Search PubMed⌕ Search

Biomedical subjects

M Ohshima

Publications and source records attributed to M Ohshima.

At least 19 recordsLinked to original sources
PubMed1999

Enhanced resistance to bacterial diseases of transgenic tobacco plants overexpressing sarcotoxin IA, a bactericidal peptide of insect.

Sarcotoxin IA is a bactericidal peptide of 39 amino acids found in the common flesh fly, Sarcophaga peregrina. Many agronomically important bacteria in Japan are killed by this peptide at sub-micro molar levels, and the growth of tobacco and rice suspension cultured cells is not inhibited with less than 25 microM. Transgenic tobacco plants which overexpress the peptide, i.e. over 250 pmol per gram of fresh leaf, under the control of a high expression constitutive promoter showed enhanced resistance to the pathogens for wild fire disease (Pseudomonas syringae pv. tabaci) and bacterial soft rot disease (Erwinia carotovora subsp. carotovora).

Animals↗
PubMed1998

Role of the isoprenyl tail of ubiquinone in reaction with respiratory enzymes: studies with bovine heart mitochondrial complex I and Escherichia coli bo-type ubiquinol oxidase.

The hydrophobic isoprene tail of ubiquinone-2 (Q2) exihibits binding specificity in redox reactions with bovine heart mitochondrial complex I (Ohshima, M., Miyoshi, H., Sakamoto, K., Takegami, K., Iwata, J., Kuwabara, K., Iwamura, H., and Yagi, T. (1998) Biochemistry 37, 6436-6445) and the Escherichia coli bo-type ubiquinol oxidase (Sakamoto, K., Miyoshi, H., Takegami, K., Mogi, T., Anraku, Y., and Iwamura, H. (1996) J. Biol. Chem. 271, 29897-29902). To identify the structural factor(s) of the diprenyl tail of Q2 governing the specific interaction with these enzymes, we synthesized a series of novel Q2 analogues in which only one of the structural factors of the diprenyl tail was systematically modified. In bovine complex I, the presence of the methyl branch and the pi-electron system in the first isoprene unit are responsible for high-affinity binding of Q2 to the ubiquinone reduction site, which results in a low Km and kcat values of Q2 reduction. The position of the methyl group in the tail is strictly recognized by the enzyme. In contrast to complex I, in bo-type ubiquinol oxidase, either of the two pi-electron systems in the tail is required for high-affinity binding of Q2H2 to the enzyme, while the presence of the methyl branch and the location of the pi-electron systems are not strictly recognized by the enzyme. We concluded that the role of the ubiquinone tail is not simply the enhancement of the hydrophobicity of the molecule and that molecular recognition of the tail by the quinone redox site differs among the respiratory enzymes.

Animals↗
PubMed1998

Essential structural factors of annonaceous acetogenins as potent inhibitors of mitochondrial complex I.

The annonaceous acetogenins are the most potent of the known inhibitors of bovine heart mitochondrial complex I. These inhibitors act, at the terminal electron transfer step of the enzyme, in a similar way to the usual complex I inhibitors, such as piericidin A and rotenone; however, structural similarities are not apparent between the acetogenins and these known complex I inhibitors. A systematic set of isolated natural acetogenins was prepared and examined for their inhibitory actions with bovine heart mitochondrial complex I to identify the essential structural factors of these inhibitors for the exhibition of potent activity. Despite their very potent activity, the structural requirements of the acetogenins are not particularly rigid and remain somewhat ambiguous. The most common structural units, such as adjacent bis-tetrahydrofuran (THF) rings and hydroxyl groups in the 4- and/or 10-positions, were not essential for exhibiting potent activity. The stereochemistry surrounding the THF rings, surprisingly, seemed to be unimportant, which was corroborated by an exhaustive conformational space search analysis, indicating that the model compounds, with different stereochemical arrangements around the THF moieties, were in fairly good superimposition. Proper length and flexibility of the alkyl spacer moiety, which links the THF and the alpha, beta-unsaturated gamma-lactone ring moieties, were essential for the potent activity. This probably results from some sort of specific conformation of the spacer moiety which regulates the two ring moieties to locate into an optimal spatial position on the enzyme. It is, therefore, suggested that the structural specificity of the acetogenins, required for optimum inhibition, differs significantly from that of the common complex I inhibitors in which essential structural units are compactly arranged and conveniently defined. The structure-activity profile for complex I inhibition is discussed in comparison with those for other biological activities.

Animals↗
PubMed1998

Characterization of the ubiquinone reduction site of mitochondrial complex I using bulky synthetic ubiquinones.

A wide variety of alkyl derivatives of Q2 (6-geranyl-2, 3-dimethoxy-5-methyl-1,4-benzoquinone) and DB (6-n-decyl-2, 3-dimethoxy-5-methyl-1,4-benzoquinone), in which methoxy groups of the 2- and/or 3-positions of the quinone ring were replaced by other bulky alkoxy groups from ethoxy to butoxy, were prepared by novel synthetic procedures. Electron-accepting activities of the bulky quinones were investigated with bovine heart mitochondrial complex I and its counterpart of Paracoccus denitrificans(NDH-1) to elucidate structural and functional features of the quinone reduction site of the enzymes. The bulky quinone analogues served as sufficient electron acceptors from the physiological quinone reduction site of bovine complex I. Considering the very poor activities of even the ethoxy derivatives as substrates for other respiratory enzymes such as mitochondrial complexes II and III [He, D. Y., Gu, L. Q., Yu, L., and Yu, C. A. (1994) Biochemistry 33, 880-884], this result indicated that the quinone reduction site of bovine complex I is spacious enough to accommodate bulky exogenous substrates. In contrast to bovine complex I, bulky quinone analogues served as poor electron acceptors with Paracoccus NDH-1. These observations indicated that bovine complex I recognizes the substrate structure with poor specificity. The substituent effects in the 2- and 3-positions of the quinone ring on the electron-transfer activity with bovine complex I differed significantly between Q2 and DB series despite having the same total number of carbon atoms in the side chain. The inhibitory effect involving Q2 due to its geranyl side chain was markedly diminished by structural modifications of the quinone ring moiety. These findings indicate that the side chain plays a specific role in the redox reaction and that the quinone ring and side-chain moieties contribute interdependently to binding interaction. Moreover, structural dependency of the proton-pumping activity of the quinone analogues was comparable to that of the electron-transfer activity with bovine complex I, indicating that the mechanism of redox-driven proton-pumping does not differ depending upon the substrate structure.

Animals↗
PubMed1998

Role of NMDA receptors in the propagation of excitation in rat visual cortex as studied by optical imaging.

To examine the role of the N-methyl-D-aspartate (NMDA) type of glutamate receptors in the propagation of information in visual cortex, optical imaging with high spatial and temporal resolution of neuronal activity was used in cortical slices of rats. Single-shock stimulation of the white matter elicited a vertical propagation of excitation toward the cortical surface simultaneously with a horizontal spread of excitation in lower layers. The horizontal spread in upper layers occurred subsequent to the vertical spread reaching these layers. The results from perfusion of Ca2+-free medium and application of an antagonist of non-NMDA receptors indicated that this intracortical propagation of signals is due mostly, if not exclusively, to the postsynaptic excitation of cortical neurons. Blockade of NMDA receptors attenuated the rising and peak phases of the upper horizontal spread, but did not affect those of the lower horizontal or vertical propagation of excitation. Perfusion with Mg2+-free solution enhanced the upper horizontal spread, but in most cases did not significantly change the spread of excitation in the other pathways. These results indicate that NMDA receptors are involved in the flow of information in the upper layers of visual cortex, and further suggest that this propagation of activity is mediated mainly by horizontal connections intrinsic to the upper layers.

2-Amino-5-phosphonovalerate↗
PubMed1998

Enhanced expression of an antimicrobial peptide sarcotoxin IA by GUS fusion in transgenic tobacco plants.

To enhance the disease resistance of plants expressing a foreign peptide, the gene for sarcotoxin IA, which is an antimicrobial peptide from an insect consisting of 39 amino acid residues, was introduced into tobacco (Nicotiana tabacum) under the control of a high expression promotor via Agrobacterium-mediated transformation. In transgenic plants, sarcotoxin IA mRNA accumulated to detectable levels, however, the amount of the peptide produced was so small that we could scarcely detect it by protein gel blot analysis, probably because of the instability of short peptides in plant cells. To improve the expression efficiency, genes for four types of amino-terminal and carboxyl-terminal translational fusions of sarcotoxin IA together with the GUS gene were introduced into tobacco. In all four types of transgenic tobacco plants, high level transcripts similar to that in the direct expression sarcotoxin IA construct were found. Protein gel blot analysis with both anti-sarcotoxin IA and GUS antibodies showed production of high levels of fusion protein in all transgenic plants. Among them, three types had abnormal membranes and phenotypes, although no such abnormalities were found in transgenic plants in which only sarcotoxin IA was expressed in a secretable form. All together, these results indicated that, for stable and effective expression of a foreign short peptide in transgenic plants, expression as a fusion protein is useful and that secretion of sarcotoxin IA outside of cells is necessary for generation of useful antimicrobial transgenic plants.

Anti-Bacterial Agents↗
PubMed1998

Circadian rhythms of hormone concentrations in alcohol withdrawal.

We investigated the circadian rhythm of hormones (cortisol, melatonin) in alcoholic patients during and 1 month after alcohol withdrawal. Patients with delirium tremens had irregular serum hormone concentration rhythms during withdrawal, which normalized after the withdrawal period. Patients without delirium tremens had normal circadian rhythms even during the withdrawal period. We speculated that the disturbance of the biological oscillator, in terms of the decline of synchronizing function or the decrease in synchronizing factors, caused abnormal circadian rhythms of hormone release during delirium tremens.

Adult↗
PubMed1998

Expression of KAI1 in paraffin-embedded normal, hyperplastic and neoplastic prostate and prostate carcinoma cell lines.

Expression of KAI1, a tumor metastasis suppressor gene, was studied with different fixatives in frozen and paraffin-embedded sections of human and rat prostate carcinoma cell lines and human prostate lesions by immunohistochemistry. Immunoreactivity of the membrane antigen in cell lines was associated with known expression levels in these lines and the fixative used. Formalin and paraformaldehyde helped maintain the immunoreactivity of cells. In human prostate, frozen sections revealed diffuse reactivity of the antigen in normal and neoplastic tissues while paraffin-embedded tissues usually showed focal reactivity, although more than 50% of cases with normal epithelium and adenocarcinomas were reactive. In some cases, pretreatment with trypsin enhanced immunoreactivity. Benign prostatic hyperplasia (BPH) showed the most intense diffuse immunoreactivity, which suggested enhanced expression. Prostatic intraepithelial neoplasia (PIN) also often expressed high levels of KAI1. Three of five metastases were reactive but two primaries and their metastases were not. Lymphocytes in primary carcinomas and lymphocytes and germinal center cells in lymph nodes were immunoreactive, while adjacent primary or metastatic prostate adenocarcinoma epithelium was not immunoreactive. Although paraffin-embedded human tissues were not optimal for determining levels of expression of KAI1, they did show immunoreactivity that could have prognostic value and showed the specific cytoplasmic localization of the protein in cells.

Adenocarcinoma↗
PubMed1998

Photosensitive seizures provoked while viewing "pocket monsters," a made-for-television animation program in Japan.

PURPOSE: To describe the recent epidemic of photosensitive seizure that occurred in relation to an episode of the television animation program "Pocket Monsters," we report four patients who experienced seizures while watching the episode in question. We also report some technical aspects of the program episode. METHODS: We investigated the clinical symptoms of the four patients and performed routine EEGs with intermittent photic stimulation (IPS). If IPS provoked no photoparoxysmal response (PPR) during the routine EEG examination, a second EEG was performed with the photic stimulator placed 10 cm from the patient's eyes. In addition, we reviewed the "Pocket Monsters" episode, focusing our attention on the visual techniques used with reference to the Independent Television Commission (ITC) guidelines. RESULTS: One patient who had myoclonic jerks before the convulsion in question was diagnosed as having juvenile myoclonic epilepsy, and the diagnosis of another patient was pure photosensitive epilepsy. The remaining two patients had their first seizures, which could be occasional seizures, and we therefore could not reach a diagnosis of epilepsy. In our four patients, only one showed PPR on the routine EEG. Two patients revealed PPR on the second EEG, and the remaining patient showed no PPR. Rapid changes in color are believed to be responsible for the photosensitive seizures because all four patients had seizures at around 18:50, when seconds of deep red and bright blue flashes, alternating at a frequency of 12 Hz, were shown. CONCLUSIONS: Regulations for technical aspects of children's programming, including the use of colors, are urgently needed in Japan to prevent a repeated incident. In addition, the IPS procedure needs to be standardized, especially for patients who are suspected to have photosensitivity.

Adolescent↗
PubMed1998

Overexpression of CD44 variant transcripts in rat transplantable thyroid carcinoma lines demonstrating lung metastasis.

Expression of CD44 isoforms has been reported to be involved in tumor invasion and metastasis in both rodents and man. We earlier documented establishment of rat transplantable thyroid carcinoma lines in vivo from primary lesions induced by a chemical carcinogen. Recently, two lines (L1a-M4 and L2a-M6) were found to spontaneously metastasize to the lung after subcutaneous transplantation. To determine whether CD44 splice variants contribute to their metastatic spread, carcinoma lines with and without lung metastasis were evaluated quantitatively and qualitatively using RT-PCR followed by hybridization and immunohistochemical analyses. The L1a-M4 and L2a-M6 metastatic lines showed significant overexpression of CD44 variant transcripts containing variant exons v4-v6 or v9-v10/v8-v10, respectively, with concomitant reduced levels of standard transcripts. Investigation of the precise composition of alternatively spliced mRNA in normal tissues and carcinoma lines using an exon-specific RT-PCR method, revealed major chain variant transcripts containing v2/v3, v4-v6, v7-v10 and v8-v10 in all specimens. Applying the same RT-PCR analysis to mRNAs derived from cultured cell lines, demonstrated essentially the same pattern. The results suggest that quantitative increase rather than qualitative change in CD44 variant isoforms is associated with the pathogenesis of lung metastasis of rat thyroid carcinomas.

Alternative Splicing↗
PubMed1997

Probing the ubiquinone reduction site of mitochondrial complex I using novel cationic inhibitors.

A wide variety of N-methylpyridinium and quinolinium cationic inhibitors of mitochondrial complex I was synthesized to develop potent and specific inhibitors acting selectively at one of the two proposed ubiquinone binding sites of this enzyme (Gluck, M. R., Krueger, M. J., Ramsay, R. R., Sablin, S. O., Singer, T. P., and Nicklas, W. J. (1994) J. Biol. Chem. 269, 3167-3174). N-Methyl-2-n-dodecyl-3-methylquinolinium (MQ18) inhibited electron transfer of complex I at under microM order regardless of whether exogenous or endogenous ubiquinone was used as an electron acceptor. The presence of tetraphenylboron (TPB-) potentiated the inhibition by MQ18 in a different way depending upon the molar ratio of TPB- to MQ18. In the presence of a catalytic amount of TPB-, the inhibitory potency of MQ18 was remarkably enhanced, and the extent of inhibition was almost complete. The presence of equimolar TPB- partially reactivated the enzyme activity, and the inhibition was saturated at an incomplete level (approximately 50%). These results are explained by the proposed dual binding sites model for ubiquinone (cited above). The inhibition behavior of MQ18 for proton pumping activity was similar to that for electron transfer activity. The good correlation of the inhibition behavior for the two activities indicates that both ubiquinone binding sites contribute to redox-driven proton pumping. On the other hand, N-methyl-4-[2-methyl-3-(p-tert-butylphenyl)]propylpyridinium (MP6) without TPB- brought about approximately 50% inhibition at 5 microM, but the inhibition reached a plateau at this level over a wide range of concentrations. Almost complete inhibition was readily obtained at low concentrations of MP6 in the presence of TPB-. Thus MP6 appears to be a selective inhibitor of one of the two ubiquinone binding sites. With a combined use of MP6 and 2,3-diethoxy-5-methyl-6-geranyl-1,4-benzoquinone, we also provided kinetic evidence for the existence of two ubiquinone binding sites.

1-Methyl-4-phenylpyridinium↗
PubMed1997

Unsuspected gallbladder cancer diagnosed by laparoscopic cholecystectomy: a clinicopathological study.

Laparoscopic cholecystectomy (LC) for gallstones has become a popular treatment modality, but LC for gallbladder cancer is not yet established and its prognosis is still unknown. We clinicopathologically studied unsuspected gallbladder cancer presenting at pathologic evaluation after LC. The entire cross section of gallbladders after 456 LCs was histologically examined. The presence of malignant lesions was confirmed in 13 (2.85%) of 456 cases. The preoperative diagnoses were gallstones in 5 patients, gallbladder polyps in 5, gallstones with gallbladder polyps in 2, and gallstones with adenomyomatosis in 1. The carcinoma had invaded the mucosa in 7, the proper muscle in 2, and the subserosa in 3; the serosa was exposed in 1. The tumor size ranged from 2 mm to the entire gallbladder. An additional resection was performed in 2 patients. During the mean follow-up period of 25.9 months, 1 patient died of recurrence at 8 months while 1 demonstrated recurrence at the port site 1 year after surgery. No other patients have had any recurrence to date. Since early gallbladder cancer removed laparoscopically can be expected to have a satisfactory outcome, diagnostic and therapeutic LC is recommended in cases where total biopsy is indicated. However, in every case, extreme caution needs to be exercised to prevent the dissemination and implantation of cancer cells at the port site.

Aged↗
PubMed1997

Interferons suppress mitochondrial gene transcription by depleting mitochondrial transcription factor A (mtTFA).

Mitochondrial gene transcription activity in organello was suppressed after culturing HeLa cells with 1000 U/ml of interferon-alpha (IFN-alpha) or IFN-gamma for 18 h. The suppression was associated with reduced levels of mitochondrial gene transcripts. Northern blot analysis of HeLa cell RNA showed marked reduction of the mRNA encoding for mitochondrial transcription factor A (mtTFA). Immunoblotting with antiserum directed against recombinant mtTFA showed a reduced level of the protein as well. Consistently, gel-retardation assay of mitochondrial extract showed reduced level of functional mtTFA, which is known to bind to both heavy and light strand promoters of bidirectionally transcribed mitochondrial DNA. We suggest that depletion of mtTFA is a pathway through which IFNs depress the mitochondrial respiration.

DNA-Binding Proteins↗
PubMed1997

An improved assay method for fibroblast gelatinolytic enzyme.

A useful gelatinolytic enzyme assay for fibroblasts, utilizing a novel sample preparation method for collagenase with dithiothreitol (DTT) treatment to inactivate endogenous collagenase inhibitors, was developed using soluble fluorescein isothiocyanate (FITC)-labeled gelatin. The substrate, gelatin was prepared by heating commercially available FITC-labeled type I collagen. The denatured collagen was cleaved with purified trypsin and partially purified fibroblast gelatinase, and the digested FITC-fragments were measured fluorometrically. The intensity of the fluorescence was in proportion to the reaction time and enzyme concentration. Both enzyme activities were measurable within the nanogram range of enzyme preparations. The enzyme activity was detected after 4-aminophenylmercuric acetate (APMA) treatment which was completely inhibited by metalloproteinase inhibitors, but not by serine- and cysteine-proteinases' inhibitors. Conditioned media of human periodontal ligament fibroblasts (PLF) and gingival fibroblasts (GF) were separately treated with DTT prior to the enzyme assay, and then the assay was performed in the presence of APMA. The enzyme activities of PLF and GF were 106- and 55-fold higher than those of the conventional gelatinase assay which was carried out without DTT treatment. This assay method allowed the measurement of gelatinolytic enzyme activity when tissue inhibitors of metalloproteinases were present in the fibroblast culture medium.

Biological Assay↗
PubMed1997

Effect of dental cyst epithelium-conditioned medium on collagenase production by periodontal ligament fibroblasts.

Seven dental cyst epithelia were cultured in vitro and the conditioned media were analyzed for periodontal ligament fibroblast collagenase production. All the cyst epithelium-conditioned media stimulated fibroblast collagenase production, indicating that dental cyst epithelium, which is considered to be identical to the cell rests of Malassez, might play a role in periodontal pocket formation.

Cells, Cultured↗
PubMed1996

A family of novel DNA-binding nuclear proteins having polypyrimidine tract-binding motif and arginine/serine-rich motif.

NP220 is a DNA-binding nuclear protein originally cloned from human cell lines. Human NP220 (hNP220) has an arginine/serine-rich motif found in non-small nuclear RNP splicing factors (SR proteins) and shares three domains (MH1, MH2 and MH3) with an acidic nuclear matrix protein, matrin 3. The MH2 domain repeats three times and has homology to the polypyrimidine tract-binding motif of heterogeneous nuclear RNP I/L. NP220 also has a DNA-binding domain and nine repeats of the sequence LVTVDEVIEEEDL (acidic repeat). We have now isolated mouse equivalents of NP220 (mNP220s) and found that NP220s form a family of proteins with four members produced by alternative splicing of a common pre-mRNA. Two longer forms (NP220alpha and NP220beta) have all functional domains mentioned above while two shorter forms (NP220gamma and NP220delta) lack the DNA-binding domain and the acidic repeat. The structural aspects of NP220s are distinct from that of the SR proteins but rather resemble U2AF and Tra2 which activate a specific 3'-splicing site of specific genes in response to differentiation-dependent signals.

Amino Acid Sequence↗
PubMed1996

A large DNA-binding nuclear protein with RNA recognition motif and serine/arginine-rich domain.

cDNA species encoding a large DNA-binding protein (NP220) of 1978 amino acids was isolated from human cDNA libraries. Human NP220 binds to double-stranded DNA fragments by recognizing clusters of cytidines. Immunofluorescent microscopy with antiserum directed against NP220 revealed a punctate or "speckled" pattern and coiled body-like structures in the nucleoplasm of various human cell lines. These structures diffused in the cytoplasm during mitosis. Western blot analysis showed that NP220 is enriched in the lithium 3,5-diiodosalicylate-insoluble fraction of nuclei. The domain essential for DNA binding is localized in C-terminal half of NP220. Human NP220 shares three types of domains (MH1, MH2, and MH3) with the acidic nuclear protein, matrin 3 (Belgrader, P., Dey, R., and Berezney, R. (1991) J. Biol. Chem. 266, 9893-9899). MH1 is a 48-amino acid sequence near the N terminus of both human NP220 and rat matrin 3. MH2 is a 75-amino acid sequence homologous to the RNA recognition motifs of heterogeneous nuclear RNP I and L. It is repeated three times in NP220 and twice in matrin 3. MH3 is a 60-amino acid sequence at the C terminus of both NP220 and matrin 3. NP220 has an arginine/serine-rich domain commonly found in pre-mRNA splicing factors. Close to the domain essential for DNA binding, there are nine repeats of the sequence LVTVDEVIEEEDL. Thus, NP220 is a novel type of nucleoplasmic protein with multiple domains.

Amino Acid Sequence↗