(pyrimidin-2-yl)piperazin-1-yl]methyl¿phenyl)cycloprop yl] acetamide (Y-39041), is a dual cytokine regulator of tumor necrosis factor (TNF)-alpha and interleukin-10 production. Lipopolysaccharide-induced TNF-alpha release in BALB/c mice was inhibited by the oral treatment with the compound at 10-100 mg/kg (about 80% suppression) while interleukin-10 release was augmented (about 10-fold increase at 30 mg/kg). In addition, Y-39041 (30 mg/kg, p.o.) completely protected mice from lipopolysaccharide-induced death by the treatment before and after lipopolysaccharide injection. The finding that Y-39041 suppresses TNF-alpha production and stimulates interleukin-10 production at the same time provides new insights for the treatment of septic shock, rheumatoid arthritis and Crohn's diseases.

Acetamides↗

PubMed2000

Biochemical and molecular biological evidence for the presence of type II iodothyronine deiodinase in mouse mammary gland.

In the present study we have obtained several lines of evidence indicating the presence of type II iodothyronine deiodinase (DII) in the mouse mammary gland. 5'-deiodinase activity in the mammary gland has an apparent K(m) value of 4.4 nM for T(4) and is inhibited by aurothioglucose but not by propylthiouracil. These characteristics are similar to those of DII in other tissues. We cloned a 1.4-kb cDNA, which contains the entire mouse DII coding region and has high homology with the rat DII cDNA, from the mammary gland and brain. Northern blot analysis showed the presence of 7.9 kb DII mRNA in the mammary gland and brain. The levels of DII activity and mRNA in lactating gland were significantly lower than those in virgin and pregnant glands, suggesting that DII is regulated at the pretranslational level. In addition, we found the low level of DII enzyme activity and transcript in various other mouse tissues.

Amino Acid Sequence↗

PubMed2000

Genomic organization, chromosomal mapping and promoter analysis of the mouse selenocysteine tRNA gene transcription-activating factor (mStaf) gene.

mStaf is a zinc-finger protein that activates the transcription of the mouse selenocysteine tRNA gene. The mStaf gene is approx. 35 kb long and split into 16 exons. All exon-intron junction sequences conform to the GT/AG rule. The transcription start site is located 83 bp upstream of the initiation codon. Chromosomal mapping localized the gene to mouse chromosome 7, region E3-F1. Sequence analysis of the proximal promoter region revealed several potential regulatory elements; these include the recognition elements of Sp1, Nkx, CP2, E2A, SIF (SIS-inducible factor), TFII-I and cAMP-responsive element (CRE), but no TATA sequences. Transfection experiments demonstrated that the 5'-flanking region (-1894 to +37) of the mStaf gene drives transcription in mouse NMuMG cells and that a construct containing a fragment from -387 to +37 showed the highest transcriptional activity. Deletion and mutation experiments suggested that four Sp1 sites played an important role for the basal promoter activity. Furthermore, electrophoretic mobility-shift assays demonstrated that Sp3 but not other Sp (specificity protein) family members binds to three of the Sp1 sites. Our present study suggests that Sp3 is involved in the basal transcriptional activation of the mStaf gene.

Amino Acid Sequence↗

PubMed2000

Expression of functional soluble human alpha-globin chains of hemoglobin in bacteria.

Individual, soluble human alpha-globin chains were expressed in bacteria with exogenous heme and methionine aminopeptidase. The yields of soluble alpha chains in bacteria were comparable to those of recombinant non-alpha chains expressed under the same conditions. Molecular mass and gel-filtration properties of purified recombinant alpha chains were the same as those of authentic human alpha chains. Biochemical and biophysical properties of isolated alpha chains were identical to those of native human alpha chains as assessed by UV/vis, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy which contrasts with previous results of refolded precipitated alpha chains made in the presence of heme in vitro (M. T. Sanna et al., J. Biol. Chem. 272, 3478-3486, 1997). Mixtures of purified, soluble recombinant alpha-globin and native beta-globin chains formed heterotetramers in vitro, and oxygen- and CO-binding properties as well as the heme environment of the assembled tetramers were experimentally indistinguishable from those of native human Hb A. UV/vis, CD, and NMR spectra of assembled Hb A were also the same as those of human Hb A. These results indicate that individual expressed alpha chains are stable in bacteria and fold properly in vivo and that they then can assemble with free beta chains to form hemoglobin heterotetramers in vivo as well as in vitro.

Globins↗

PubMed2000

Screening for abdominal aortic aneurysms during a basic medical checkup in residents of a Japanese rural community.

To determine the prevalence of abdominal aortic aneurysms (AAA) and the size distribution of the abdominal aortic diameter in the Japanese population, 1,591 residents of a farming community were screened by ultrasonography during the annual basic medical checkup arranged by the town. The mean diameter of the abdominal aorta was 17.2 +/- 2.2mm and four AAAs were found (0.3%), all in men aged 60 years or older, three of whom were hypertensive. The mean diameter of the common iliac artery was 10.2 +/- 5.1mm and five iliac arterial aneurysms were found (0.3%). One man had both aortic and iliac arterial aneurysms. The iliac arterial aneurysms were also found in men, four of whom were aged 60 years or older and had a smoking habit. We were able to keep the cost of screening low by combining the program with an annual basic medical checkup. These findings indicate that it is important to examine the iliac artery as well as the abdominal aorta because the incidence of aneurysm is similar in both. Screening for AAAs using ultrasonography is accurate enough to detect them before rupture, and can be effectively performed in men aged 60 years or older during their annual basic medical checkup.

Adult↗