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M Shirai

Publications and source records attributed to M Shirai.

At least 19 recordsLinked to original sources
PubMed2006

Does central nitric oxide chronically modulate the acute hypoxic ventilatory response in conscious rats?

AIM: Hypoxia initiates an increase in ventilation (VE) through a cascade of events of which central nitric oxide (NO) has been implicated as an important neuromodulator. There have not been any reports describing the consequences of long-term imbalances in the central NO pathways on the modulation of the acute hypoxic ventilatory response (HVR). Chronic hypoxia (CH) can potentially modify the HVR, and so we hypothesized that central NO may be involved. In this study we describe the long-term role of central NO in the modulation of HVR before and after CH. METHODS: Male Sprague-Dawley rats (BW c. 200-320 g; n = 21) were implanted with an osmotic pump for continuous intracerebroventricular administration of either artificial cerebrospinal fluid (control), Nomega-nitro-L-arginine methyl ester (L-NAME) (150 microg kg(-1) day(-1)) or the NO-donor, 3-[4-morpholinyl]-sydnonimine-hydrochloride (SIN-1) (100 microg kg(-1) day(-1)). The VE response to acute poikilocapnic hypoxia (8% O2 for 20 min) was measured by plethysmography seven days after surgery, in normoxia, and again after 14 days of exposure to CH (CH = 12% O2). RESULTS: The magnitude of the HVR (c. 230% increase in VE) was unaltered by centrally infusing either L-NAME or SIN-1 for 1 week. CH did not modify the HVR, although baseline VE and HVR were shifted downward by L-NAME during CH - because of a reduction in the frequency component. CONCLUSIONS: These results suggest that long-term alterations in central NO levels may not alter the HVR under moderate CH, presumably because of the onset/development of compensatory mechanisms. However, NO appears to be an important component of the HVR following CH.

Acute Disease↗
PubMed2004

Serine-arginine-rich nuclear protein Luc7l regulates myogenesis in mice.

Using a gene trap technique, we identified a murine homologue of the yeast LUC7-like gene (Luc7l), which is a serine-arginine-rich protein (SR protein) that localizes in the nucleus through its arginine-serine-rich domain (RS domain) at the C-terminus and shows a speckled distribution pattern. Although its transcripts are widely expressed in embryos and adults, they are rarely detected in adult skeletal muscle, and Luc7l expression was found to be negatively regulated during the course of development of limb skeletal muscle, as well as during in vitro differentiation of the myoblast cell lines Sol8 and C2C12. We also demonstrated that forced expression of Luc7l protein inhibited myogenesis in vitro. Based on our results, Luc7l is thought to play an important role in the regulation of muscle differentiation.

Animals↗
PubMed2003

Downregulation of androgen, estrogen and progesterone receptor genes and protein is involved in aging-related erectile dysfunction.

We hypothesize that downregulation of sex hormone receptors (androgen, estrogen and progesterone receptors) is involved in aging-related erectile dysfunction. To test this hypothesis, we investigated the expression of sex hormone receptors in penile crura of aging rats. A total of 40 rats were divided into four groups based on age (6, 12, 18 and 24 months), and the erectile function was analyzed by the measurement of intracavernous pressure. Gene and protein expressions of sex hormone receptors were analyzed by RT-PCR and immunostaining, respectively. The mean intracavernous pressures of 6-, 12-, 18- and 24-month-old rats were 110.1, 89.6, 73.5 and 42.7 cm H(2)O, respectively. Gene and protein expressions for androgen receptor, estrogen receptor-beta and progesterone receptor were present in similar levels in 6-, 12- and 18-month-old rat crura, but significantly lower or absent in 24-month-old crura. This is the first study to demonstrate that downregulation of sex hormone receptors in aging rat crura is associated with erectile dysfunction.

Aging↗
PubMed2003

Lack of evidence for a role of Epstein-Barr virus in the increase of lung cancer in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is known as an independent risk factor for lung cancer. Because Epstein-Barr virus (EBV) may be involved in the genesis of IPF as well as certain malignancies, we investigated whether EBV contributes to the increased incidence of lung cancer in IPF The formalin-fixed and paraffin-embedded lung sections were prepared from 22 lung cancer patients with IPF and 22 lung cancer patients without IPF All ofthe IPF patients pathologically showed usual interstitial pneumonia. In situ hybridization for EBV-encoded small non-polyadenylated RNAs failed to show positive signals in the cancer tissues of either IPF or non-IPF patients. This study did not provide evidence for an etiologic role of EBV in the development of lung cancer in IPF.

Adenocarcinoma↗
PubMed2003

Geldanamycin, an inhibitor of Hsp90, sensitizes human tumour cells to radiation.

PURPOSE: The effects of the heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) were examined on the radiosensitivity and signal transduction pathways in human tumour cell lines. MATERIALS AND METHODS: Two human cell lines, SQ-5 and DLD-1, derived from lung carcinoma and colon adenocarcinoma, respectively, were incubated for 16 h at 37 degrees C in medium containing 0.2 microM GA. The cells were then irradiated with X-rays and incubated with GA for a further 8 h. Radiation sensitivity was determined by clonogenic assays and protein levels were examined by Western blotting. RESULTS: GA radiosensitized both cell lines, but potentiated X-ray sensitivity more in SQ-5 than in DLD-1 cells. It was found that GA depleted EGFR and ErbB-2 in DLD-1 cells and depleted only ErbB-2 in SQ-5 cells. GA also reduced the expression of Akt and phosphorylated Akt (pAkt) expression in SQ-5 cells. In addition, the ratio (%) of apoptotic cells and poly [ADP-ribose] polymerase cleavage increased in SQ-5 but not in DLD-1 cells after exposure to GA and X-ray irradiation. The findings suggest that GA enhances the radiation sensitivity of human tumour cells by inhibiting the EGFR signal transduction system and the Akt signalling pathway. CONCLUSION: Targeting Hsp90 with GA provides a promising experimental strategy for radiosensitization of carcinoma.

Adenocarcinoma↗
PubMed2002

The 5'-upstream cis-acting sequences of a cyanobacterial psbA gene: analysis of their roles in basal, light-dependent and circadian transcription.

Transcription of the psbA2 gene in the unicellular photosynthetic cyanobacterium Microcystis aeruginosa K-81 is modulated by light and follows a circadian rhythm. In this study, we further characterized psbA transcription using a series of 5'-upstream deletions and mutant promoters which were tested in both photosynthetic and non-photosynthetic bacteria. Specific psbA2 transcripts were obtained from a minimal promoter sequence (-38/+14) with Escherichia coli RNA polymerases (RNAPs) both in vivo and in vitro, indicating the presence of a common regulatory mechanism for basal transcription. A DNase I footprinting assay showed that the E. coli RNAP, which is structurally similar to that of cyanobacteria, specifically binds to a large segment (from -115 to +23) of the sequence upstream of psbA2. In cyanobacteria, the -10 sequence (TAGTAT), but not the -35 motif (TTTACA), is essential for basal transcription by homologous and heterologous RNAPs that contain the major sigma factor. Each of the conserved thymidine nucleotides at positions -12 and -7 (underlined above) was essential, and both an insertion and a deletion in the spacer region of the promoter caused reductions in transcription. RNAP was able to bind to a mutant promoter lacking the -10 sequence, though this did not actually lead to transcription. Interestingly, a high level of arrhythmic circadian transcription was observed in mutants lacking the -35 region. In contrast, a mutation in the AU-box mutation, which controls the stability of the psbA2 mRNA, did not affect the circadian pattern of transcription. These findings demonstrate that light-dependent psbA2 expression is controlled at the transcriptional and post-transcriptional levels, whereas the circadian pattern of expression is regulated at the transcriptional level.

Base Sequence↗
PubMed2001

K(ATP) channels predominantly regulate conduit vessel tone in normoxic cat pulmonary arteries in vivo.

Through our investigations of the intact pulmonary circulation, we aimed to find out whether K(ATP) channels contribute to regulating basal vascular tone and to clarify which vascular segments dilate during K(ATP) channel activation under basal tone conditions. Using an X-ray television system on anesthetized cat lungs, we measured internal diameter (ID) responses to two K(ATP) channel inhibitors (glibenclamide and 4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl-hydrochloride (U-37883A)) and to an activator (levcromakalim) in normoxic pulmonary arteries. In conduit arteries (800-3000 microm ID), the inhibitors and activator induced larger ID constrictions (14-17%) and dilatations (29-32%), respectively. However, in resistance arteries (<500 microm), the constriction response was negligible and the dilatation response relatively small (5-10%). The data suggest that K(ATP) channels are active and capable of regulating basal vascular tone primarily within conduit pulmonary arteries even though these channels are present in all pulmonary arteries.

Adamantane↗
PubMed2001

Retraction.

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Retraction Notice↗
PubMed2001

An AU-box motif upstream of the SD sequence of light-dependent psbA transcripts confers mRNA instability in darkness in cyanobacteria.

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. The expression of the psbA2 transcript has been shown to be light-dependent as assessed under light and dark (12/12 h) cycling conditions. We aligned the 5'-untranslated leader regions (UTRs) of psbAs from different photosynthetic organisms and identified a conserved sequence, UAAAUAAA or the 'AU-box', just upstream of the SD sequences. To clarify the role of 5'-upstream cis-elements containing the AU-box for light-dependent expression of psbA2, a series of deletion and point mutations in the region were introduced into the genome of heterologous cyanobacterium Synechococcus sp. strain PCC 7942, and psbA2 expression was examined. A clear pattern of light-dependent expression was observed in recombinant cyanobacteria carrying the K-81 psbA2 -38/+36 region (which includes the minimal promoter element and a light-dependent cis-element with the AU-box), +1 indicating the transcription start site. A constitutive pattern of expression, in which the transcripts remained almost stable under dark conditions, was obtained in cells harboring the -38/+14 region (the minimal element), indicating that the +14/+36 region with the AU-box is important for the observed light-dependent expression. Point mutations analyses within the AU-box also revealed that changes in number, direction and identity (as assayed by adenine/uridine nucleotide substitutions) influenced the light-dependent pattern of expression. The level of psbA2 transcripts increased markedly in CG- or deletion-box mutants in the dark, strongly indicating that the AU- (AT-) box acts as a negative cis-element. Furthermore, characterization of transcript accumulation in cells treated with rifampicin suggests that psbA2 5'-mRNA is unstable in the dark, supporting the view that the light-dependent expression is controlled at the post-transcriptional level. We discuss various mechanisms that may lead to altered mRNA stability such as the binding of factor(s) or ribosomes to the 5'-UTR and possible roles of the AU-box motif and the SD sequence.

5' Untranslated Regions↗
PubMed2001

In situ EXAFS study on nickel metal particles dispersed on Loy Yang brown coal.

The structure of nickel catalysts supported on Loy Yang brown coal by an ion-exchanging method during thermal treatment was studied by a XAFS technique. The exchanged nickel species aggregated to metal particles during pyrolysis and hydrogasification. The aggregation behaviour of nickel species depended on the amount of nickel loaded and/or treatment atmospheres.

Journal Article↗
PubMed2001

XANES analysis of sulfur form change during pyrolysis of coals.

Three kinds of coal and their density separated samples were rapidly pyrolyzed in a free-fall reactor under a nitrogen stream at a terminal temperature of 1253 K. X-ray absorption near edge structure analysis was applied to directly determine the sulfur forms in the coal and pyrolysis char. The results showed that the low-density group contained sulfide and sulfoxide, which could contribute to the high extent of organic sulfur removal. Thermally stable thiophenic sulfur was observed in the higher density groups as a major form of organic sulfur.

Journal Article↗
PubMed2001

Cloning and characterization of adenylate kinase from Chlamydia pneumoniae.

Chlamydiae proliferate only within the infected host cells and are thought to be "energy parasites," because they take up ATP from the host cell as an energy source. In the present study, we isolated from Chlamydia pneumoniae the gene encoding adenylate kinase (AK). Using the enzyme produced in Escherichia coli, its properties were characterized. K(m) values for AMP and for ADP of the purified C. pneumoniae AK (AKcpn) were each 330 microm, which is significantly higher than the reported values of other AKs, whereas K(m) for ATP was 24 microm, which was rather lower than others. AKcpn contains 1 g atom of zinc/mol of 24,000-dalton protein. Mass spectrometric analysis of AKcpn and analysis of properties of mutated AKcpn strongly suggested that zinc is associated with four cysteine residues in the LID domain of the enzyme. The apo-AKcpn that lost zinc retained AK activity, although K(m) for AMP of apo-AKcpn increased about 2-fold and V(max) decreased about one-half from that of holo-AKcpn. The apo-AKcpn was more thermolabile and sensitive to trypsin digestion than the holo-AKcpn. Moreover, the recovery in vitro of the AK activity during the renaturation process of the denatured apo-AKcpn was dependent on zinc. A mutated protein in which cysteine residues in the LID domain were substituted by other amino acids lost both zinc and enzyme activity. The mutated protein was more sensitive to protease than the apo-AKcpn. These results indicate that zinc in AKcpn, although not essential for the catalysis, stabilizes the enzyme and probably plays a crucial role in proper folding of the protein. Furthermore, the catalytic properties of AKcpn suggest a distinctive regulatory mechanism in the metabolism compared with AKs in other organisms.

Adenylate Kinase↗
PubMed2001

Impaired development of HIV-1 gp160-specific CD8(+) cytotoxic T cells by a delayed switch from Th1 to Th2 cytokine phenotype in mice with Helicobacter pylori infection.

Th1 and Th2 cells play a central role in immunoregulation during infection. We show that Helicobacter pylori induces Th1 cytokine responses early (2 weeks) but predominantly Th2 responses later (6 weeks) in infection. The switch is principally mediated by urease-specific CD4(+) T cells, and correlates with a loss of urease-specific high-avidity JNK(+) Th1 and gain of low-avidity JNK(-) (possibly Th2) cells at the later stage of infection, concomitant with a 100-fold higher colonization level of H. pylori at 6 weeks than at 2 weeks that might tolerize high-avidity Th1 cells. Furthermore, differentiation of HIV gp160-specific CD4(+) Th and CD8(+) cytotoxic T lymphocytes (CTL) into effector cells is impaired in 6-week H. pylori-infected mice immunized with vaccinia expressing gp160, and serum IL-12 stimulated by vaccinia infection is barely detectable. Adoptive transfer of urease-specific Th2 cells to mice infected only with gp160-expressing vaccinia abrogates Th1 polarization of the gp120 response, down-modulates virus-specific CTL responses, and delays virus clearance. Therefore, the H. pylori urease-mediated immunoregulation in the switch from JNK(+) Th1 to JNK(-) Th2 phenotype, and the preceding low IL-12 response, are likely critical steps in the impairment of antiviral immunity.

Animals↗
PubMed2001

Allelic exchange mutagenesis of rpoN encoding RNA-polymerase sigma54 subunit in Helicobacter pylori.

The rpoN gene, encoding the alternative sigma factor (sigma54) of Helicobacter pylori, was amplified from genomic DNA. H. pylori rpoN has an overall similarity to the rpoN of other bacteria, but lacks a glutamine (Q)-rich region in region I and an acidic region in region II. When the rpoN gene was disrupted, the mutant was found to be completely nonmotile. Because the flaB gene has an rpoN consensus sequence in its promoter region, we assessed the transcriptional activity of the flaB gene, using xylE transcriptional fusion. In the isogenic mutant of rpoN, transcription of the flaB gene was severely affected, but transcription of the ureA gene (control) was intact. In late stationary phase, the rpoN mutant showed marked decreases in viability: i.e., the number of colony-forming units (CFU) at 100 h was 4 log lower in the rpoN mutant than in the wild-type strain. By morphological examination with acridine orange staining, the rpoN mutant showed green and faintly orange-stained irregularly shaped cells with a few orange-stained rod/spiral cells. In contrast, the wild-type strain and the non-flagella flgE mutant (control) contained many orange-stained rod/spiral and coccoid cells. These results indicated that in H. pylori, RpoN is involved not only in motility but also in viability, through the morphological changes in the stationary phase.

Alleles↗
PubMed2001

Analysis of the serological response to Chlamydia pneumoniae in patients with ischemic heart disease by recombinant MOMP-ELISA.

To investigate the humoral immune response to the major outer membrane protein (MOMP) of Chlamydia pneumoniae, a fusion protein, thioredoxin-(His)6-MOMP (rMOMP) was produced in Escherichia coli and purified; this served as an antigen to establish an enzyme-linked immunosorbent assay (ELISA). Specific IgG and IgA antibodies against rMOMP were determined in sera from patients with ischemic heart disease. The findings were compared with those obtained by ELISA using the outer membrane protein complex (Hitazyme). The positivity rates for IgG antibody by rMOMP-ELISA were low (28%) compared with those by Hitazyme (72%). However, the positivity rates of IgA antibody by rMOMP-ELISA were similar to those by Hitazyme (76%). Interestingly, antigen positivity by immunohistochemical staining in the atherosclerotic lesions of coronary arteries was high in the groups with a high IgA titer of rMOMP-ELISA.

Aged↗
PubMed2001

Expression of vinexin alpha in the dorsal half of the eye and in the cardiac outflow tract and atrioventricular canal.

Vinexin, a recently identified cytoskeletal protein, contains three SH3 domains and plays important roles in regulation of cytoskeletal organization and signal transduction. Using whole-mount in situ hybridization, we showed here that expression of vinexin alpha, the longer vinexin transcript, is strictly regulated, although the shorter transcript, vinexin beta, is expressed almost ubiquitously during embryonic development in mice. Expression of vinexin alpha was limited to within part of the eye and heart in 10.5 dpc embryos. Analysis of cryosections of 10.5 dpc embryos showed that vinexin alpha was expressed in a dorsal half of the retinal pigment epithelium and in the outflow tract and atrioventricular canal of the heart. Furthermore, we also found that vinexin alpha was expressed in the gonad and in a ventral part of the pons of 12.5 dpc embryos. These results indicated that the expression of vinexin alpha is strictly regulated in a temporally and spatially restricted manner.

Animals↗
PubMed2001

Inhibitory effect of a quercetin metabolite, quercetin 3-O-beta-D-glucuronide, on lipid peroxidation in liposomal membranes.

To study the antioxidant activity of quercetin 3-O-beta-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after intake of quercetin-rich food, the inhibitory effect of Q3GA on lipid peroxidation was estimated using phosphatidylcholine large unilamellar vesicles (PC LUV) as a biomembrane model. Iron ion, an aqueous peroxyl radical generator, a peroxynitrite generator, or lipoxygenase was used as the inducer of lipid peroxidation. In all cases, Q3GA inhibited lipid peroxidation significantly, although its inhibitory effect was lower than that of quercetin aglycon. The ultrafiltration of PC LUV containing Q3GA revealed that Q3GA has low but significant affinity with the membranes of phospholipid bilayers. It is therefore likely that Q3GA acts as an efficient antioxidant in membranous lipid peroxidation through its localization in the phospholipid bilayer. This conjugated quercetin metabolite seems to retain the ability to protect cellular and subcellular membranes from peroxidative attack by reactive oxygen species and peroxidative enzymes.

Arachidonate 15-Lipoxygenase↗