Search PubMed⌕ Search

Biomedical subjects

S Ohta

Publications and source records attributed to S Ohta.

At least 631 records · Page 35Linked to original sources
PubMed1991

[Amplified DNA detection of HPV types 16 and 18 DNA in cervical scrapes by polymerase chain reaction].

Cervical scrapes from 43 patients (12 with chronic cervicitis, 13 with dysplasia, 11 with carcinoma in situ and 7 with invasive carcinoma) were examined by the polymerase chain reaction (PCR), to detect the amplified E7 gene of the HPV types 16 and 18 DNA sequence. HPV types 16 and/or 18 DNA were detected in 25 of 43 cases by the PCR (type 16 in 16, type 18 in 8, and both types 16 and 18 in 1). In comparison with the results for histological grades, the positive rate significantly increased as the grade of cervical dysplasia became higher. All cases were also examined with a HPV detection kit, the "Vira Pap" which contains 32P-labeled mixed RNA probes complimentary to HPV types 6, 11, 16, 18, 31, 33, 35 DNA. Six of 18 cases in which HPV DNA were not detected by the Vira Pap were positive for HPV types 16 and/or 18, so that the PCR was a highly sensitive method compared to the Vira Pap. The PCR has some advantages: 1. it requires only a small amount of specimens and 2. paraffin-embedded sections can also be used. We suggest that the PCR is a useful method for the screening and for retrospective investigation of HPV infection.

Adult↗
PubMed1990

A point mutation in the mitochondrial tRNA(Leu)(UUR) gene in MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes).

Mitochondrial myopathy, encephalopathy, lactic acidosis and strokelike episode (MELAS) is a major group of heterogeneous mitochondrial disorders. To identify the defective gene, mitochondrial DNA from a patient with MELAS was sequenced by using amplified DNA fragments as sequencing templates. In 14.1 kbp determined out of 16.6 kbp of the whole mitochondrial gene, at least 21 nucleotides were different from those of a control human mitochondrial DNA. One of the substitutions was a transition of A to G in the tRNA(Leu) (UUR) gene at Cambridge nucleotide number 3,243. This nucleotide is conserved not only in many mitochondrial tRNAs but in most cytosolic tRNA molecules. An Apa I restriction site was gained by the substitution of this nucleotide. The Apa I digestion of the amplified DNA fragment revealed that all independent 6 patients had G at nucleotide number 3,243 in their mitochondrial DNAs, but none of 11 control individuals had G at this position. This result strongly suggests that the mutation in the mitochondrial tRNALeu gene causes MELAS.

Acidosis, Lactic↗
PubMed1990

Augmentation for intratumoral accumulation and anti-tumor activity of liposome-encapsulated adriamycin by tumor necrosis factor-alpha in mice.

The effect of recombinant human tumor necrosis factor-alpha (rhTNF-alpha) on anti-tumor activity of adriamycin entrapped in small unilamellar liposomes (ADM-Lip) has been studied using BALB/c mice bearing subdermal Meth-A fibrosarcoma. Accumulation of i.v. injected ADM-Lip, but not of free ADM, into tumor tissue was augmented by a single i.v. injection of rhTNF-alpha. The maximum effect of rhTNF-alpha was expressed when it was injected 1 hr before ADM-Lip. This rhTNF-alpha treatment did not result in increased accumulation of ADM-Lip in normal organs. Based on these results, we treated mice bearing Meth-A tumor with rhTNF-alpha and then with ADM-Lip 1 hr later. By 2-cycle treatments using this regimen, tumor growth was markedly inhibited, and 5 out of 13 mice tested were cured of tumor, while treatment with rhTNF-alpha alone, ADM alone, ADM-Lip alone or rhTNF-alpha plus ADM resulted neither in prolongation of survival time nor in cure of mice. Our results indicate that ADM-Lip can express a potent and systemic anti-tumor activity after pretreatment of animals with TNF, but only when administration of these drugs is appropriately timed.

Animals↗
PubMed1990

Enhancement of foreign gene expression by a dicot intron in rice but not in tobacco is correlated with an increased level of mRNA and an efficient splicing of the intron.

The first intron of castor bean catalase gene, cat-1 was placed in the N-terminal region of the coding sequence of the beta-glucuronidase gene (gusA) and the intron-containing gusA was used with the cauliflower mosaic virus (CaMV) 35S promoter. Using this plasmid, pIG221, the effect of the intron on expression of beta-glucuronidase (GUS) activity was examined in transgenic rice calli and plants (a monocotyledon), and transgenic tobacco plants (a dicotyledon). The intron-containing plasmid increased the level of GUS enzyme activity 10 to 40-fold and 80 to 90-fold compared with the intronless plasmid, pBI221, in transgenic rice protoplasts and transgenic rice tissues, respectively. In contrast, the presence of the intron hardly influenced the expression of the GUS activity in transgenic tobacco plants. Northern blot analysis showed that the catalase intron was efficiently spliced in rice cells while transgenic tobacco plants contained both spliced and unspliced gusA transcripts in equal amounts. Furthermore, the level of the mature gusA transcript in transformed rice calli was greatly increased in the presence of the intron. The catalase intron was removed at the same splice junctions in transgenic rice and tobacco plants. These findings indicate that the stimulating effect of the intron on GUS expression is correlated with an efficient splicing of pre-mRNA and an increased level of mature mRNA.

Base Sequence↗
PubMed1990

The alpha 1 beta 1 heterodimer of ATP synthase.

The alpha 3 beta 3 hexamer was reconstituted from the alpha and beta subunits of TF1 portion of ATP synthase of thermophilic bacterium (Kagawa et al. (1989) FEBS Lett. 249, 67). The alpha 1 beta 1 heterodimer of ATP synthase was isolated by high performance liquid chromatography (HPLC) of the alpha 3 beta 3 hexamer in the presence of AT(D)P-Mg. On polyacrylamide gel electrophoresis, both bands corresponding to the dimer and hexamer showed ATPase activity. The alpha 1 beta 1 dimer was dissociated into the equal amounts of the alpha and beta monomers by sodium dodecyl sulfate. The alpha and beta monomers were practically inactive. The alpha 2 and beta 2 homodimers were not detected by electrophoresis and HPLC.

Bacteria↗
PubMed1990

Small-angle neutron scattering from the reconstituted TF1 of H(+)-ATPase from thermophilic bacterium PS3 with deuterated subunits.

Subunits alpha, beta and gamma of adenosine triphosphatase (H(+)-ATPase) from the thermophilic bacterium PS3 (TF1) have been over-expressed in Escherichia coli. alpha and beta subunits deuterated to the level of 90% were obtained by culturing E. coli in 2H2O medium. Both the subunits and the reconstituted alpha beta gamma complex, TF1, which contain the deuterated components in various combinations, were studied in solution by small-angle neutron scattering. The individual shapes of the subunits and their organization in the alpha beta gamma-TF1 complex were examined using the techniques of selective deuteration and contrast variation. The alpha and beta subunits are well approximated as ellipsoids of revolution having minor semi-axes of 20.4(+/- 0.4) and 20.0(+/- 0.2) A, and major semi-axes of 53.0(+/- 1.4) and 55.8(+/- 0.9) A, respectively. In the TF1 complex, three beta subunits are aligned to form an equilateral triangle, with their major axes tilted by 35 degrees with respect to the 3-fold axis of the complex. The beta-beta distance is about 53 A. Three alpha subunits are similarly arranged, positioned between the beta subunits, and with their direction of tilt opposite to that of the beta subunits. The centers of the alpha and beta subunits lie in the same plane, forming a hexagon. Adjacent subunits overlap in this model, suggesting that they are not simple ellipsoids of revolution.

Bacteria↗
PubMed1990

Epidermal growth factor stimulates the anchorage-independent growth of human squamous cell carcinomas overexpressing its receptors.

We examined the effects of epidermal growth factor (EGF) on the anchorage-dependent and -independent growth of four human squamous carcinoma cell lines that overexpress EGF receptors. While EGF inhibited anchorage-dependent growth, it stimulated anchorage-independent growth of all four cell lines tested. The results suggest that the proliferative responses to EGF are characterized by a preference for anchorage-independent, rather than -dependent growth, in cells overexpressing EGF receptors. Moreover, as EGF has been shown to stimulate the in vivo growth of squamous carcinoma cells overexpressing EGF receptors, it is also suggested that the in vitro EGF responsiveness of these cells in soft agar, but not in monolayer, better correlates with the in vivo EGF responsiveness.

Carcinoma, Squamous Cell↗
PubMed1990

Sialylpentaosylceramide detected with anti-GM2 monoclonal antibody. Structural characterization and complementary expression with GM2 in gastric cancer and normal gastric mucosa.

The ganglioside fraction of human gastric mucosa was analyzed with a newly established anti-GM2 monoclonal antibody KM531. Using this antibody, accumulation of GM2 was observed in all of four cases of gastric carcinoma. In all ganglioside fractions extracted from normal gastric mucosa obtained from eight cases of peptic ulcer GM2 itself was not detected, but three kinds of glycolipid showing slower mobility than GM2 on thin-layer plates were detected by immunostaining with KM531. These glycolipids were assigned as NGM-1, -2, and -3. They were completely lost in all carcinoma tissues and in non-cancerous gastric mucosa from two cases of gastric cancer, and they were also not detected in the ganglioside fraction of small or large intestine. Of these glycolipids, the major one, NGM-1, was isolated from the pooled ganglioside fraction of normal gastric mucosa obtained from cases of peptic ulcer. The structure was determined by proton nuclear magnetic resonance, negative ion fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry, and treatment with exoglycosidases and mild acid hydrolysis. The structure was GalNAc beta 1----4(NeuAc alpha 2----3) Gal beta 1----4GlcNAc beta 1----3 Gal beta 1----4Glc beta 1----1Cer, which has the same terminal sequence as GM2 but has internal neolacto series structure. This epitope was previously identified as Cad blood group antigen. The decrease of this glycolipid and the increase of GM2 was considered to be a cancer-associated change in gastric mucosa.

Antibodies, Monoclonal↗
PubMed1990

Novel regulatory enhancer in the nuclear gene of the human mitochondrial ATP synthase beta-subunit.

The system coordinating expressions of nuclear coded mitochondrial proteins was investigated by examination of the 5'-flanking region of the human mitochondrial ATP synthase beta-subunit gene. The promoter activity was measured by a transient expression of a chloramphenicol acetyltransferase (CAT) gene connected with various 5'-deletion mutants of the 5'-flanking region. In this experiment, at least two regions enhanced this promoter activity and at least one region repressed it. In one of the enhancing regions, a consensus sequence was found for the genes of other mitochondrial proteins such as those for cytochrome c1 (Suzuki, H., Hosokawa, Y., Nishikimi, M., and Ozawa, T. (1989) J. Biol. Chem. 264, 1368-1374) and the pyruvate dehydrogenase alpha-subunit (Maragos, C., Hutchison, W. M., Hayasaka, K., Brown, G. K., and Dahl, H.-H. M. (1989) J. Biol. Chem. 264, 12294-12298; Ohta, S., Endo, H., Matsuda, K., and Kagawa, Y. (1989) Ann. N. Y. Acad. Sci. 573, 458-460). The characteristics of this enhancing element were examined by introducing a synthetic oligonucleotide element into the CAT plasmid with a deleted enhancing element. The resulting plasmid showed full recovery of promoter activity, and this activity was independent of the orientation or location of the insert. Therefore, this is an enhancer that may be common to the nuclear genes of some mitochondrial proteins involved in energy transduction.

Base Sequence↗
PubMed1990

Marked decrease of mitochondrial DNA with multiple deletions in a patient with familial mitochondrial myopathy.

Muscle mitochondrial DNA (mtDNA) from a patient with mitochondrial myopathy was examined by Southern blotting. Her family history suggests autosomal dominant inheritance of this disorder. In contrast to other cases of this myopathy that are associated with a compensatory increase of mitochondria, in this patient, the content of mtDNA was markedly decreased (15% of controls), whereas protein of the ATP synthase beta-subunit was not decreased appreciably as judged by Western blotting. In addition, the mitochondrial DNA had multiple deletions, which were located between the replication origins of the heavy strand and light strand.

Adult↗
PubMed1990

Effects of ethidium bromide treatment of mouse cells on expression and assembly of nuclear-coded subunits of complexes involved in the oxidative phosphorylation.

Using mouse cell lines 5P and 5PEr (ethidium bromide-resistant derivative of 5P), we examined the influence of blocking expression of mitochondrial-gene products with ethidium bromide on the expression and assemblies of nuclear-coded subunits of the complexes involved in the oxidative phosphorylation. The results suggest that in the absence of mitochondrial-coded products, the expressions of subunit VIc of complex IV and beta-subunit of F1-ATPase are not affected, but that most nuclear-coded subunits other than alpha- and beta-subunits of F1-ATPase cannot be assembled nor inserted into the inner membrane.

Actins↗
PubMed1990

Primary structure of a constituent polypeptide chain (AIII) of the giant haemoglobin from the deep-sea tube worm Lamellibrachia. A possible H2S-binding site.

The deep-sea tube worm Lamellibrachia, belonging to the Phylum Vestimentifera, contains two giant extracellular haemoglobins, a 3000 kDa haemoglobin and a 440 kDa haemoglobin. The former consists of four haem-containing chains (AI-AIV) and two linker chains (AV and AVI) for the assembly of the haem-containing chains [Suzuki, Takagi & Ohta (1988) Biochem. J. 255, 541-545]. The tube-worm haemoglobins are believed to have a function of transporting sulphide (H2S) to internal bacterial symbionts, as well as of facilitating O2 transport [Arp & Childress (1983) Science 219, 295-297]. We have determined the complete amino acid sequence of Lamellibrachia chain AIII by automated or manual Edman sequencing. The chain is composed of 144 amino acid residues, has three cysteine residues at positions 3, 74 and 133, and has a molecular mass of 16,620 Da, including a haem group. The sequence showed significant homology (30-50% identity) with those of haem-containing chains of annelid giant haemoglobins. Two of the three cysteine residues are located at the positions where an intrachain disulphide bridge is formed in all annelid chains, but the remaining one (Cys-74) was located at a unique position, compared with annelid chains. Since the chain AIII was shown to have a reactive thiol group in the intact 3000 kDa molecule by preliminary experiments, the cysteine residue at position 74 appears to be one of the most probable candidates for the sulphide-binding sites. A phylogenetic tree was constructed from nine chains of annelid giant haemoglobins and one chain of vestimentiferan tube-worm haemoglobin now determined. The tree clearly showed that Lamellibrachia chain AIII belongs to the family of strain A of annelid giant haemoglobins, and that the two classes of Annelida, polychaete and oligochaete, and the vestimentiferan tube worm diverged at almost the same time. H.p.l.c. patterns of peptides (Figs. 4-7), amino acid compositions of peptides (Table 2) and amino acid sequences of intact protein and peptides (Table 3) have been deposited as Supplementary Publication SUP 50154 (13 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.

Amino Acid Sequence↗
PubMed1990

Primary structure of a linker subunit of the tube worm 3000-kDa hemoglobin.

The deep-sea tube worm Lamellibrachia contains two giant extracellular hemoglobins, a 3000-kDa hemoglobin and a 440-kDa hemoglobin. The former consists of four heme-containing chains (AI-AIV) and two linker chains (AV and AVI) for the assembly of the heme-containing chains. The 440-kDa hemoglobin consists of only four heme-containing chains (Suzuki, T., Takagi, T., and Ohta, S. (1988) Biochem. J. 255, 541-545). The complete amino acid sequence of a linker subunit (chain AV) has been determined by automated Edman sequencing of the peptides derived by digestions with lysyl endopeptidase and endoproteinase Asp-N. The chain is composed of 224 amino acid residues, and the molecular mass for the protein moiety was calculated to be 24,894 Da. An Asn-X-Thr sequence which is possible as a glycosylation site was suggested at positions 108-110. A computer-assisted homology search showed that the sequence shows no notable homology with any other globins and proteins. However a careful alignment of the linker sequence with a heme-containing chain sequence suggested that there is a slight, but significant homology between the two sequences. The alignment also suggested that the linker resulted from gene duplication of a heme-containing chain with a three exon-two intron structure, and that the first exon of domain 1 and the last exon of domain 2 had been lost during evolution. In our alignment, domain 1 has the heme-binding proximal histidine, but domain 2 does not. This is the first linker subunit to be sequenced completely.

Amino Acid Sequence↗
PubMed1990

Confirmation of the enantiomers of 1-methyl-1,2,3,4-tetrahydroisoquinoline in the mouse brain and foods applying gas chromatography/mass spectrometry with negative ion chemical ionization.

With the aid of a new chiral derivatizing reagent and a sensitive and specific assay using capillary gas chromatography/negative ion chemical ionization mass spectrometry, the proportions of R and S enantiomers of 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) in mammalian tissues and foods were studied. R- and S-1MeTIQ enantiomers derivatized with a chiral derivatizing reagent, perfluoro-2-propoxypropionylchloride, were clearly separated. The ratio of enantiomers was R/S = 0.24, 0.55 and 0.60 in wine, cocoa and mouse brain. S-1MeTIQ predominated in all samples. This result suggested that 1MeTIQ could be formed at least partially through an enzymatic mechanism.

Animals↗
PubMed1990

Regulation of mitochondrial ATP synthesis in mammalian cells by transcriptional control.

1. Mitochondrial ATP synthesis (oxidative phosphorylation) is mainly regulated by the membrane potential (respiratory control) and protein synthesis (transcriptional control). 2. According to the current view, genes for enzymes of oxidative phosphorylation are classified as housekeeping genes that are transcribed constitutively. These genes have been sequenced. 3. Four complexes of oxidative phosphorylation (complexes I, III and IV, and ATP synthase) are exceptional oligomers that contain subunits encoded by mitochondrial DNA. The remaining subunits of these complexes, as well as thousands of other mammalian enzymes are encoded by nuclear DNA. 4. It is proposed that ATP synthase (F0F1) and these oligomers supplying energy to F0F1, though they are housekeeping, are under some coordinated transcriptional control. Not transcription, but translation of mitochondrial DNA is mainly regulated. 5. Recently, studies on cloned human genes for the FoF1 beta subunit and 7 enzymes related to ATP synthesis revealed coordinated transcription. Moreover, a novel common cis-element (enhancer) was discovered in the 5'-upstream region of genes for the F1 beta subunit, cytochrome c1 and pyruvate dehydrogenase E1 alpha subunit. 6. In contrast to heme control and catabolite repression of yeast via trans-acting elements such as HAP and GP, signal transduction and coordinated transcription of human oxidative phosphorylation is not directed by CP1 (a HAP homolog), but may be closely related to cell differentiation.

Adenosine Triphosphate↗