Biomedical subjects

S Maeda

Publications and source records attributed to S Maeda.

At least 253 records · Page 14Linked to original sources

PubMed1998

Promoter structure of human sonic hedgehog gene.

Sonic hedgehog (Shh) is a secreted signal transducer responsible not only for patterning of the anterior-posterior axis during early limb and neuronal development, but also for generating cell-type diversity at later developmental stages. To elucidate the mechanism regulating human Shh gene expression, we cloned the 5'-flanking region of the human Shh gene and characterized it by transient transfection studies. Two transcription start sites were identified by primer extension analysis. Two TATA-boxes, a CCAAT-box and a palindrome-like structure constituted the basic promoter structure. Furthermore, two continuous E-boxes and a putative homeodomain containing an ATTA-box were located around 350-450 bp upstream of the upper TATA-box. Consensus binding sites of the RA, estrogen, D3 and glucocorticoid/progesterone receptors were not found within the cloned sequence. Short-term treatment with TPA increased luciferase activity up to 2.1-fold; on the other hand, treatment with dibutyryl-cyclic AMP decreased it to 0.8-fold. Retinoic acid (RA), vitamin D3, dexamethazone (DEX) and estradiol (E2) had no effect on the luciferase activity. Since the zebrafish Shh promoter contains two closely spaced axial (HNF3beta) binding sequences on its basic promoter, the palindrome-like structure located in the corresponding site of the human Shh promoter may be a crucial binding domain regulating human Shh gene expression.

Base Sequence↗

PubMed1998

Analysis of 5'-flanking region of human Smad4 (DPC4) gene.

Among the transducers of the transforming growth factor (TGF)-beta/bone morphogenetic protein (BMP) receptor signaling proteins, Smad4 and Smad1 act together in BMP 2/4 signaling pathways, and Smad4 and Smad2 act in TGF-beta/activin signaling. To investigate how the Smad4 gene is regulated at the transcriptional level, we cloned and characterized its 5'-flanking region. The major transcription start site mapped by primer extension analysis was 132 bp upstream of the translation start site. The promoter region lacked canonical TATA and GC boxes; it did, however, contain a TATA-like structure (TAAAAT) 32 bp upstream of the transcription start site. A consensus sequence for homeoprotein HoxA-5 (TTTAAAAATTA) was identified at 171 bp upstream of the transcription start site. Within 600 bp upstream of the transcription start site, two Pit-1 and four F2F binding sites were found. One putative AP-1 site was located at -1129. These findings suggest that these homeoproteins could conduct their signals specifically by controlling both inter- and intracellular signal transduction pathways.

Base Sequence↗

PubMed1998

Disruption of sexual behavior in male aromatase-deficient mice lacking exons 1 and 2 of the cyp19 gene.

Aromatase is known to be involved in the control of morphological, neuroendocrine, and behavioral sex differences in the brain. To study the detailed functions of aromatase in mouse brain, we generated aromatase-deficient mice that lack exons 1 and 2 and the proximal promoter region of the cyp19 gene by homologous recombination. The genotypes of offspring from heterozygous matings conformed to the Mendelian rule. The aromatase knockout (ArKO) male and female mice displayed no significant external morphological abnormalities, although the uteri of adult ArKO female mice were remarkably underdeveloped. Male-typical sexual behavior directed toward females was, however, strongly modified in the ArKO male mice. The ArKO male mice showed significantly prolonged latencies to mount and decreased numbers of mounts in response to receptive stimulus females. These results suggest an important participation of the aromatase gene in the reproductive behaviors.

Animals↗

PubMed1998

Extra-chromosomal telomere repeat DNA in telomerase-negative immortalized cell lines.

We found novel extra-chromosomal telomere repeat (ECTR) DNAs in telomerase-negative immortalized KMST-6 cells, by staining these cells with a (TTAGGG)n probe using both cycling oligonucleotide-primed in situ synthesis and by fluorescence in situ hybridization. Relatively small amounts of ECTR DNAs were also observed in telomerase-negative VA13 and SUSM-1 cells, but not observed in telomerase-positive immortalized HeLa cells. The ECTR DNAs existed mainly in the nucleoplasm with a small amount in the cytoplasm. The nucleoplasm ECTR DNAs were co-stained with an antibody directed to the telomeric-repeat binding factor 1 (TRF1), suggesting that they exist as a complex with TRF1. In consistent with these cytological studies, Southern blot analysis showed the existence of small telomere repeat DNAs. The ECTR DNA may provide an insight into the elucidation of the mechanisms responsible for the maintenance of telomeres in telomerase-negative immortalized cells.

Cell Line↗

PubMed1998

Homologous p35 proteins of baculoviruses show distinctive anti-apoptotic activities which correlate with the apoptosis-inducing activity of each virus.

The anti-apoptotic activity of p35s from two baculoviruses, Autographa californica nucleopolyhedrovirus (AcNPV) and Bombyx mori NPV (BmNPV), was compared in mammalian cells. AcNPV p35 efficiently blocked apoptosis induced by caspase overexpression, but BmNPV p35 did so very poorly. Analysis of chimeric p35s and in vitro cleavage of wild type p35s suggest that the cleavage efficiency of p35 correlates with the blocking activity. Single amino acid substitutions of BmNPV p35 with those observed in AcNPV p35, however, resulted in significant loss of its anti-apoptotic activity. We speculate that sequences flanking the cleavage site have uniquely evolved during baculovirus evolution.

Amino Acid Sequence↗

PubMed1998

Molecular cloning and analysis of the 5'-flanking region of the human bone morphogenetic protein-6 (BMP-6).

We cloned a genomic fragment of the 5'-flanking region of the gene encoding bone morphogenetic protein-6 (BMP-6) and assessed its promoter activity. Primer extension revealed the presence of one major transcription start site 178 bp upstream of the translation start site. The promoter region lacked a canonical TATA box but did contain a GC-rich region. A putative tramtrack responsive element, a Drosophila transcriptional factor regulating the segment polarity, was found in the promoter region. Known steroid hormonal responsive elements, however, were not found. Although BMP-6 is classified as a member of the vgr-1 family, the structure of the promoter was similar to that of BMP-2 and 4.

Base Sequence↗

PubMed1998

Prostaglandin D2 inhibits inducible nitric oxide synthase expression in rat vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) as well as macrophages have been shown to generate a substantial amount of NO in inflammatory vascular lesions. Prostaglandin (PG) D2 (PGD2) is produced by inflammatory cells, including mast cells and macrophages. We investigated whether PGD2 modulates NO metabolism in rat VSMCs. PGD2 at a concentration of 10(-7) mol/L or greater dose-dependently inhibited nitrite accumulation in the medium of cultured VSMCs stimulated with interleukin 1beta (IL-1beta). In a dose-response analysis of IL-1beta and nitrite accumulation, PGD2 was seen to decrease the maximal ability of VSMCs to generate NO, arguing against competition by PGD2 at cytokine receptors. Northern analysis showed that PGD2 suppresses induction of inducible NO synthase (iNOS) mRNA in IL-1beta-stimulated VSMCs, with consequent inhibition of iNOS protein expression in Western analysis. A thromboxane A2 (TXA2) analogue, U46619 (10(-5) mol/L), produced less inhibition of NO generation than did PGD2. Neither the PGI2 analog carbaprostacyclin nor PGE1 showed any inhibition. PGD2 dose-dependently inhibited NO generation despite the addition of the TXA2 antagonist SQ29548. PGJ2, delta12-PGJ2, and 15-deoxy-delta12,14-PGJ2, all metabolites of PGD2, were as potent as or slightly stronger than PGD2 in the inhibition of NO generation. These data suggest that PGD2 suppresses NO generation in VSMCs by inhibiting iNOS mRNA expression, most likely through the cascade of the PGJ2 series rather than through the TX receptor or cAMP upregulation. Such action makes it likely that PGD2 regulates NO metabolism in vascular lesions.

Animals↗

PubMed1998

Polyhedron-like inclusion body formation by a mutant nucleopolyhedrovirus expressing the granulin gene from a granulovirus.

The polyhedrin gene in Bombyx mori nucleopolyhedrovirus (BmNPV) was replaced with the granulin gene of Trichoplusia ni granulovirus (TnGV). The substitution was verified by Southern hybridization, and expression of granulin by the mutant virus, BmGran, was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by amino acid sequencing of the predominant protein of BmGran inclusion bodies (IBs). Light and electron microscopy examination of BmGran-infected B. mori and BmN cells revealed large, cuboidal, polyhedron-like IBs in the nucleus and cytoplasm, but granules were not seen. IBs contained small, parallel, electron-dense streaks, which defined the geometric pattern of crystallization. Geometric patterns of nuclear IBs were frequently disrupted by occlusion of polyhedron envelope fragments, resulting in IB instability and fracturing. Virions were not embedded in most of the polyhedron-like IBs, but accumulated with polyhedron envelope fragments. Some virions were coated with matrix protein and were partially wrapped by polyhedron envelope. These results suggested that (1) the amino acid sequence of granulin insufficient for determining IB morphology in TnGV-infected cells, and TnGV may have genes, not present in BmNPV, that control granule formation, and (2) interactions among the virion, the IB envelope, and the matrix protein may be important in virion occlusion and IB morphology and stability.

Adipocytes↗

PubMed1998

Interaction of the Imidazole Ring with Surface Anchored Polypyrrole Latex Particles

Sterically stabilized polypyrrole latex particles attached to Au substrates were observed to adsorb imidazole and histidine from aqueous solutions by ellipsometry. In initial studies the imidazole ring adsorption was mainly irreversible, but after electrochemical reduction-oxidation cycling of the particles in HCl solution only reversible adsorption of the heterocyclic compounds was observed. Adsorption experiments on control surfaces containing the different components of the latex particles identified iron species incorporated during synthesis as the probable irreversible adsorption sites. The reversible adsorption sites were possibly due to the acidic properties of the polypyrrole core in the particles. Copyright 1998 Academic Press. Copyright 1998Academic Press

Journal Article↗

PubMed1998

Polymerase chain reaction amplification of Trichomonas vaginalis DNA from Papanicolaou-stained smears.

DNA from Papanicolaou-stained smears was successfully amplified using the polymerase chain reaction (PCR) to investigate if it could be used for retrospective genome studies, such as for the detection of Trichomonas vaginalis. DNA was isolated from 20 archival Papanicolaou-stained smears (19 cervical and 1 seminal fluid samples) and purified by treatment with proteinase K, phenol/chloroform, and ethanol. The method of recovering DNA from the Papanicolaou-stained smear is very important in the successful amplification of Trichomonas DNA in archival cytological smears. We used a specific sequence and successfully amplified DNA in cytological smears of T. vaginalis using PCR. We conclude that the diagnostic accuracy of the detection of T. vaginalis in cervical smears is enhanced by using a combination of a Papanicolaou-stained smear and PCR.

Adult↗

PubMed1998

pp60c-src activation in hepatocellular carcinoma of humans and LEC rats.

For the related Src kinases, a close correlation exists between elevated tyrosine kinase activity and cell transformation. However, the involvement of pp60c-src in hepatocellular carcinoma (HCC) remains obscure. The aim of this study was to evaluate whether pp60c-src tyrosine kinase activity is elevated in HCC. We analyzed the kinase activity of pp60c-src in normal liver tissue, chronic hepatitis liver tissue, and tumorous and adjacent nontumorous portions of HCC tissue from patients and Long-Evans cinnamon (LEC) rats that are known to develop liver cancer spontaneously. The kinase activity of pp60c-src was rarely detected in the normal human liver tissue and chronic hepatitis liver tissue, but it was elevated in tumorous and nontumorous portions of HCC tissue. Furthermore, the kinase activity of pp60c-src was significantly elevated in tumorous tissues compared with nontumorous tissues. The kinase activity of pp60c-src was also higher in poorly differentiated HCC. In addition, the kinase activity of pp60c-src increased proportionately with the development of HCC of LEC rats. Our results suggest that activation of the protooncogene product pp60c-src may play an important role in the malignant transformation of hepatocytes in human and LEC rats, and that it may be closely related to the histopathological grading of human HCC.

Animals↗

PubMed1998

Mesenteric schwannoma.

Schwannoma is a benign neurogenic tumor arising from the sheath of peripheral nerves. It occurs very rarely in the mesentery, where it is difficult to diagnose. Herein we describe a case in which contrast-enhanced CT and gadolinium-DTPA-enhanced MR showed a locally enhanced well-defined tumor with a cystic component just anterior to the duodenum. These findings corresponded well to the resected specimen.

Contrast Media↗

PubMed1998

Idiopathic benign biliary stricture.

A rare case of idiopathic benign biliary stricture is reported. A 50-year-old man with liver dysfunction underwent ultrasonography, which revealed dilation of the intrahepatic bile ducts, and endoscopic retrograde cholangiopancreatography, which revealed a short, ring-like stenosis at the junction of the left and right hepatic ducts. Although a benign stricture was suspected, radiologic tests alone were insufficient to make a firm diagnosis. Therefore, a cholecystectomy and resection of the extrahepatic biliary tract were performed. Microscopic examination of the resected specimen demonstrated no evidence of malignancy. The final diagnosis was mild, localized, chronic cholangitis. The patient had not had previous biliary tract surgery, choledocholithiasis, nor did he have a congenital abnormality of the biliary tract, bile duct carcinoma, or pancreatic disease. Since there was no evidence of primary sclerosing cholangitis, the stricture was considered to be idiopathic.

Anastomosis, Surgical↗

PubMed1998

Detection of Mycoplasma genitalium in patients with urethritis.

PURPOSE: We attempted to detect Mycoplasma genitalium in urethral swab specimens by a polymerase chain reaction based assay to determine the prevalence of M. genitalium in patients with urethritis. MATERIALS AND METHODS: We examined a total of 171 Japanese men who presented to our hospital from February 1995 through January 1997. Of these men 150 had symptoms and signs compatible with acute urethritis and 21 had no symptoms or signs of urethritis. Urethral swab specimens were used to culture Neisseria gonorrhoeae, to detect Chlamydia trachomatis by an enzyme immunoassay and to detect M. genitalium by a polymerase chain reaction based assay. RESULTS: Gonococcal urethritis was diagnosed in 74 symptomatic men, and nongonococcal urethritis was diagnosed in 76 symptomatic men. Of the 74 cases of gonococcal urethritis 3 (4.1%) were positive for M. genitalium, and 14 (18.9%) were positive for C. trachomatis. Of the 76 cases of nongonococcal urethritis 10 (13.2%) were positive for M. genitalium, and 42 (55.2%) were positive for C. trachomatis. While only 1 of the 42 cases with chlamydial nongonococcal urethritis (2.4%) was positive for M. genitalium, 9 of the 34 chlamydia negative nongonococcal urethritis cases (26.5%) were positive for the mycoplasma. In contrast, all 21 cases men were negative for N. gonorrhoeae, M. genitalium, and C. trachomatis. CONCLUSIONS: The prevalences of M. genitalium in patients with gonococcal urethritis and nongonococcal urethritis who attended our clinic were 4.1 and 13.2%, respectively. M. genitalium was detected significantly more often in men with nongonococcal urethritis than in asymptomatic men. In addition, its prevalence in men with chlamydia negative nongonococcal urethritis (26.5%) was significantly greater than in those with chlamydia positive nongonococcal urethritis (2.4%). These findings suggest that M. genitalium may be associated with the development of nongonococcal urethritis independent of C. trachomatis.

Adolescent↗

PubMed1998

Down-regulation of ET(B) receptor, but not ET(A) receptor, in congestive lung secondary to heart failure. Are marked increases in circulating endothelin-1 partly attributable to decreases in lung ET(B) receptor-mediated clearance of endothelin-1?

Receptors for endothelin (ET)-1, a potent vasoconstrictor peptide, have two isoforms, i.e. ET(A) receptors and ET(B) receptors. We previously reported that an ET(A) receptor antagonist greatly ameliorated pulmonary hypertension due to congestive heart failure (CHF) in rats. In the present study of rats with pulmonary congestion secondary to CHF, we determined not only ET(A) receptor mRNA expression but also ET(B) receptor mRNA expression in the congestive lung because lung ET(B) receptors are reported to be important for the clearance of circulating ET-1. We also measured lung ET-1 and circulating ET-1 levels. The expression of ET(B) receptor mRNA in the lung was significantly lower in rats with CHF than in age-matched control rats, while the expression of ET(A) receptor mRNA did not differ between the two groups. The protein level of ET(B) receptor, determined by Western blot, in the lung was lower in the rats with CHF than in the control rats, while the protein level of ET(A) receptor did not differ between the two groups. The lung ET-1 level and plasma ET-1 level were significantly higher in the rats with CHF than in the controls by 1.4-fold and 5.3-fold, respectively. Thus, in the rats with CHF, ET-1 was increased to a much greater extent in plasma than in the lung. The present findings suggest that selective down-regulation of ET(B) receptor, but not ET(A) receptor, occurs in the congestive lung. Since lung ET(B) receptors play a role in the clearance of circulating ET-1, we propose that down-regulation of lung ET(B) receptors partly contributes to marked increases in circulating ET-1 and that increased ET-1 in the circulating plasma as well as in the lung is involved in the progression of pulmonary hypertension in CHF.

Animals↗

PubMed1998

Peripheral type benzodiazepine receptor in T lymphocyte rich preparation.

Some types of mood disorders and drugs are suggested to affect peripheral type benzodiazepine receptors (PBR), but their mechanisms are unclear. The isolation of pure lymphocytes is requisite for the investigation of the function of PBR on lymphocytes, since platelets and monocytes also have many PBR. The objective of this study was to establish a method of binding assay for PBR using pure T lymphocytes. Mononuclear cells and T lymphocytes were prepared by using a density gradient material and magnetic beads, respectively. The cells were analyzed using flow cytometry and a counting chamber. Binding studies were performed using T lymphocytes from 10 normal volunteers. The T lymphocytes were incubated with [3H]PK11195, harvested on glass fiber filters, and counted with a plate scintillation counter. The binding data were analyzed by the Scatchard method. With the magnetic bead technique, pure T cells were selected that contained only 1.5% monocytes and platelet/cell ratio of 1.4. The Scatchard plot of the data indicated that only one type of specific binding site was involved in the binding. The dissociation constant (Kd) was 3.8+/-1.3nM (mean+/-SD), and the Bmax was 379+/-124 fmol//10(6) cells (mean+/-SD). The density gradient- magnetic beads technique can be used as an appropriate method of preparation of T cells for PBR binding assay.

Binding Sites↗

PubMed1998

Chitin-graft-poly(2-methyl-2-oxazoline) enhanced solubility and activity of catalase in organic solvent.

Catalase from bovine liver was lyophilized from an aqueous solution containing chitin-graft-poly(2-methyl-2-oxazoline) (3), which was synthesized by the reaction of 52% deacetylated chitin (1) with living poly(2-methyl-2-oxazoline) (2). The rate of consumption of H2O2 in chloroform catalyzed by the lyophilized catalase with 3 was enhanced more than 10 times that by catalase without 3. The dispersibility and solubility of lyophilized catalase with 3 in chloroform were improved in comparison with catalase itself.

Animals↗

PubMed1998

Isolation and characterization of calmodulin in the pheromone gland of the silkworm, Bombyx mori.

Production of the sex pheromone bombykol in the silkworm, Bombyx mori, is regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN). It has been suggested that the external signal of PBAN in this species is transmitted to the intracellular cascade reactions consisting of Ca2+/calmodulin (CaM) complex and phosphoprotein phosphatase. To demonstrate the molecular mechanisms regulated by PBAN, we attempted to characterize CaM in the pheromone gland of B. mori. By using ion-exchange and RP-HPLC, B. mori CaM was purified from the cytosolic fraction of the pheromone gland. The primary structure was deduced by composition/sequence analysis and mass spectrometric analysis of the fragment peptides obtained from enzymatic and chemical fragmentations. The amino acid sequence of B. mori CaM was identical with Drosophila CaM deduced from the CaM gene of D. melanogaster, suggesting that insects have well conserved the molecule of CaM.

Amino Acid Sequence↗